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ALICEA-CRUZ, GIL MARIE,  Pontifical Catholic University of Puerto Rico, MARC Honor Program Student;   Castilloveitia-Rosa, Alma Y., Biology, Pontifical Catholic University of Puerto Rico; Ruiz, Eileen; Hernández-Muñiz, Wilfredo; Biochemistry, Ponce School of Medicine

The Association Between CC16 Polymorphism (A38G) and Asthma in the Puerto Rican Population

Asthma is a chronic inflammatory disease of the airways that is characterized by airway obstruction, airway inflammation, and bronchial reactivity. It is caused by combination of genetic and environmental factors. The prevalence of asthma is increasing worldwide. The identification of genetic risk factors for asthma will help to select predisposed subjects for allergen avoidance or immunoprophylaxis measures during infancy, to define the molecular basis of clinical subtypes of the disease, and to improve its pharmacological management, targeting specific abnormalities expressed by the genes involved. Most studies on the genetics of asthma have focused on candidate genes suspected to be involved in the pathogenesis of the disease. This investigation focused on Clara Cell Secretory Protein (CC16) and its association with asthma in Puerto Rican population. CC16 is 16- KD protein primarily expressed in the respiratory tract by noncilated bronchiolar secretory cells, accounting for 7% of the total protein content in the bronchoalveolar lavage fluid. 60 asthmatic individuals were analyzed by restriction enzyme digestion of PCR product for the presence of the polymorphism (A38). There were 82 controls.  Although linkage with the CC16 polymorphism and asthma were found previously in the Australian population, our studies conclude that there is no definite link between this gene and asthma in the Puerto Rican population.

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ALMODÓVAR, WANDA; Martínez Cruzado, J.C., UPR-MAYAGÜEZ, Departamento de Biología

Contribución de los Distintos Haplogrupos en el Genóma Puertorriqueño A Través de la Línea Paterna 

El propósito a largo plazo del estudio es determinar la contribución de los distintos grupos continentales, africanos, europeos e indígenas, al genoma puertorriqueño.  Como un segundo paso siguiendo a la caracterización del DNA mitocondrial, el cual se hereda por vía materna, estamos analizando la proveniencia en la población puertorriqueña del cromosoma Y.  El cromosoma Y se hereda por la vía paterna y, al igual que el DNA mitocondrial, tiene una gran porción que nunca se recombina.  Esto permite la acumulación progresiva de mutaciones cuyo estudio, al combinarse con conocimientos sobre la distribución geográfica de los mismos, reconstruye la historia de migraciones de poblaciones humanas.  Analizamos para el cromosoma Y, usando técnicas de PCR y restricción enzimática, 14 muestras de DNA de estudiantes de la Universidad de Puerto Rico-Mayagüez, procedentes de distintas áreas de la isla como lo son Yauco, Villalba, Utuado, San Sebastián, Guaynabo, Carolina, Camuy y Caguas.  De las muestras, un 43% resultaron pertenecer al haplogrupo 1C, que comprende el 68% de los españoles y el 33% de los indios de América.  Las restantes muestras están siendo analizadas.  De éstas, un 21% parecen ser 1C y un 14% se inclina hacia los haplogrupos africanos y sur africanos, pero podrían ser europeos o sureuropeos.

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ALMODÓVAR, JORGE; Sánchez, Aixa; Diffoot, Nanette, Prof., UPR-MAYAGÜEZ, Laboratory of Virology, Department of Biology

 Determination of Encapsidation Pattern of LuIII and MvMi Chimeric Plasmid

LuIII and MvMi are single-stranded DNA viruses with palindromic termini belonging to the family Parvoviridae. Because of their ability to infect their host without apparent pathogenic symptoms, parvoviruses are considered good vectors for gene therapy. In order to determine proper protocols for future applications the mechanisms of replication of the virus must be known. Studies comparing LuIII and MvMi show an 80% identity between the genomes of the viruses. This phenomenon contrasts with the fact that 99% of the assembled MvMi particles have a negative polarity genome, while LuIII encapsidates both strands with equal frequency. In order to determine what part of the genome determines the strand to be encapsidated we have employed the use of chimeric plasmids which contain the genome of LuIII and the termini of MvMi. It is suggested that the signals necessary for replication and encapsidation reside in the termini of the viral genome, yer whether they determine the encapsidation pattern is not known. Hela cells have been transfected with CC6 to determine if the plasmid is capable of replicating and if so, which viral strand is encapsidated. Hela cells transfected with a known replicating plasmid have been used as a positive control of replication. Southern Blot analysis was employed to detect the replication of viral genome in the cells and determine the encapsidation pattern of CC6.

BATISTA CAMACHO, MIGDALIA; Gutiérrez Sánchez, Jaime, UPR-MAYAGÜEZ, Departamento Ciencias Sociales-Psicología

Demografía, Actitudes y Patrones de Movilidad de los Usuarios de Carro Público en el Área Metropolitana de San Juan

En este estudio se mira al transporte público desde la perspectiva de quienes con frecuencia viven la experiencia del carro público como modo de transporte en el Área Metropolitana de San Juan. También, el estudio es importante  para planificar y establecer cuáles características deben ser atendidas y / o consideradas para ofrecer una transferencia eficiente de los carros públicos al sistema de transporte del Tren Urbano. Los propósitos de este estudio son 1) Describir el perfil demográfico de los usuarios de los carros públicos. 2) Medir las actitudes de los usuarios de carros públicos hacia el carro privado y el carro público. 3) Conocer los patrones de comportamientos en términos de la movilidad y la accesibilidad de los usuarios de carros públicos. 4) Señalar cuáles son las áreas que este sistema de transporte público debe mejorar desde la percepción de los usuarios para proveer un servicio inter-modal eficiente. Se entrevistaron a 100 usuarios de carro público en el área de Bayamón, Río Piedras y Centro Médico.

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BAUTISTA, KARYM; Massol, Arturo, Prof., UPR-MAYAGÜEZ, Department of Biology

Studies and Identification of Microbial Infections in Lepidoptera spp., Danaus plexippus 

Danaus plexippus is the scientific name given to monarch butterflies. These butterflies, like other moths, undergo four different stages: egg, larva, pupa, and adult. One factor likely to regulate the abundance of monarchs is their interactions with natural enemies. The purpose of our research is to study and identify the causes of death in caterpillar (larva) cultivated in a butterfly garden (CasaPueblo, Adjuntas). It is thought that some microbial infection is the major cause of these abnormalities. We are trying to identify the microorganism that is causing the infection by hemolymph analysis of infected larva. Bacillus thuringiensis is the principal suspect; because this microorganism is very specific when it infects Lepidoptera spp. The isolation of a bacillus from the hemolymph of larvae demonstrates the presence of a Gram (+), endospore forming bacilli present only in infected individuals. This culture was applied to milkweeds from which the larva fed.  One of the systems was used as control which consisted of healthy larva feeding on milkweed soaked with distilled water.  The experimental system consisted of milkweed which was initially treated with a dilution of the isolated bacilli in distilled water. Our results showed a 100% death rate for the larvae feeding on infected milkweed while all larvae from non infected system developed normally.

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BONILLA-VÉLEZ, JOHNNY; Arrocho-Vega, Yanice; Ersan-Said, Sufyan; Babilonia-Marichal, Maritere; Muñiz-Perez, Darlene; Reyes-Quiñones, Tania; Jusino-Atresino, Rafael,  UPR-AGUADILLA, Natural Sciences, Natural Science

Study of Biodiversity and Foraging Behavior of Ants at Mata dePlatano Reserve in Arecibo, Puerto Rico 

At the Mata de Platano reserve, in front of the Culebrones cave in Arecibo, Puerto Rico, a study about the clasification of ant genera  and their foraging behavior took place. To collect the data we used three diferents kind of traps (bait traps, pitfall and underground traps). The bait trap experiment lasted 24 hours where every 3 hours a trap was pleced on the ground and pased 15 minutes are pick up. The pitfall traps were placed level with the surface up to seven days. and the undergroun ones just for 6 hours. After the traps were collected, they were placed in a cooler to preserve the insects, so it could arrive to the laboratory. In the the laboratory the ants are cleaned and placed on vials with etanol for preserve it. The ants were observed under a stereoscope so thy  could be classified by their genera. Betwen the most abundant genderswe found Paratrechina, Wasmannia, Pheidole and Brachymyrmex. We could observe some foraging behavior like niche partition betwen some of the genders found.

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CABÁN, HÉCTOR, UPR-HUMACAO, Department of Biology; Planas, José, Prof., UPR-AGUADILLA, Department of Biology

Determination of Allelic Variation of Immunoglobulin Vh1 Family Genes by Single Sperm Analysis

The human immunoglobulin heavy chain locus is on chromosome 14q32.3 and consists of ~120 VH gene segments, ~20 DH, 6 JH and 11 CH gene segments. One hundred and twenty three VH gene segments have been identified and are subdivided into seven families (VH1-VH7) based on their degree of sequence identity. The purpose of this project is to study the allelic variation of VH gene segments at each locus for the gene segments in the VH1 family by using single sperm cells. Semen samples will be collected from unrelated healthy donors with highly diversified genetic backgrounds to allow us to have an overview of the VH region diversification in the entire human population. Three different primers (F forward, R reverse and N nested) were designed to amplify gene segments in the VH1 family which allowed us to amplify the genic region together with 5’ regulatory elements and recombination signal sequences. Single sperm are obtained by laser microdissection. PCR with a step involving nested primers is performed to amplify specific gene segments from single cell. PCR products are purified and further sequenced to determine allelic variation between different individuals.

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CABRERA, MIGUEL, A., UPR-CAYEY, General Science Program, Ayala-Torres, Sylvette, Universidad Central del Caribe, Department of Pharmacology 

Age-associated Damage to Brain Mitochondrial DNA and In Vivo Induction of Damage after 3-NPA 

Reactive oxygen species (ROS) cause damage to DNA, primarily to mitochondrial DNA and this damage can lead to mitochondrial dysfunction. Chronic exposure to ROS has been associated with several degenerative diseases including aging, the molecular basis of which remains unknown.  Increasing evidence implicates oxidative damage to mitochondrial DNA (mtDNA) as a major contributor to the age associated loss of brain function in the mouse.  We sought to test the hypothesis that aging brain will exhibit greater basal levels of mtDNA damage than younger brain and that the mitochondrial neurotoxin 3-NPA will lead to an increase in oxidative mtDNA damage in the brain.  We used a quantitative polymerase chain reaction assay to assess the relative amounts of DNA damage in the mitochondrial genome in brain obtained from C57Bl/6 mice of different ages injected with 3-NPA and the aged matched controls.  Our results show an age associated increase in basal levels in brain mtDNA damage and that 3-NPA leads to an increase in oxidative mtDNA damage preferentially in young brains.  Our results raise the possibility for a role for mtDNA damage in brain aging and neurodegeneration.

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CABRERA, ARELYS; Quiñones, José L.; García, José E., Prof., UPR-RÍO PIEDRAS, Departamento de Biología        

Un Posible Rol de la Fibronectina en la Regeneración del Intestino de Holothuria glaberrima 

Fibronectina es una glucoproteína que se encuentra en la materia extracelular en donde lleva a cabo varias funciones asociadas a la regulación, adhesión, migración y crecimiento celular. La molécula de fibronectina tiene una secuencia conocida como RGD (Arg-Gly-Asp),  la cual es la  necesaria para el reconocimiento  por integrinas en la superficie celular, y así mantener una comunicación entre componentes de la materia extracelular y el citoesqueleto.  El pepino de mar, Holothuria glaberrima, es un invertebrado que tiene la capacidad de regenerar sus visceras luego de eviscerar las mismas. Estudios previos han demostrado que durante la regeneración u organogénesis ocurre una remodelación de la matrix extracelular, así como cambios en la composición de la misma. Uno de los procesos que aparentemente sucede en la formación del nuevo órgano  es la migración celular.  Para estudiar un posible rol de la fibronectina en la regeneración intestinal de H. glaberrima,  se trataron animales con pétidos sintéticos que compiten con la secuencia de RGD, por la interacción con integrina.  Los péptidos utilzados fueron GRGDTP ( Gly-Arg-Gly-Asp-Thr-Pro) y RGD (Arg-Gly-Asp) en concentraciones entre 0.8 – 10 mg/ml. Grupos controles fueron inyectados con una solución salina (PBS) o con un  péptido control que no compite por la secuencia de RGD. Las estructuras regeneradas se analizaron mediante inmunohistoquímica para colágeno y músculo, y se midieron la cantidad de células presentes. Los resultados preliminares indican que los animales que fueron inyectados con RGD demuestran un atraso en la formación del músculo y en la migración celular dando como indicio que la fibronectina tiene una función importante en la regeneración intestinal de equinodermos. Financiado por NSF, NIH-MBRS, AMP y la Universidad de Puerto Rico.

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CANTRES, ONIX; Colón, Wilma; Pérez-Chiesa, Ivette, Prof., UPR-RÍO PIEDRAS, Departamento de Biología

Secuenciación y caracterización parcial de los genes  Adh y Adhr de Drosophila cardini 

El gen que codifica para la enzima deshidrogenasa de alcohol (ADH) en Drosophila ha sido analizado extensamente en estudios de evolución molecular.  La enzima tiene una función importante en la detoxificación de alcoholes.   El propósito de este estudio es secuenciar los genes Adh y Adhr de la especie D. cardini para contribuir al entendimiento de la evolución del gen Adh en el género Drosophila y para describir las relaciones filogenéticas entre los miembros del grupo cardini.  Se extrajo DNA usando el GNOME DNA Kit (Bio 101).  Se utilizaron “primers” específicos para amplificar, mediante PCR, dos fragmentos que abarcan desde el exón 1 de Adh hasta el exón 1 de Adhr; y desde el exón 3 de Adh hasta el exón 2 de Adhr.   Los productos de PCR fueron secuenciados usando el equipo Automated DNA Sequencer Model ABI373A (Applied Biosystem) y el PRISM DyeDeoxy Terminator Kit.  Las secuencias resultantes fueron analizadas para describir la estructura de los genes Adh- Adhr, y describir los cambios evolutivos de la región al compararla con la de especies cercanas.  La secuencia va a ser utilizada posteriormente en la construcción de un “gene-tree” para aclarar las relaciones filogenéticas entre los miembros del grupo cardini.

Auspiciado por FIPI y Howard Hughes

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CASTILLOVEITIA ROSA, ALMA; Alicea Cruz, Gil Marie, PUCPR, Department of Biology, MARC Honor Program; Ruiz, Eileen; Hernández Muñiz, Wilfredo, Prof., PUCPR, Ponce School of Medicine, Biochemistry    

Lack of Association Between Il-4 Polymorphism (C589T) and Asthma in the Puerto Rican Population 

Asthma is a chronic inflammatory disease of the airways that is characterized by airway obstruction, airway inflammation, and bronchial reactivity. It is caused by combination of genetic and environmental factors. The prevalence of asthma is increasing worldwide. The identification of genetic risk factors for asthma will help to select predisposed subjects for allergen avoidance or immunoprophylaxis measures during infancy, to define the molecular basis of clinical subtypes of the disease, and to improve its pharmacological management, targeting specific abnormalities expressed by the genes involved. Most studies on the genetics of asthma have focused on candidate genes suspected to be involved in the pathogenesis of the disease. This investigation focused on human interleukin-4 (IL-4) and its association with asthma in Puerto Rican population. They examine a large population of asthmatics for association between the genetic variant in the IL-4 promoter (C589T) and asthma severity. 98 asthmatic individuals were analyzed by restriction enzyme digestion of PCR product for the presence of the polymorphism (C589T).  Although linkage with the IL-4 polymorphism and asthma were found previously in the Caucasian and African American population, our studies conclude that there is no definite link between this gene and asthma in the Puerto Rican population.

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COLLAZO, GRETCHEN, UPR-HUMACAO, Department. of Biology; Muller, Rafael, Prof., UPR HUMACAO, Department of Physics

Image Capture of ”Comet Cells” with an inexpensive, high-resolution CCD camera 

An inexpensive, high-resolution CCD camera is coupled to a microscope optimized for fluorescence. The system is used to capture images of snail mantle cells that have been subject to electrophoresis. The cells, which have been subjected to chemical agents, might show different types of damage to their DNA when exposed to fluorescence. It has been difficult to obtain high-resolution images of these so called “comet cells”, and very expensive microscopy/imaging systems have been required for such a task. We have succeeded in obtaining high-resolution images of DNA comet cells with an inexpensive high-resolution CCD camera optimized for astronomy coupled to a microscope optimized for fluorescence. We present the results obtained with our inexpensive system.

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COLÓN VILLAFAÑE, OLVIA C.; Martínez Cruzado, Juan C., Prof., UPR-MAYAGÜEZ, Arts and Sciences, Department of Biology

Identification of Population Specific Y-Chromosome Haplogroups to Trace Puerto Rican Ancestry 

A common interest to uncover Puerto Rican heritage has recently been addressed by the identification specific polymorphisms within the Y chromosome.  The unchanging portion of the Y-chromosome that is transmitted from father to son is only exposed to mutations that in occasions have been geographically confined known as haplogroups[1].  Usefulness of the Y chromosome for evolutionary studies has been subject to the findings of common population polymorphisms[2].  A collection of previously identified polymorphisms in the Y-chromosome was gathered in order to detect their presence within the male Puerto Rican population.  A philogenetic tree has been constructed in order to methodically test male oral samples by the use of targeted PCR reactions and enzyme restrictions.  Preliminary results indicate that 38% of the samples collected belong to the IC haplogroup, which has been identified in European or Native American populations, over Eastern European 1D and Iberian 22 groups.  Currently, 23% of the samples are being tested for 1F haplogroup by the identification of mutation in the RPS4Y locus characteristic of Native American population and PN2 polymorphism leading to haplogroup 4, which has been identified in some European and African populations.  Data presented is part of a work in progress.

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COLÓN WILMA; Cantres, Onix, Pérez-Chiesa, UPR-RIO PIEDRAS, Natural Sciences, Biology Department 

Secuenciación y Caracterización de los Genes Adh y Adhr de Drosophila cardini

El gen que codifica para la enzima deshidrogenasa de alcohol (ADH) en Drosophila ha sido analizado extensamente en estudios de evolución molecular.  La enzima tiene una función importante en la detoxificación de alcoholes.   El propósito de este estudio es secuenciar los genes Adh y Adhr de la especie D. cardini para contribuir al entendimiento de la evolución del gen Adh en el género Drosophila y para describir las relaciones filogenéticas entre los miembros del grupo cardini.  Se extrajo DNA usando el GNOME DNA Kit (Bio 101).  Se utilizaron “primers” específicos para amplificar, mediante PCR, dos fragmentos que abarcan desde el exón 1 de Adh hasta el exón 1 de Adhr; y desde el exón 3 de Adh hasta el exón 2 de Adhr.   Los productos de PCR fueron secuenciados usando el equipo Automated DNA Sequencer Model ABI373A (Applied Biosystem) y el PRISM DyeDeoxy Terminator Kit.  Las secuencias resultantes fueron analizadas para describir la estructura de los genes Adh- Adhr, y describir los cambios evolutivos de la región al compararla con la de especies cercanas.  La secuencia va a ser utilizada posteriormente en la construcción de un “gene-tree” para aclarar las relaciones filogenéticas entre los miembros del grupo cardini.

CORTÉS, CARLA;  Tossas, A.,  Departamento de Biología, UPR-RÍO PIEDRAS; Fumero, José, Escuela Graduada De Biología, Universidad de Puerto Rico; Elvia Meléndez-Ackerman, Instituto De Ecosistemas Tropicales-Biología, UPR-RÍO PIEDRAS       

Breeding System in Populations of Pitcairnia angustifolia (Bromiliaceae) 

Outcrossing rates should be favored when selection acts to reduce inbreeding depression or by virtue of maintaining high levels of genetic diversity there by increasing the evolutionary potential of the species.  Self-pollination may evolve under a number of conditions. From the ecological point of view it should be favored when the pollinators are scarce and from the evolutionary point  of view it should be favored in the absence of inbreeding depression. Local populations of  Pitcairnia angustifolia show significant differences in their pollinator abundance. Measures of reproductive success will be higher for pollinations and seeds derived from selfed pollen in P.angustifolia provided that there is inbreeding depression. Inbredding depression will be more intense in the population of Rio Abajo, but less intense or else absent in El Verde and Maricao. We tested this hypothesis by comparing the performance of plants produced by artificial self- and cross- pollination for three populations of P.angustifolia. We tested differences in treatments for fruit set, seed weight, number of seeds, seed germination and mortality.  Growth rate was also determined between treatments for each population. Paired-t test was made to determine differences between treatments among populations. We found significant reduction in the performance of selfed progeny in the population of Rio Abajo and increased fruit set of self-pollination at El Verde. There was no significant evidence to conclude that plants derived from selfed pollination express better fitness that those derived from cross pollination.

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COSME BLANCO, WILFREDO; Arroyo, Nancy; Diffoot, Nanette, Prof., UPR-MAYAGÜEZ, Department of Biology

Insertion of a Consensus Sequence of a Yeast ARS in Various Plasmids to Increase the Efficiency of Transformation of Saccharomyces cerevisiae 

The autonomous replicating sequence (ARS) confers the capacity of autonomous replication to non-replicating DNA.  The analysis and determination of the structure and function of the ARS was initially studied in S. cerevisiae. Previous studies indicate that the presence of an A/T rich sequence, 47 bases long, in parvovirus LuIII could function as an ARS - like sequence (unpublished results). The 47 base pair sequence was inserted into a vector derived from puc19, pGN3, which has the URA 3 gene from S. cerevisiae as an auxotrophic marker. The resulting clone, named pURA-LuA/T, was shown to transform yeast cells and replicate autonomously. In comparative DNA studies of the parvovirus LuIII and a yeast ARS consensus sequence (5’ - T/A TTTA T/C A/G TTT T/A - 3’) a 10 base pair sequence (TTTTATTATTTT) was found to have a near perfect match (91 %) with the yeast consensus sequence. The 10/11 sequence in LuIII, located downstream of the A/T rich sequence was inserted into the pURA-LuA/T, transformation of this clone. These resulted in variable results in terms of the colony morphology. My specific role in this project is to attempt to insert an 11/11 sequence into previously constructed plasmids to try to increase the efficiency of transformation of S. cerevisiae. This 11/11 will be inserted into pURA-Lu88-100 (pGN3 with maps units 88–100 of the LuIII genome) and pURA-LuA/T. The plasmids have been digested with Bam HI. The 11/11 sequence was inserted into the respective plasmids and ligated accordingly. Once constructed, the plasmids were used to transform competent E.coli DH5a. The DNA of the resulting transformants were separated by gel electrophoresis and transferred for Southern Analysis.  Clones containing the appropriate units will be used to transform  S. cerevisiae with the clones above and study the efficiency of transformation.

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CRUZ, FERNANDO, UPR-MAYAGÜEZ, Artes y Ciencias,UPR-MAYAGÜEZ; Gregory, Meredith; Pérez, Víctor; Ksander, Bruce Schepens, Eye Research Institute, Harvard Medical School

Role of Membrane and Soluble Fas Ligand in Corneal Immunity

CUMBA, JAIMARI, UPR-CAYEY, Biology; Ferrer, Ivan, Ph.D.; Serrano, Adelfa E., PhD., Department of Microbiology and Medical Zoology, UPR-School of Medicine, San Juan

Plasmodium yoelii and Plasmodium berghei: Identification of Point Mutations Associated with mdr1 Gene 

Amplification, mutations, or overexpression of th pfmdr1 gene have been associated with multiple drug resistance in some strains of Plasmodium falciparum.

In order to better undertand the role of the mdr genes in drug resistance, we are using the in vivo murine malaria models P. berghei and P. yoelii. The objectives of our research are to characterize the mdr1 gene and to determine its relationship with drug resistance.

In this research the investigators used PCR reaction to amplified mdr1 gene from P. yoelii and P. berghei genomic DNA. These fragments were cloned and DNA sequence determined and analyzed. Preliminary experiments suggest that there are no point mutations in mdr1 genes related to drug resistance in any of the P. yoelii and P. berghei resistant lines.

DE LEÓN DÍAZ, LEE A; Rodríguez, Armando, Prof., UIA-BAYAMON, Artes y Ciencias, Departamento de Ciencias Naturales y Matemáticas

Patrones de Actividad y Selección de Alberge del Murciélago de los Techos (Molossus molossus) 

El propósito de la investigación es determinar los patrones de actividad del murciélago Molossus molossus y las condiciones térmicas del alberge.  Para esto se realizó un conteo de los murciélagos a la hora en la que salen, en una casa donde el techo es de zinc, ubicada en el barrio Higuillar de Dorado.  Se midió la temperatura en el albergue de manera continua y se estudió el comportamiento del murciélago a diferentes temperaturas en una incubadora.  Molossus molossus busca las temperaturas más bajas en su albergue y su patrón de salida está asociado a la puesta del sol. 

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DE LEÓN DÍAZ, LEE A; Rodríguez, Armando, Prof., UIA-BAYAMON, Artes y Ciencias, Departamento de Ciencias Naturales y Matemáticas

Identification of Cross-Reacting Antigens between Plasmodium falciparum and Ascaris suum 

Plasmodium falciparum a parasitic protozoan of the blood, is the cause of a terrible disease, with a high number of cases and mortality known as Malaria.  In the endemic areas were Malaria can be found, it can co-exist with other parasitic infections endemic in the same region, as in the case with the intestinal nematode of man Ascaris lumbricoides. Since it is well known that Ascaris lumbricoides share many antigenic components with  other species of parasitic organisms, and is quite difficult to obtain samples from persons infected with this parasite; instead were using the intestinal nematode of  pigs Ascaris suum. 

The life cycle of both Ascaris lumbricoides and Ascaris suum are quite similar, eggs are ingested, the larvae migrate to the lungs, after two weeks are re-ingested and the adults live in the small intestine. 

Both share common antigenic components, and Ascaris suum is easily obtained from slaughterhouses. The purpose of this study is the identification of homologous antigenic components between Plasmodium falciparum and Ascaris suum. SDS-PAGE, Western blot and Silver Stain Techniques were used to attain this. The advantage of identifying the antigenic components shared between Plasmodium falciparum and Ascaris suum can help to identify specific components, so a more sensitive diagnostic procedure could be applied in the future.    

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DELGADO, MANUEL; Suárez, Melvin; Quiles, Yasmín; Puente Rolón, Alberto, Prof., UIA-ARECIBO, Departamento de Ciencias y Tecnología

Comparison of External Bacterial Flora between Newborns and Adults of the Puerto Rican Boa (Epicatres inornatus) 

The Puerto Rican boa (Epicrates inornatus) is the largest native species of snake in the Island.  This species was included in the federal list of endangered species in October 13, 1970.  Sterile swabs and phosphate buffered saline (PBS) were used to take samples from the ventral, cloacae, oral (in adults) and dorsal area, using aseptic techniques.  The identification process of microorganisms was throughout biochemical tests and rapid identification systems such as Micro Scan and Enterotube.  A total of forty-seven bacteria were found at this moment in adults and newborns of the Puerto Rican Boa. The most common bacteria found was Staphylococcus and Bacillus, between others. It is important to emphasize that larger quantities of bacterium were found on the dorsal and cloacal area.  Identification still in process.

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ESCALONA, YMA; Barbosa, Juan M., Prof., UIA-BAYAMON, Arts and Sciences, Department of Natural Sciences and Mathematics

Identification of Cross-Reacting Antigens Between Plasmodium falciparum and Ascaris suum

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FAJARDO HEREDIA, DOUGLAS; Bendezú, Pedro, Prof., UIA-METRO, Artes y Ciencias, Departamento de Biología

Micropropagación en  Violeta Africana

Con esta investigación se pretende instruir y enseñar a la audiencia estudiantil  y la particular de los procedimientos adecuados  que requiere una micropropagación en este caso de la  violeta Africana.

Para comenzar se necesitó  buscar la solución adecuada a la  planta.  A pesar de que en el mercado se consigue el alimento liquido para la misma no se siguió con ese parámetro y se comenzó  a hacer   distintas  formulas  con diferentes micronutrientes  y macronutrientes de las cuales se obtuvieron once.

 El 11-10-01 se colocó cada pistilo de la planta en las once soluciones para entrar en observación. El 22-10-01 se comenzó a observar raices en la solución cinco y cinco dias después en solución nueve. Con esto se verificó el mejor “stock”y se prosiguió  a  concentraciones altas y bajas para tener alta eficacia de la preparación del  agar-agar se está manteniendo en la actualidad en ésta observación y   para la fecha de marzo se pretende haber conseguido la micropropagación para dicha exposición y a la vez continuar con la investigación durante todo el año para tejidos de cultivo y la forma de  que la comunidad tome provecho .

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FLECHA-FLECHA, SARILVETH; Tremblay, Raymond L., Prof., UPR-HUMACAO, Ciencias Naturales, Departamento de Biología

Vigor del Híbrido en un Cruce Entre Dos especies de Orquídeas, Lepanthes rupestris y Lepanthes woodburyana en Medios Asimbióticos

La hibridización en orquídeas, se da como una práctica con fines mayormente comerciales y también para estudios biológicos. Un híbrido es el resultado de un cruce de dos especies o variedades diferentes. Para determinar si un híbrido en orquídeas del género Lepanthes tiene mayor vigor que las especies parentales, se realizó un cruce entre Lepanthes rupestris y Lepanthes woodburyana. Estas semillas fueron sembradas en medio asimbiótico para observar su crecimiento y se comparó con un cruce entre dos individuos de Lepanthes rupestris y dos de Lepanthes woodburyana. Las semillas de las especies parentales fueron sembradas por separado y todas fueron observadas por un periodo de 6 meses. Los resultados obtenidos sugieren que existe mayor vigor del híbrido.

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FLORES, JACQUELINE; Barbosa, Juan M., Prof., UIA-BAYAMON, Arts and Sciences, Department of Natural Sciences and Mathematics

Identification of the Secretory – Excretory Components of Ascaris suum

Ascaris suum (A. suum) is a parasitic nematode whose adult stage lives in the pig intestine. Unlike females, whose size is 40 cm. the size of the males is of 15 – 25 cm. Its life cycle is very similar to that parasitic nematode present in human beings, Ascaris lumbricoides. Once the infective eggs are ingested, the larvae lodge in the small intestine. Immediately, after the larvae perforate the intestinal wall, they pass to the circulatory system and are transported to the lungs. In the lungs they suffer a molt, mature after two weeks in the lung’s tissue and are re – ingested. Finally, they develop as adults in the small intestine, completing their life cycle.

The purpose of this research relies on the Identification of the Secretory – Excretory (S/E) Components of A. suum. The adults were obtained from pig’s intestines that were sacrificed in the slaughterhouse La Muda, in Caguas, P.R. Once in the laboratory, the adults were rinsed several times with 0.85% saline solution and then their uterus were dissected with the purpose of obtaining their fertilized eggs. These were dispersed in the same solution with 1 – 2% of Formaline to avoid contamination and growth of other microorganisms. In a period of two weeks we observed the different stages of the eggs development.

Subsequent to the larvae’s development, these were rinsed with sterile 0.85% saline solution and were homogenized to obtain its soluble products by extracting at 4  C overnight (WLE: Whole Larvae Extract). The larvae were cultured for two weeks in sterile saline to obtain the S/E products.

The WLE and the S/E were analyzed via the SDS – PAGE technique. Future analysis will involve infecting mice with the infective eggs, inoculating intramuscular tissue with the WLE and the S/E products to obtain their serum and analyze the patterns via the Immunoblot technique.

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FONTÁN NAVARRO, LUDALIS Z.; Seda Miró, Jasmine; Pérez Santos, Noemí, UPR-ARECIBO, Department of Biology; Méndez, Abel, Prof., UPR-ARECIBO, Department of Physics and Chemistry

Planetary Microbial Ecology: Microbial Growth Kinetics in Near-surface Planetary Environments

Microorganisms are subject to extreme physical fluctuations at short spatial and temporal scales in natural environments. Factors such as temperature, humidity and radiation levels affect their survival and growth rates. In particular, daily temperature oscillations of soils control microbial growth rates. Most experiments regarding this issue have been done under constant laboratory conditions. Here we report preliminary experiments and techniques to measure the growth rate of bacteria under the temporal influence of natural physical variables. We are constructing an experimental setup to continuously measure the growth rate of bacteria exposed to the daily natural temperature fluctuations of soils but otherwise enclosed in nutrient rich batch cultures. A laser-based technique will be used to monitor the optical density of the culture medium  at some shallow depth. We already tested this technique with success in the laboratory by measuring the growth rate of E. coli at many constant temperatures. The experiments will generate growth curves for some bacteria during one or more days in the soil. We will attempt to model their growth rate under such conditions. We also plan to extent our model by simulating other planetary conditions in the laboratory to estimate the habitability of other none terrestrial surfaces such as Mars. This is important to estimate backward and forward contamination risk between Earth and other planetary bodies.

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FONTANES, VANESSA, UPR-MAYAGÜEZ, Arts and Sciences; Margaret, Zupancic, Plant and Microbial Biology, University of California – Berkeley; Hofmeister, Antje, Plant and Microbial Biology, University of California – Berkeley

Mechanisms by which Chromosomal Gene Position Regulates Cell-Cell Communication in Bacillus subtilis Sporulation

Sporulation in Bacillus subtilis involves communication between the two cells involved in the process, the forespore and the mother cell.  This communication is essential for establishing the program of mother cell-specific gene expression.  Activation of the mother cell transcription factor sE is linked to sF activity in the neighboring forespore by means of a signal transduction pathway initiated by sF-directed transcription of the spoIIR gene.  It has been shown that chromosomal gene position of spoIIR affects the timing and level of activity in the neighboring cell.  A strain was constructed by moving the spoIIR gene away from its normal position, causing a delay in sE activation, and consequently, a deficiency in sporulation.  This strain was subject to chemical mutagenesis, selected for its ability to sporulate, and screened for sE activity.  Cells were also fixed for microscopy to determine formation of disporics.  Mutants were classified into two groups:  mutants with partially restored sporulation that retain defects in activity which indicate that a defect in sE activity does not necessarily cause a sporulation deficiency; and mutants whose sporulation defect is not fully accounted for by disporics.  This second group suggests the possibility of a chromosomal misorientation which causes genes to be transcribed at different times than normally, generating other sporulation defects than disporic formation.

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FONTÁNEZ, YARITZA; González, Lisandy; González, Millie; Hernández, Carmen; Casillas, Lilliam, UPR-HUMACAO, Biology Department;   Nieves, Deborah, UPR-HUMACAO, Microbiology Department

A Novel Bacillus isolated from the Cabo Rojo Solar Salterns 

Analysis of the several salt ponds located in the Cabo Rojo saltern indicated a large concentration of commonly limiting nutrients such as phosphorous and nitrogen and significant chemical oxygen demand (COD).  From such productive ecosystem we cultured a variety of aerobic organisms using plates supplemented with different concentrations (from 5 to >30%) of NaCl.  In the plates supplemented with 5 - 15% NaCl the most abundant organisms were spore formers from the Bacillus genus.  One of the bacilli cultures exhibited distinctive colony morphologies that were characterized by highly elevated colonies.  Phylogenetic characterization of our isolate indicated a 98% identity to B. licheniformis.  Further characterization of the colonies by transmission electron microscopy indicated the production of an exo-polymeric substance.  We are currently characterizing the chemical nature and function of this polymeric substance. 

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GARCÍA ALTIERI MAURO; Lázaro Pena, María; Vega Sepúlveda, Yaritza; González Vargas, Carlos I., UPR-RIO PIEDRAS,  College of Natural Sciences,  Department of Biology

Understanding the Role of the HRP1/DSE Complex in the Nonsense-Mediated mRNA Decay Pathway in Saccharomyces cerevisiae

The rate of protein synthesis in eukaryotes is determined by multiple regulatory events at different levels of gene expression. mRNA turnover plays a major role in determining the final level of a protein. Many studies have demonstrated that mRNA turnover and translation are intimately linked. One pathway that clearly exemplifies this link is the nonsense-mediated mRNA decay (NMD) pathway. In both prokaryotes and eukaryotes, nonsense mutations in a gene can accelerate the decay of the mRNA transcribed from that gene. In Saccharomyces cerevisiae, several factors and sequences involved in this pathway have been identified. Results from these studies have demonstrated that, in addition to a nonsense-codon, downstream sequence elements (DSE) located 3' from the stop codon are required to promote NMD pathway. Further, mutations in UPF1, UPF2, and UPF3 result in an increased accumulation of nonsense-containing mRNAs while having no effect on the abundance of most wild-type transcripts. More recently, we have identified the RNA binding protein, Hrp1p, as a factor that directly interacts with the DSE and modulates the activity of the NMD pathway. These results led us to propose a model for the mechanism of NMD. Based on these results, we are further characterizing, at both the molecular and biochemical levels, the role of the HRP1/DSE complex in the NMD pathway. In addition, we are currently investigating several putative DSEs located in the 3’-untranslated region of the CYC1-512 transcript. The results from these experiments will help us determine how the NMD pathway recognizes and degrades aberrant mRNAs. 

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GONZÁLEZ, EMANUEL; Martínez, Juan C., Prof., UPR-MAYAGÜEZ, Department of Biology

Contribución Étnica al Acervo Genético del Cromosoma Y en la Población Puertorriqueña

Polimorfismo en el cromosoma Y de lo seres humanos es utilizado como un marcador para rastrear el origen de ciertas poblaciones. Este cromosoma es heredado entre los varones de esa población. En este proyecto utilizamos estos polimorfismos para determinar los orígenes genéticos en la población puertorriqueña por la línea paterna.  Se conoce que la población puertorriqueña es una mezcla de varias poblaciones (caucásica, indígena y africana).  Estos métodos nos permiten estimar la aportación genética en el linaje paterno de los puertorriqueños de estos grupos continentales. Al realizarle la reacciones de PCR para amplificar ciertos fragmentos diagnósticos de cromosoma Y de Puerto Rico y ser cortados con diferentes enzimas de restricción se puede determinar si existe en ese cromosoma alguna mutación característica de algún haplogrupo. Los haplogrupos dan información importante sobre el origen continental del cromosoma Y. Aún se estan realizando pruebas para poder lograr este objetivo.  Sin embargo, la tendencia de las muestras es que muchas de estas son del haplogrupo 1C el cual se encuentra en el 68% de los españoles.

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GONZÁLEZ, ISADORA, UPR-CAYEY, Department of Biology; Heetae, Kim; Yongmin, Liu; Powel, Brown, Breast Center, Baylor College of Medicine, Houston, TX

Estrogen Can Induce Gene Expression in MCF7 Breast Cancer Cells by Activating the AP-1 Transcription Factor 

The Activating Protein-1 (AP-1) complex is a critical regulator of transcription that induces the expression of various genes controlling cell proliferation, differentiation and transformation. In breast cells, the AP-1 transcriptional factor is activated by growth factors such as estrogen, IGF, EGF and heregulin. Estrogen can activate gene through one of two pathways: the classical pathway, in which estrogen binds to the estrogen receptor, and the complex binds to the ERE promoter, or the nonclassical pathway, in which estrogen binds the estrogen receptor, and this complex then binds to the AP-1 transcription factor complex triggering AP-1 dependent transcription. According to this hypothesis; estrogen can stimulate both pathways. To determine whether the two pathways are activated by estrogen in breast cancer cells, I measure ER-dependent and AP-1 dependent gene expression in estrogen treated MCF7 cells. To measure estrogen-induced gene expression I starve MCF7 cells of estrogen and transfected with two different constructs: ERE-luc and Col-Z-luc. ERE-luc has a estrogen receptor binding site in its promoter, and therefore measure activation of the “classical pathway”.  I then transfected the cells with the Col-Z-luc construct. Col-Z-luc has an AP-1 binding site in its promoter, and therefore measure activity of the nonclassical Pathway. I stimulated the cells with estrogen or with vehicle (EtOH) for different time periods (0h, 12h, 24h) and then measured the Luciferase activity in cell lysates using the Dual Luciferase Reporter Assay (Promega). A Renilla Luciferase construct (PRL-tK) was also co-transfected and Renilla activity was measured and used to normalize the data.

The results of these study showed in the classical pathway a higher activity of transcription with the stimulation of estrogen and in the nonclassical pathway a low activity of AP-1 transcription. Estrogen can induce the ERE-dependent gene expression and the AP-1 gene expression by the classical.

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HERNÁNDEZ, ROSALIE; Serrano, Yolanda, Prof., UIA-BAYAMON, Arts and Sciences, Department of Natural Sciences and Mathematics

Determinación de Propiedades Antibacteriales del Latex de Euphobia milii, Ficus benjamina y Allamanda cathartica 

El latex es un compuesto secundario producido por numerosas angiospermas en células especializadas llamadas laticíferos. Esta substancia lechosa es una mezcla de compuestos químicos que incluyen proteínas, azúcares, sales minerales, alcaloides, aceites y otros. Se cree que su función es la de proteger la planta contra depredadores y sellar heridas producidas a ésta. La mayoría de los latex son irritantes a nuestra piel y algunos pueden ocasionar la muerte si son ingeridos. Se utilizó el latex de Euphorbia milii (corona de Cristo), Ficus benjamina (ficus) y Allamanda cathartica (canario amarillo). Las bacterias Bacillus cereus, Bacillus subtilis, Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, Micrococcus luteus, Pseudomonas aeruginosa y Staphylococcus aureus fueron analizadas con el propósito de observar si el latex exhibía propiedades antibacteriales en éstas. Se usaron céspedes bacterianos, dilución en serie y curva de crecimiento para determinar si el latex inhibía el crecimiento bacterial. Además se usó la técnica de gota colgante para determinar si el latex afectaba el movimiento bacterial. No se observó inhibición en ningun césped bacteriano tratado con los diversos latex. En su lugar se observó una mayor  cantidad de baterias alrededor del disco impregnado con el latex. Pseudomonas aeruginosa y M. luteus fueron las bacterias más afectadas en su movimiento por el latex de F. benjamina y E. milii respectivamente. Se observó una fase logarítmica más lenta en la curva de crecimiento de la bacteria de E. coli tratada con el latex de E. milii.

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JIMÉNEZ, MARÍA; Semidey, Marisol; Figueroa, Maricelys; Ortiz, Almarie; Asencio, Carmen, Prof., PUCPR, Departamento de Biología         

Biología Reproductiva de Zamia pumila L. 

Zamia pumila es la primera especie de Zamia descrita por Lineo en 1763.  Es una especie caribeña, limitada al centro de Cuba, República Dominicana y al sur de Puerto Rico.  El objetivo de este trabajo preliminar es determinar la fenología reproductiva de Z. pumila.  El área de las canteras del barrio Cuevitas en la carretera #PR552 en Juana Díaz se visitó dos veces al mes durante el período de agosto a diciembre de 2001.  Se identificaron las plantas y se coleccionaron los siguientes datos: número de conos por planta, largo y ancho de los conos, sexo, producción de semillas y abortos.  Los resultados hasta ahora indican que Z. pumila puede producir entre uno y cuatro conos rojizo-marrones por planta.  Además se ha encontrado una tasa muy alta de abortos, mayor de 90%.  Esta alta tasa de abortos podría, entre otros factores explicar su limitada distribución en Puerto Rico.

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LLANES, JOAN, UPR-MAYAGÜEZ, Arts and Sciences, Department of Biology; López, Joseph P.; Clark, Emily S.; Shepherd, Virginia L., Vanderbilt University, School of Medicine, Pathology, Deparment of Veterans Affairs

The Effect of Surfactant Protein-A on Mycobacterial Clearance in Murine Alveolar Macrophages

Surfactant protein A (SP-A) is an important component of the innate immune system. SP-A acts as one of the first lines of defense against microbial invasion in the lungs. It has been demonstrated previously that SP-A enhances the clearance of Mycobacterium bovis Bacillus Calmette-Guerin (BCG) by means of enhancing BCG ingestion and nitric oxide (NO) production in rat bone marrow-derived macrophages (RBMM). Since BCG infection is localized in the lungs, it is important to conduct the experiments in alveolar macrophages to further examine the physiological roles of SP-A. A murine alveolar cell line known as MH-S is thought to be a possible candidate for this study. MH-S cells were treated with BCG and BCG pre-opsonized with SP-A to measure the amount of NO produced. Surprisingly, the cells did not produce NO in response to the treatments. The MH-S cells were then treated with fluorescently-labled BCG opsonized or not with SP-A. While BCG was readily taken up by the cells, there was no noticeable difference of ingested BCG between the two treatments. Western blot analyses for total tyrosine phosphorylation were also done on MH-S after 5 and 15 minute treatments with BCG and BCG+ SP-A. As in the other experiments there was no noticeable difference between the two treatments, although both stimulated tyrosine phosphorylation of many proteins. The results demonstrate that the findings obtained in rat bone marrow-derived macrophages cannot be duplicated in this particular cell line.  This may be explained by the nature of the cells as virally-transformed alveolar macrophages that may not produce specific SP-A receptors and are thus unable to respond to SP-A.   

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LLENIN, GABRIELA S.; Carrasquillo, Mara Z.; Vargas, María, Prof., UPR-MAYAGÜEZ, Arts and Sciences, Department of Biology

Mycoflora Present in the Habitat Where the Tilapia spp. Live in the Center of Investigation and Development of Aquiculture of Puerto Rico 

Being decomposer of organic material, the Fungi Kingdom plays a vital role in life’s cycle, although they can affect humans, plants, and animals. Some of the Tilapia spp. in the CILAC presents symptoms that indicate its infection with fungi. The investigation proved that there are fungi present in the water and soil where the Tilapia spp. grows. We took samples of water and soil in certain ponds and used them to make dilutions of 1/100, 1/1000, and 1/10000 concentration. We placed them in petri plates with Potato Dextrose Agar, and incubated them for one week under 25ºC. With the help of slide culture, we found in the water Aspergillus glaucus group, Aspergillus niger, Cladosporium, Curvularia, Fusarium, Geotrichum, Nigrospora, Paecilomyces, Penicillium, yeasts and sterile mycelia. In the soil, only Fusarium was identified. With this investigation, we concluded that the Tilapia’s environment in the CILAC provides the ideal conditions for these fungi species to develop. It has been also proved that most of the species are common contaminants. To determine the impact of these species of fungi in the Tilapia spp., we need to study the internal organs, the skin, and the eyes of the fishes. During this semester, we are incubating some of those internal organs so that we can compare them with the obtained results. After we have reached the final results, we will know if the Tilapia that we receive is infected, or just grows in an environment along with others microorganisms, such as fungi, without getting infected.

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LÓPEZ-BAQUERO, RAFAEL; Rubin, Michael, Prof., UPR-CAYEY, General Science Program, Department of Biology

PCR Amplificaton of Gene Fragments Encoding Synapsin From Rhesus Macaca mulatta

The synapsins are a family of neuronal phosphoproteins associated with synaptic vesicles and regulate neurotransmitter release. Past studies have shown that expression of synapsins correlates temporally with synapse formation, but there has been no direct evidence that they are involved in synaptogenesis. The experimental goal of this work is to amplify synapsin specific fragments from Rhesus macaca mulatta genomic DNA. The Rhesus monkey shares many genes with humans and the synapsins have not been identified in this model organism. We designed PCR primers and used them to amplify synapsin specific gene fragments from Macaca mulatta genomic DNA. Results showed amplification of synapsin fragments required both up and down primers. Synapsin fragments were amplified from Rhesus genomic DNA and a plasmid containing rat synapsin IIb cDNA. Putative synapsin fragments 425 bp, 240 bp and 170 bp in length were detected in Rhesus genomic DNA. These fragments will be purified, cloned and sequenced. The sequence of Rhesus synapsins will help to determine the structure and function of this important gene family.

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MALAVEZ, YADIRA; Rodríguez, Fidel J.; Del Llano, Ana; Sastre, Miguel, UPR-HUMACAO, Departamento de Biología; Muller, Rafael, Departamento de Física, UPR-HUMACAO

Análisis Cuantitativo del Grado de Fragmentación del DNA Utilizando el Programa Vis Comet y el Microscopio de Epifluorecencia

El ensayo cometa es un método rápido y sensitivo utilizado para analizar daños en el DNA a nivel celular/molecular. Dos de las aplicaciones que se pueden realizar con esta técnica son la detección de rompimientos de cadena sencilla (“single strand breaks”) y la detección de apoptosis. Utilizando un microscopio de epifluorescencia clasificamos visualmente los núcleos en cuatro clases según la cantidad de DNA que ha migrado del núcleo hacia el lado positivo de la bandeja de electroforesis (esta migración se asemeja a la cola de un cometa). La clasificación está relacionada a la cantidad de rompimientos de cadena sencilla. Las células apoptóticas, detectadas sin realizar electroforesis, poseen una zona de difusión ancha muy característica. Esta técnica de clasificación visual provee un conteo rápido, económico y sencillo para detectar rompimientos de cadena sencilla y apoptosis; sin embargo, es un método cualitativo y subjetivo. El propósito de este trabajo es establecer la relación entre la clasificación visual y el análisis de imágenes computarizado. De esta manera podemos corroborar nuestros resultados visuales. Utilizamos el programa VisComet para analizar cuantitativamente la cantidad de fluorescencia observada bajo el microscopio.

En los núcleos sometidas a electroforesis encontramos que los no apoptóticos poseen mucha más fluorescencia total que los apoptóticos. En aquellos sometidos a electroforesis encontramos que la clasificación visual corresponde al porciento de DNA en la cola. Nuestro trabajo confirma que existe una relación estrecha entre el análisis visual de daño y el análisis de imágenes computarizado.

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MALDONADO, FRANCISCO, UPR-MAYAGÜEZ, Artes y Ciencias, Biotecnología Industrial; Martínez, Juan C., Prof., UPR-MAYAGÜEZ, Departamento de Biología

Análisis de Cromosoma Y Para Determinar la Ascendencia Puertorriqueña 

El puertorriqueño es producto de años de desarrollo y crecimiento e interacción de tres grupos continentales primordiales: europeo, africano e indígena.  La meta en esta investigación es determinar las contribuciones relativas de estos distintos grupos.  Para investigar la herencia puertorriqueña de estos grupos por vía paternal, estamos estudiando el cromosoma Y, el cual se transmite de padre a hijo varón.  La mayor parte de las secuencias de este cromosoma resisten recombinación.  Por tal razón, las mutaciones presentes en este cromosoma son acumulativas, permitiendo un análisis filogenético simple que al combinarse con información geográfica arroja información sobre las migraciones del Homo sapiens que lo llevaron a colonizar el planeta.  Se usa el término haplogrupo para denominar un grupo de cromosoma Y que comparten una combinación de mutaciones y por lo tanto un origen común.  Estos haplogrupos son característicos de cada grupo étnico.  Por lo tanto, la identificación del haplogrupo al cual pertenece un cromosoma Y es un primer paso a la identificación del origen de ese cromosoma.  Un total de 16 muestras de un grupo de estudiantes fueron tomadas y analizadas mediante amplificación de ciertas regiones del cromosoma Y, seguido de análisis de restricción o fraccionamiento electroforético directo para la identificar los haplogrupos a los cuales pertenecen.  Los resultados apuntan a la posible presencia de haplogrupos caucásicos y africanos en la población estudiada y a una muy escasa o inexistente presencia de haplogrupos indígenas.

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MARTÍNEZ COSME, ILYANA, UPR-CAYEY, Departamento de Biologia; Staray, Vincent, Department of Bacteriology, University of Wisconsin-Madison; Escalante-Semerena, Jorge C., Department of Bacteriology, University of Wisconsin-Madison

Elucidating the Role of cobB Sirtuin in Salmonella enterica Propionate Catabolism

The cobB was recently identifies in Salmonella enterica. We are interested in this gene because it encodes a member of the SIR2 family of proteins (aka, sirtuins). Proteins in this family play a significant regulatory role in eucaryotic gene expression. Specifically, sirtuins have enzymatic activities that are relevant to the processes of genetic silencing and cell aging. To date, no other physiological role has been assigned to these enzymes. The aim of the present study was to investigate the role of CobB sirtuin during the catabolism of propionate in Salmonella enterica. In this bacterium, this sirtuin is required for growth on propionate as the sole source of carbon and energy. In the absence of CobB, the cell fails to grow on propionate. Genetic evidence obtained in our laboratory indicated that the need for CobB can be bypassed. There appear to be least two alternative pathways. One involves propionyl kinase. The propionyl kinase-dependent pathway requires phosphotransacetylase activity encode by the pta gene, strongly suggesting that CobB is somehow involved in the conversion of exogenous propionate into propionyl_CoA. A strain carrying a deletion of the cobB gene was mutagenized to isolate derivatives of it that activated the second alternative pathway. We tested whether or not the alternative pathway required the involvement of the Pta enzyme. Our results showed that cobB revertants lacking pta function retained the ability to grow on propionate, suggesting that pta function was not part of the second alternative, CobB-independent pathway. Future work will define identify the locus affected by the gain-of-function mutations that activate the second alternative pathway. Knowledge of these functions will help define the role of the CobB sirtuins in metabolism.

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McLEAN, ELIZABETH, L.; Alston, Dallas E; Cabarcas-Nuñez, Alexis; Ojeda, Eduardo; Quintero, Herbert; García, Samuel, UPR-MAYAGÜEZ, Department of Marines Sciences

Factors Influencing Visible Patterns Present on Tropical Fish Scales

Stock assessment of marine fish is important to scientists, marine related industries, and resource administrators. Accuracy and precision are needed to estimate stock assessment of fish populations. Groupers, a tropical carnivore, are long-lived, growing slowly after they become adults. Analyses of rings formed on calcareous structures (such as otoliths) are the most frequently used method for determining their age and growth. Other methods include length-frequency analyses or tag-recapture. In temperate climates, fish scales or other bony structures usually indicate a year-mark reflecting slow growth during colder months and faster growth during warmer periods. Temperature variations are one of the strongest environmental influences on growth. However, changes are minimal in the tropics and fish scales typically do not have year-marks. Scales collected from 41 groupers (Epinephelus fulvus, coney; and E. cruentatus, graysby) caught with hook and line six km south of La Parguera indicated little variance due to seasonal changes. Of these, 29% of the scales presented obvious patterns, including marks and shadows, with some repetition within each scale. Patterns were consistent on scales pertaining to same fish. Because temperature changes are not considered to be important, other possible factors could influence secretion of growth hormones altering ring formation. These include low oxygen, fluctuating light regime, and food availability. Oxygen is abundant year-round at the collection site. The light regime, while important, is not considered to be a major contributing factor to scale growth. Food availability may not be constant, thus may be an important variable affecting growth and hormone secretion.

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MEDINA, ÁNGEL; Uscian, John, UPR-MAYAGÜEZ, Biology Department

Purification and Characterization of Trypsin From Intestinal Tissues of the Black Wing Snapper, Lutjanus buccanella 

The abundance of digestive proteases in fish digestive organs will vary with diet.  On the idea that relative trypsin abundance may therefore function as an environmental indicator, trypsin from the black wing snapper, Lutjanus boccanella, was purified and characterized via standard enzyme analytical procedures.  Using tissues obtained from fish caught by commercial anglers of Puerto Real, PR, a homogenate was produced.  Subsequent centrifufation and ammonium sulfate fractionation procedures performed on this homogenate resulted in highest trypsin activity being detected in the 50% ammonium sulfate fraction.  Current investigations are focused on purifying the enzyme from this fraction through use of Benzamidine "Hi-Trap" columns (Amersham-Biosciences), which are affinity columns specific for trypsin.

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MEDINA, YESSICA M.; Rodríguez, Sandra; Quiñonez, Edwin; Soto Vélez, Idelisse; Puente Rolón, Alberto, Prof., UIA-ARECIBO, Departamento de Ciencias y Tecnología

Densidad y Diversidad de Bacterias en la Cueva de los Culebrones, Arecibo, Puerto Rico

La presencia del ser humano en el ambiente subterráneo es uno de las fuentes inevitables de daños en las cuevas.  El propósito de este trabajo es identificar las  especies de bacterias presentes en la Cueva de los Culebrones, determinar la densidad y diversidad de microorganismos, así como la patogenicidad de los mismos con respecto a la fauna existente.  Además, nos permitirá establecer una base de datos para el monitoreo del impacto de la actividad antropogénica a largo plazo.  En total, dieciséis (16) muestras  fueron obtenidas del interior de la cueva localizada en la Reserva Mata de Plátano, Arecibo, Puerto Rico.  Hasta el presente se han identificado organismos pertenecientes a los géneros:  Klebsiella spp., Pseudomonas spp., Bacillus spp., Staphylococcus spp., Proteus spp. y Micrococcus spp.  

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MÉNDEZ CINTRÓN, MARÍA DE L.; Tremblay, Raymond L., Prof., UPR-HUMACAO, Ciencias Naturales, Departamento de Biología

Presencia de Homogamia en la Orquídea Lepanthes woodburyana, Stimson

Las orquídeas, presentan varios mecanismos para evitar la autopolinización, uno de estos es la dicogamia.  Es importante que no ocurra autopolinización, ya que esto lleva a que se reduzca la variabilidad genética dentro de una población, y se obtengan altas probabilidades de endogamia.  La protoandria es uno de los mecanismos de dicogamia, esto es cuando un organismo presenta una condición funcional masculina (androecio) antes que se desarrolle el estado funcional femenino.  Para determinar la existencia de este mecanismo, hemos realizado varias polinizaciones con flores de edades distintas, tres y seis dias.  La probabilidad de producir frutos con flores es de 14.7 por ciento en las de tres días y en las de seis días es de 16 por ciento.  Los resultados obtenidos muestran que esta especie no presenta protoandria como mecanismo de prevención de autopolinización.

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MÉNDEZ SILVAGNOLI MARLA, PUCPR, Departamento de Biología; Ricart Morales, Carlos, Prof., UPR-CAYEY, Departamento de Biología

Comparative Analysis of the Heavy Metal Presence in Water, Sediment, and Seagrass(Thalassia) Tissue in the Guayanilla and Montalva Bays. 

The study examines the heavy metal bioaccumulation and bioconcentration in the sea grass, Thalassia testudinum  in polluted areas along the Guayanilla bay (i.e., experimental site) and a less polluted area in Montalva bay (i.e., control site). Samples of water, Thalassia leaves and rhizomes, and soil sediments were taken from three stations on each area. For several decades, the area in the Guayanilla bay received different types and sources of chemical and biological pollution including discharges from  oil refineries, sewage plants and activities related to urban development.  The samples were analyzed for heavy metals detection and quantification by the inductively coupled plasma spectroscopy methodology following the protocol established by the Environmental Protection Agency (EPA). The results show that the metal concentrations obtained in the experimental area  are significantly higher than those obtained in the control area. In addition, the presence of chromium, silver, vanadium and lead was detected in the Guayanilla bay samples. The higher concentrations, of 182.5 mg/L of chromium, were found in T. testudinum  rhizomes  at la Garza station.  In contrast, the samples in Montalva Bay contained only silver, chromium and cadmium.  The higher concentrations, up to 1.07 ppm of silver, were obtained from the water samples at Frio station. Silver concentrations exceed the Environmental Quality Board water standards in both areas.

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MENDOZA, KARLA, UPR-HUMACAO, Microbiologia;   Wang Yuh-Hwa Bioquímica, UMDNJ

Clonacion de "FRA16B DNA" Para Caracterizar su Habilidad de Montar Nucleosomas

Los sitios frágiles son discontinuidades en los cromosomas y están implicados en muchos desórdenes mecánicos,  Resultados experimentales han demostrado que ciertos sitios frágiles exhiben la habilidad inherente de excluir la formación de nucleosomas.  FRA16B DNA es inducido por ciertos químicos como "distamycin A"y "berenil".  Este sitio frágil está localizado en el cromosoma 16q22.1.  El mismo, fue amplificado utilizando "Polymerase Chain Reaction"(PCR), transformado en células competentes de E. coli, purificado y analizado para determinar su preferencia en el montaje de nucleosomas.  Entendiendo hasta que punto el FRA16B DNA se enlaza con las histonas para montar los nucleosomas, nos permitira investigar si existe alguna anormalidad relacionada a este sitio frágil.

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MERCADO-GARCÍA, BENJAMÍN; Rodríguez, Carmen; Arbelo, José G., Prof., UPR-ARECIBO, Department of Biology/Microbiology

Tolerance Studies of the Strain Bacillus sp. Isolated from the Aquatic Plant Water Hyacinth in Culture Media with Trace Elements

The aquatic plant water hyacinth (Eichhornia crassipes) may be used as a sensitive biological indicator for continuously monitoring certain types of pollutants in aquatic systems. This behavior may be a result of the microbiota associated with the plant. In this study, the exposure of the strain Bacillus sp. isolated from the rhizosphere of the water hyacinth to media containing copper (Cu), vanadium (V) and arsenic species (As^III and As^V) was evaluated in terms of the bacterium tolerance to these elements. The microorganism was exposed separately to each element at concentrations ranging from 0.635 ppm to 6.35 ppm. Biochemical tests were also performed under these conditions. The results obtained indicate that the microorganism showed resistance to Cu and V at concentrations less than 6.35 ppm, respectively. In the case of the arsenic species, the motility of the bacterium was significantly affected at the concentration of 6.35 ppm. The biochemical tests showed that the presence of the elements did not alter the bacterium’s metabolism. Future research work includes heavy metals uptake and bioaccumulation experiments with this and other bacteria isolated from the water hyacinth.

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MORALES, NADYA; Ríos-Velázquez, Carlos, Prof., UPR-MAYAGÜEZ, Artes y Ciencias, Departamento de Biología

Optimización de Técnicas Específicas Para el Aislamiento de ADN de Suelo para Desarrollar Bibliotecas Metagenomas en Distintas Regiones de Puerto Rico

El estudio de procariotas de interés clínico y biotecnológico se ve limitado por falta de técnicas de aislamiento, cultivo e identificación adecuadas en el laboratorio. Se estima que sólo se conoce cerca del 0.1% de la microflora que existe en el suelo. EL propósito de este estudio es  el desarrollar bibliotecas metagenómicas de distintos tipos de suelo en Puerto Rico.  Este semestre se estuvieron practicando y optimizando distintos métodos para el aislamiento de ADN genómico y se desarrolló un protocolo para el aislamiento de ADN de suelo.  Además, se identificó y ordenó como parte del equipo de laboratorio, un disruptor de células para incrementar la calidad y cantidad de ADN de las muestras.  Se pretende como próximo paso, el uso de técnicas de ingeniería genética, para el clonaje y la expresión del ADN aislado

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MORENO QUIÑÓNEZ, AIXADELLISE, UPR-CAYEY, Departamento de Biología;   Zayas-Aponte, Carmen L,. Department of Bacteriology, University of Wisconsin-Madison; Escalante-Semerena, Jorge C., Department of Bacteriology, University of Wisconsin-Madison        

Analysis of the Last Step of the De Novo Adenosylcobinamide-Phosphate Biosynthetic Pathway of Salmonella enterica

Cobalamin (Cbl, vitamin B12) is the largest, most structurally complex cofactor with biological activity. Cbl is an essential nutrient for animals, including humans, and procaryotes are the only organisms that synthesize this important macromolecule. Salmonella enterica synthesizes adenosylCbl (AdoCbl, coenzyme B12) de novo only under anaerobic growth conditions. AdoCbl biosynthesis is a major biosynthetic pathway involving 25 gene products. To further our understanding of this complex pathway, we focused on the last step of the de novo corrin ring biosynthetic branch, i.e., the branch of the pathway responsible for the assembly of the cobalt-containing cyclic tetrapyrrole.  This step of the pathway is thought to be catalyzed by the cobinamide synthase (CbiB) enzyme. We have begun the genetic characterization of the cbiB gene. For this purpose, we used localized, chemical (hydroxylamine) mutagenesis to isolate cbiB alleles encoding mutant proteins that lack enzymatic activity.  cbiB mutant strains were identified by their inability to use cobyric acid (the proposed substrate of the enzyme) as a precursor of AdoCbl. Using this approach, 17 cbiB mutant strains were isolated, and the identification of the nature of the mutations is currently under way.  Computer analysis of the predicted primary amino acid sequence strongly suggests the CbiB protein is an integral membrane protein with several membrane-spanning domains.  Thus, it is predicted that null alleles of this gene will either prevent localization of the protein to the membrane, or will block catalysis.  Knowledge of the localization of the mutations we isolated, will shed light on this questions.

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NEGRÓN, BRENDALÍ; Robles, Iris Vannesa; Rivera, Evasomary; Candelas, Graciela, Prof., UPR-RÍO PIEDRAS, Natural Sciences, Department of Biology

 Screening, Sequencing and Characterization for the Glycine tRNA Genes of the Orb-Web Spider Nephila clavipes

The stimulation of the large ampullate glands into active fibroin synthesis, through the removal of the spider’s stored silks, elicits a series of prelude macromolecular syntheses, one of which serves to enrich the glands with selected tRNAs through the upgraded expression of their respective genes.  Upgraded are specifically, those tRNAs cognate to the protein’s preponderant aminoacids: glycine, alanine and proline.  Glycine tRNAs, cognate to the most abundant aminoacid in the fibroin product of the glands, display the highest accumulation.  The cloning of the genes encoding these molecules is critical for understanding the transcriptional controls underlying the differential accumulation of glycine tRNAs within the process of fibroin production.  Southern analyses were performed on genomic DNA samples digested with a series of restrictions endonucleases using as probe a pBR322 derivative containing the sequence of a glycine tRNA gene from Bombyx mori, donated by A. Fournier from the Claude Bernard University at Lyon.  The studies reveal several hybridization signals, among which a 2.7 kb fragment outstands for its intensity, suggesting the possible clustering of some of these genes.  A PCR strategy, relying in the high degree of conservation displayed by tRNAs, was employed in cloning a 1.0 kb fragment.  Sequence analysis of the fragment revealed a cluster of four tightly clustered genes.   The genes are 78-90 bp apart and are arrayed in the same orientation.  Although they display a high degree of similarity in their sequence, differences are observed in both, coding and flanking regions. Currently, we are in the process of subcloning the members of the cluster in order to assay their transcriptional properties in a cell-free transcription system derived from the posterior silkglands of B. mori. This research has been supported by Institutional Funds and NIH grant #RRO03641 to G.C.

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PERDOMO, SHARLIM, UPR-HUMACAO, Departamento de Biología; Visscher, PT, Marine Science Dept., Universidad de Connecticut; Hernández, C., Biology Dept., UPR HUMACAO; Nieves- Méndez, D., Universidad del Este; Casillas-Martínez, L., Biology Dept., UPR HUMACAO    

Physicochemical Conditions and Microbial Abundance of the  Cabo Rojo Solar Saltern

Although members of the Archaea domain dominate hypersaline environments, the discovery of novel groups of bacteria is now challenging such idea. Studies regarding the in situ activities, abundance and distribution of such bacteria are limited.  Consequently, we have analyzed the physicochemical conditions and microbial abundance of several hypersaline ponds located in Cabo Rojo.  At such ponds, salinity levels were up to 520 practical salinity units (PSU). Further analysis of the ponds indicated a large concentration of commonly limiting nutrients such as phosphorous and nitrogen and significant chemical oxygen demand (COD). Using microelectrodes we determined O₂ profiles of several salt ponds. Oxygen penetrated to 3 mm depth, through an area of active precipitation into the salt crust. Interestingly, the O₂ concentration in the water column of salt ponds 1, 2 and 4 was below air saturation, suggesting a sink (i.e. aerobic organisms) in the water column or bottom. Indeed we cultured a variety of aerobic organisms using plates supplemented with different concentrations (from 5 to >30%) of NaCl. The most abundant organisms cultured were spore formers from the Bacillus genus. Other heterotrophic bacteria cultivated had S. arlettae and S. captilis as their closest representatives. The phototrophic bacterium Rhodospirillum salinarum was also reported in abundant numbers. In the future we plan to study the contributions and geological transformations of such organisms in the site.

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POMALES-HERNÁNDEZ, GRIZEL, UPR-HUMACAO, Ciencias Naturales - MAVS; Tremblay, Raymond L., Departamento de Biología, UPR-HUMACAO   

Determinación de la Presencia de Protandria como Posible Mecanismo de Prevención de la Autopolinización en Dos Especies de Orquídeas: Lepanthes rupestris y L. rubripetal

En las plantas con flores hermafroditas es posible que existan mecanismos, como la protandria, para evitar la autopolinización.  La protandria es la maduración del androecium antes del gynoecium en las flores.  Con el propósito de probar si existe la protandria, determinamos la edad de las flores y tomamos en cuenta el día preciso en que las flores abren.  Se polinizó de manera cruzada las flores abiertas de tres y de seis días, tomando en cuenta si se producían frutos.  El resultado fue no significativo para Lepanthes rupestris y significativo para Lepanthes rubripetala, donde se obtuvo un 32 por ciento de frutos por polinización al sexto día y de 10 por ciento en las de tres días.  Esto nos indica que para L. rubripetala está presente la protandria que podría ser un mecanismo que evite la autopolinización de sus flores.

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QUIÑONEZ, BIANCA, UPR-MAYAGÜEZ, Artes y Ciencias; Massol-Deya, Arturo, Departamento de Biología, UPR- MAYAGÜEZ

Aislamiento y Caracterización de Bacterias Epífitas que Degradan Tolueno

La superficie de las hojas de las plantas representa un ambiente hostil que puede ser colonizado sólo por microorganismos capaces de adaptarse a este ambiente y convertirlo en su hábitat natural.  Las bacterias que crecen en la superficie de las hojas son llamadas epífitas.  El objetivo de este trabajo es estudiar bacterias epífitas encontradas en helechos, que poseen la capacidad de crecer en presencia de tolueno.  Para el experimento se colectó follaje del sotobosque del Bosque del Pueblo en Adjuntas, Puerto Rico.  Las hojas se agitaron por 1 hora a rapidez mediana en 50 mL de caldo de sales mínimas y se hicieron diluciones.  Luego se inoculó este caldo en platos Petri que contenían agar de sales mínimas.  Estos platos se enriquecieron con vapores de tolueno y se cultivaron a temperatura ambiente por 20 días.  También se cultivaron las muestras en platos Petri que contenían agar de R2A y fueron incubados sin tolueno a 25 ^oC por 7 días.   Las colonias encontradas fueron aisladas y purificadas.  Estas fueron preliminarmente caracterizadas mediante pruebas fenotípicas.  En los platos cultivados sin tolueno se obtuvo un crecimiento que fluctuaba en un rango de 7 UFC/cm2  hasta 1 x 104 UFC/cm2.   Mientras que en los platos cultivados con tolueno, el rango fluctuó entre 6.09 UFC/cm2 hasta 9 x 102 UFC/cm2.    En los cultivos con tolueno se obtuvo un crecimiento de un 3 a un 35% del total de las colonias obtenidas en los platos cultivados sin tolueno.  Los fenotipos más comunes entre las colonias que crecieron en tolueno son cocos y bacilos Gram +, mientras que en las colonias que fueron cultivadas sin tolueno predominaban cocos y bacilos Gram –.  Una evaluación inicial nos indica que la flora microbiana epífita es abundante y diversa en el bosque sub-húmedo tropical.

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RESTO, KARINA, UNIV. CATOLICA, Ponce School of Medicine; Flores, Idhaliz, Microbiology, Ponce School of Medicine; Santiago, Olga; Santiago, Cariluz; Appleyard, Caroline B., Physiology, Ponce School of Medicine

Investigation of Pathophysiological Mechanisms in an Intestinal Endometriosis Model

Endometriosis is characterized by the presence of benign endometrial implants outside the uterus.  Symptoms of intestinal endometriosis can mimic many gastrointestinal disorders leading to misdiagnosis.  Aim: To utilize an animal model to investigate the gastrointestinal complications of endometriosis and the underlying pathophysiology.  Methods: Intestinal endometriosis was surgically induced by suturing uterine horn implants next to the mesentery of the small intestine in sexually mature female Sprague-Dawley rats.  (controls: sutures without implants).  Animals were sacrificed sixty days after surgery, and the peritoneal cavity was examined for endometrial tissue. Peritoneal fluid and tissues were collected from all animals, the implants were classified in grades of growth, and the colons were examined for macroscopic damage.  Results:  In the experimental animals, vesicles were found at the site of the implants in at least 50% of cases.  They were usually oval and contained a high number of white blood cells (wbc).  Experimental animals had significantly more wbc in the peritoneal fluid compared to controls (5.75 ± 0.85 vs 3.35 ± 0.15 wbc/high power field; p<0.05), and their colons were significantly more damaged (1.12 ± 0.16 vs 0.61 ± 0.09; p<0.05).  Conclusions:  Our preliminary data demonstrate that this model of endometriosis has similarities to that of the human condition, and that these animals have intestinal involvement.  Future studies will help identify molecular markers that can differentiate between the symptoms of intestinal endometriosis and those of other gastrointestinal diseases. Supported in part by NIH, NRSA GM07732 from NIGMS and MBRS.

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RIVERA, CAROL, PUCPR, Department of Biology; Khorasanizadeh, Sepideh, Prof., The Univ. of Virginia, Biochemistry and Molecular Genetics

Cloning and Puryfing Proteins: HP1 (Heterochromatin-Associated Protein 1)

Chromosomes are made of chromatin. Chromatin is a complex, dynamic structure determined mainly by protein binding. There are two types: euchromatin or active chromatin and heterochromatin or inactive chromatin. In the chromosomes are two types of proteins (chromosomal proteins) histones and nonhistones proteins.  The HP1 protein (heterochromatin-associated protein 1) is an example of a nonhistone protein. Represents the best-characterized heterochromatin-associated nonhistone chromosomal protein family in the eukaryotic kingdom. There are some properties intrinsic to HP1, including gene silencing, and there are three types in humans: HP1 a, HP1 b, and HP1 g. Our laboratory studies the three-dimensional structure and dynamics of proteins to elucidate function of biological molecules. We prepared samples for such studies implementing tools of molecular biology and biochemistry (cloning and protein purification). For cloning we used the pET-11a vector and HP1 a, HP1 b, and HP1 g inserts cut with two restriction enzymes: Bam H1 and Nde 1. We had four positive colonies (one for HP1 b and three for HP1 g) and we sent they for sequencing because they were expressing very well. Then we made the protein purification (affinity chromatography) and finally we stored the protein at -20  C for future experiments. We are studing HP1 because it interacts with the methylated tail of Histone 3 (H 3) for gene silencing

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RIVERA ADROVET, CARLOS, UPR-HUMACAO; Roth, Monica J., Department of Biochemistry, UMDNJ; Wood, Robert, Johnson Medical School; Planas Rivera, José, UPR-AGUADILLA

Mutational Analysis in the Integrase Protein of Moloney Murine Leukemia Virus

A critical step in the retroviral life cycle is the integration of viral DNA into a host chromosome by the integrase protein (IN), encoded in the 3' end of the viral pol gene. This enzyme performs two catalytic steps, 3' processing and strand transfer. The 3' processing removes two deoxynucleotides from both 3' termini of the viral long terminal repeats (LTRs), sequences created during reverse transcription. These 3' ends are joined to 5' staggered sites in the target DNA by strand transfer reaction. Previous studies have shown that IN consists of 3 regions: the N-terminal, central core and C-terminal domains. The N-terminal domain has a very well conserved HHCC zinc finger domain that is require for viral DNA integration in vivo. In various other proteins, these HHCC repetitions are involved in DNA binding activity, but this has not been reported for the Moloney Murine Leukemia Virus (M-MuLV) IN. In these studies we attempt to mutate specific nucleotides of the N-terminal domain of M-MuLV IN to determine if they are necessary for viral DNA integration in vitro and their possible contribution to DNA binding activity.

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ROCHE RÍOS, MARLY, UIA-SAN GERMAN, Departamento de Biología; Ferrer Marrero, Tirsa; Perez-Chiesa, Ivette, Prof., UPR-RÍO PIEDRAS, Departamento de Biología

Alcohol Dehydrogenase Activity in Larvae and Adults of Drosophila polymorpha

The Drosophila Adh gene codes for alcohol dehydrogenase (ADH; E.C. 1.1.1.1) which catalyzes the oxidation of alcohols and the concurrent reduction of NAD+ to yield aldehydes or ketones and NADH. The enzyme detoxifies the alcohols ingested by the flies.  To contribute to the understanding of the evolution of this gene-enzyme system, we studied the activity of ADH in D. polymorpha of the cardini group as compared to D. melanogaster and D. nigrodunni. Flies tested were grown in banana medium supplemented with live yeast at 25 C.  Agarose gel electrophoresis (1% in TBE, 100V, 2hrs.) was done for larvae and adult homogenates (in 15  l of glycerin in TBE 1X, pH 8.6). Bands of ADH activity were detected by exposing the gel to a mixture of TBE, NBT, PMS,  NAD and 2-propanol (as substrate). Gels were fixed in acetic acid (5%).  Internal organs and carcass of D. polymorpha and D. melanogaster larvae were dissected and exposed to the same mixture ( 37  C for 7 minutes) to detect tissue-specific activity. Zymograms of D. polymorpha larvae and adults, show the same  band of ADH activity but its mobility differs from that of D. melanogaster and D.nigrodunni. We found differences in tissue- specific activity.  These results suggests that D. polymorpha has only one functional gene for larvae and adults, and that changes have occured in both coding and regulatory regions of the gene as compared to the species used as control.

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RODRÍGUEZ, MARÍA, UPR-MAYAGÜEZ, Arts and Sciences, Department of Biology; Kuppuswamy, Dr.; Willey, Chris, Cardiology; Ramírez, Doris, Prof., UPR-MAYAGÜEZ, Arts and Sciences, Department of Chemistry          

c-Src signaling in Cardiac Hypertrophy Models

Cardiac Hypertrophy is an adaptive response to hemodynamic overload.  Because it is known that FN and VN assemble on the surface of cardiocytes during pressure overload with accompanying cytoskeletal (CSK) assembly of focal adhesion proteins including the tyrosine kinase c-Src, we created a three dimensional cell culture model using type I collagen which included an RGD peptide based on the integrin binding motif of FN and VN.  RGD stimulation of cardiocytes in collagen triggered focal complex assembly of proteins.  Additionally, adenoviral constructs of c-Src were used to over express c-Src prior to embedding the cardiocytes in collagen.  Examination of  CSK bound c-Src  revealed that at least a 4mg/ml concentration of RGD peptide is required for its assembly to the cytoskeleton although Src activation is not a prerequisite for its movement to the CSK.  However, c-Src activation may bring additional proteins to the CSK.  With this model we have recreated an environment similar to the pressure overload situation.  Therefore, this model will enable us to study the mechanism for focal complex assembly as well as its role in cardiac hypertrophy.

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RODRÍGUEZ, JOSÉ, PUCPR, MARC U*STAR Program; McCollum, Andrea M.; Ganko, Eric; McDonald, John F., Prof., UGA Athens, Genetics 

Presence of Burdock LTR-Retrotransposon in the cathD Gene of Drosophila melanogaster

Tranposable elements (TEs) are DNA sequences with the ability to replicate in host genome. TEs have the capasity to insert in randomly in different genomic locations. Retroelements are Class 1 TEs and have been found in all the genome of eukaryotes, thus far analyzed where they have amplified to high copy numbes over evolutionary time. Based on genomic analysis, we have identifid a full-length retroelement (Burdock) that is present in the cathD gene in Drosophila melanogaster. PCR assays were used to test for the presene of this Burdock element in the cathD gene in flies representing a broad distribution of populations of D. melanogaster from around the world. Our results indicate that the insertion into the cathD gene is not conserved across populat ions surveyed in this study.

I ackwnolege the MARC U*STAR Program for given me the opportunity to present and built my future.

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RODRÍGUEZ CRUZ, EVA NILDA; García Soto, Arlene; Cajigas Rodríguez, Ivelisse; Rodríguez Cruz, Eva Nilda; Nadal, Eduardo; González, Alexis; Candelas, Graciela, Prof., UPR-RÍO PIEDRAS, Natural Sciences, Department of Biology

Search for Activities Preluding Fibroin Synthesis:  Upgrading of U Small Nuclear RNA’s

We present a retrospective and updated report on the series of timed molecular syntheses, which optimize or differentiate the large ampullate glands of the orb-web spider, Nephila clavipes for the production of its tissue-specific product, the strongest known natural fiber. Monitoring of the elicited process through time sequence studies has revealed, as previously described, four peaks of time molecular synthesis, small RNAs are produced within two bouts, one precedes and the other succeeds the synthesis of fibroin’s template by 15 minutes, respectively. Fractionation of small RNAs by denaturing polyacrilamide gel electrophoresis reveals that during the earlier of these events ( reproducibly of higher magnitude) a variety of small RNA moieties migrating in the domain of small nuclear RNA of the U series are accumulated. The involvement of these species in mRNA splicing and the timing of their accumulation, within the series of events preluding fibroin production, suggest their involvement in a post-transciriptional regulatory axis. Northern blot analyses of the expression levels of UsnRNA have revealed a differential accumultion of U1, U5, U6, and U2 sn RNA, thus far. The last member of the UsnRNA family , involved in spliceosome assemby, the U4 is currently under scrutiny. The data from these studies will veer toward analyzing these upgradings at the level of transcriptional regulation and searches for regulatory elements underlying coordinated gene expressions during the process of fibroin synthesis. Supported by Institutional Funds and NIH grant #RRO03641 to G.C.

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RODRÍGUEZ CRUZ, SANDRA I., UIA-BAYAMÓN, Natural Sciences; Pérez Chiesa, Ivette, Prof., UPR-RÍO PIEDRAS, Department of Biology

Molecular Analysis of the Sod Gene of Drosophila nigrodunni

The Sod gene of Drosophila melanogaster codifies for Cu/Zn superoxide dismutase [Superoxide: superoxide oxireductase; SOD (E.C. 1.15.1.1)]. The gene has two exons and one large intron. The enzyme is highly important for survival, it catalyzes the convertion of O₂ - , generated by the cell's metabolism, into H₂O₂ . This in turn, is converted to H₂O and O₂ by catalase. The evolution of the gene in the genus Drosophila has been studied by comparing the nucleotide sequence of numerous species.  Species of the Tripunctata radiation have not been included in these studies.  Our objective is to sequence Sod from D. nigrodunni of the cardini group (subgenus Drosophila) as a representative of this radiation, and compare the nucleotide sequence with that of D. cardini (another member of the group), D. melanogaster (subgenus Sophophora) and other species of the genus . Genomic DNA was extracted with the  GNOME Bio Kit 101 and fragments of the genes were amplified with PCR.  Primers used were those of Hudson et al., (1994). PCR conditions were: hot start (94 C/ 5 min); denaturation (94 C/ 30s); annealing (57 C/ 30 min) and polymerization (72 C/ 30s). Amplicons were purified from low melting point agarose gels (1.2% in TAE, pH 8.3) with gelase and sequenced according to the method of Sanger with Big Dye Terminator Reaction Kit (PE Applied Biosystems) in a ABIPRISM 377 Automated DNA Sequencer (Perkin Elmer).  Two fragments of 253 bp and 800 bp, which cover almost the entire gene, were obtained.  As compared to D. melanogaster,  D. nigrodunni appears to have a smaller intron and based on the  253 bp fragment sequenced so far, codon bias appears to be lower. As expected, there is a higher degree of identity between members of the same group and subgenus than with D. melanogaster.

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RODRÍGUEZ MARCANO, BÁRBARA; Tremblay, Raymond L., Prof., UPR-HUMACAO, Ciencias Naturales, Departamento de Biología

Estudios de Patrones de Paternidad en la Orquídea Lepanthes rupestris Usando la Técnica AFLP

La técnica de huellas digitales AFLP (amplified fragment length polymorphism) se ha convertido en una de las herramientas primordiales para el estudio genético de organismos.  Esta técnica posee la capacidad de ampliar un conjunto limitado de fragmentos de DNA independientemente de su origen y complejidad.  AFLP ha resultado ser útil debido a que puede aplicarse a una amplia variedad de organismos y requiere pequeñas cantidades de DNA genómico.  En un estudio comparando la diversidad genética entre plantas detectadas por RFLP, RAPD, AFLP y SSR, AFLP proveyó la mayor diversidad en bandas polimórficas siendo de 10 – 100 veces más sensitiva que otras técnicas de huellas digitales.  El objetivo de esta investigación es evaluar la eficiencia de AFLP como técnica de huellas digitales en la orquídea litofítica Lepanthes rupestris con el propósito de contestar preguntas de base ecológica haciendo uso de las diversas técnicas moleculares.

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ROSA MORALES, REY JESÚS, UPR-RÍO PIEDRAS, Natural Sciences; Quiñones Rivera, José L.; García Arrarás,  José E., UPR-RÍO PIEDRAS, Department of Biology

Inhibition of Metalloproteases Intervene with the Process of Intestinal Regeneration in Holothuria glaberrima

Metalloproteases are enzymes that intervene in the degradation of extracellular matrix components. Their intervention plays an important role in cellular differentiation as in tissue regeneration in some systems. Our study focus on the possible role of metalloproteases in the degradation of extracellular matrix components during intestinal regeneration of Holothuria glaberrima. We have used 1,10-phenantroline, a chelant  agent for the zinc ion to inhibit metalloproteases during different stages of intestinal regeneration. The results were analyzed using the immunohistochemical technique for collagen, finding that the regeneration process of the intestine was slower during the first seven days when compared with a control group injected with a saline solution. Moreover, intestinal growth was quantified by measuring the regenerate area, and finding that in animals injected with 1,10-phenantroline, the regeneration was slowed approximately five days. Experiments in which a recovery period was allowed following the drug injection reject the possibility that intestinal growth inhibition has been a product of toxicity or death. Our results suggest that metalloproteases are important during the intestinal regeneration process. In a preliminary set of experiments, a group of animals was injected with the drug 1,10-phenantroline at days 6, 8 and 10 and dissected at day 12. At these stages inhibition of growth was not observed, suggesting that the inhibition effect is stage dependent. Our results emphasize the important role that metalloproteases and extracellular matrix play in the tissue regeneration. Funded by NIGMS-SCORE,FIPI and AMP

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SÁEZ, LORENA, UPR-PONCE; Hernández, Gerardo; Appleyard, Caroline B., Prof., Ponce School of Medicine, Physiology Department        

Bacteria Peptide Exacerbate Colitis in Acute Model of Inflammatory Bowel Disease

We have previously demonsrated that bacterial load plays an important role in the pathogenesis of IBD in two different animal models of acute colitis (FASEB Journal 2001 15(5):A821). The aims of this study were to investigate the role of the bacterial peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP)and to evaluate the secretory responses of the intestine in an acute animal model of colitis.Methods: Rats were divided into three different groups; the experimental groups (TNBS+fMLP) and the control groups (TNBS+DMSO & ETOH+fMLP). Acute colitis was induced in male Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol intracolonically). Twenty four hours after the initial induction of colitis (2.5) of fMLP in 6% DMSO was administrated intracolonically. Two hours later the rats were sacrificed and distal colon was removed and scored for macroscopic damage. Representative sections of the colon were taken for measurement of myeloperoxidase (MPO) activity and secretory responses using Ussing Chambers. Results: The average macroscopic damage score for TNBS+fMLP and TNBS+DMSO groups was significantly higher (11.94±0.53)and (9.84±1.7)respectively than the ETOH+fMLP group (5.55±0.61)(n=5;*p<0.05). MPO activity was significantly different between the normal and the damage tissue in all groups (n=5;*p<0.05). No significant differences were found between the groups in terms of short circuit current at diferrent acetylcholine concentrations. Conclusions:Bacterial peptides may exarcebatethe colitis during acute phase after the initial damage caused by TNBS but this may not affect the tissue viability. These results sugest that enteric microflora peptides may contribute to the exacerbation of colitis after an initial triggering by an unknown factor. Supported in part by MBRS.

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SANABRIA VALENTÍN, EDGARDO L., UPR-MAYAGÜEZ, Arts and Sciences, Department of Biology; Darwin, Andrew, Prof., NYU- School of Medicine         

Analysis of Variant Toxin Genes in Yersinia species

Yersinia enterocolitica is a Gram-negative coccobacilli and a known GI tract pathogen. We have identified Y. enterocolitica serotype O:8 genes that may encode an AB type toxin with ADP-ribosyltransferase activity. We used PCR to determine whether homologous toxAB genes are present in the genomes of other serotypes of Y. enterocolitica (O:3 and O:9), and other Yersinia species . toxAB homologues were found in the genome of Y. enterocolitica O:3 and Y. intermedia. However, the predicted ToxA and ToxB proteins of these strains are almost to each other, and to the proteins from Y. enterocolitica O:8. We constructed expression plasmids to study the products of the variant tox genes. Attempts were then made to overexpress and characterize the proteins in Escherichia coli K-12.

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SANTANA, MILDRED; Rodríguez, José L.; Corrales, Gabriela; Monllor,Verónica; Sastre, Inés, Prof., UPR-MAYAGÜEZ, Artes y Ciencias, Departamento de Biología

A Restauration Program to Increase Moss Diversity in Some Secondary Forest in Puerto Rico

In Puerto Rico many forest areas that have recuperated from diturbances show a low moss diversity eventhough shade conditions have been re-established. We propose a program to reintroduce species to increase moss diversity.This program consist on determination of population genetic diversity levels using RAPDs, propagation and plant establishment. Neckeropsis distichia and N.undulata are two mosses that commonly occurr in humid lowland forest. Five populations of N. disticha of different forest areas were selected. This will help us to identify best population source for species re-introduction.At this point we are working on DNA protocol extraction for mosses. Also we are testing three moss propagation methods.

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SANTIAGO,  FÉLIX; Massol Deya, Arturo, Prof., UPR-MAYAGÜEZ, Biology Department

Descripción de los Efectos Causados por la Acción Depredadora de Protozoarios Bacterívoros en la Degradación de Tolueno por Bacterias Endógenas de Suelos Tropicales

La depredación es una interacción ecológica que ejerce una presión selectiva determinante sobre aquellos organismos que sirven de presa. En el caso de las bacterias, al ser presa de protozoarios bacterívoros, éstas modifican su población presentando polimorfismo, cambios fisiológicos, metabólicos y físicos. Determinar si los efectos de depredación bacteriana por el protozoario Tetrahymena pyriformis posee algún efecto en la degradación de tolueno proveerá información útil en en el desarrollo de estrategias biorremediativas. Poblaciones bacterianas fueron aisladas de muestras de suelo tropical y se selecionaron aquellas capaces de utilizar tolueno como fuente única de carbono y energía.  Para estudiar el efecto de la interacción ecológica en la degradación del tolueno se preparó un cultivo de protozoarios libre de bacterias. Sistemas con representación de ambas poblaciones de microorganismos (bacterias y protozoarios) fueron preparados para confirmar la capacidad depredadora del protozoario. Tetrahymena pyriformis demostró ser un depredador activo de las bacterias degradadoras de tolueno. Al momento, los sistemas de depredación en presencia de tolueno están en progreso. Al finalizar ésta última etapa se espera documetar claramente la influencia de la depredación en la degradación de tolueno y entonces desarrollar otras técnicas que promuevan la degradación de contaminantes por bacterias endógenas en suelos tropicales contaminados.

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SANTIAGO MARTÍNEZ, EDGARDO; Reyes Lope-de Haro, Guy; Santiago Cortés, Alma I., Prof., PUCPR, Departamento de Biología         

Contribution of DPB1 Locus to Susceptibility to Type 1 Diabetes

Genetic and environmental factors are believed to share approximately equal roles in the development of type 1 diabetes.  These factors cause a 200-fold worldwide difference in the incidence of the disease.  The class II genes (DQ and DR) of the Human Leukocyte Antigen complex (HLA), are the greatest contributors to the genetic susceptibility to type 1 diabetes.  Molecular analysis of these polymorphic genes worldwide has identified many alleles that contribute to susceptibility or protection against the disease. A possible role of the DPB1 locus in the HLA region in the susceptibility to type 1 diabetes has been reported in some populations.  To determine its role in Puerto Ricans 91 diabetic patients and 86 controls were molecularly typed.  Polymerase Chain Reaction (PCR) and reverse blot were used for the DNA-based typing of the DPB1 locus.  Our results point to DPB1*0301 and DPB1*1701 as susceptibility alleles to type 1 diabetes, whereas a trend towards protection is seen in the DPB1*0101, the DPB1*1301 and DPB1*5001 alleles.

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SCHENK, CHRISTIAN E.; García Arrarás, José E., Prof., UPR-RÍO PIEDRAS, Natural Sciences, Department of Biology      

A Cell Phenotype Associated with ECM Remodeling During Intestinal Regeneration of the Sea Cucumber Holothuria Glaberrima

The extracellular matrix plays an important role in regeneration processes.  Recent studies from our laboratory have shown drastic changes in ECM components during intestinal regeneration in the sea cucumber Holothuria glaberrima.  Using immunocytochemistry we have now identified cell population s that seem to be involved in these ECM changes.  The cells, recognized by three different monoclonal antibodies, are about 20-40 um in size and are present within the internal connective tissues of the holothurian intestine.  They have colorless spherules in the cytoplasm that vary in size (10.0 um) and can only be seen with antibody labeling.  In terms of morphology and abundance these cells could be labeling what has been previously seen in classical histology as morula cells.  During early regeneration the cells appear in great numbers in the connective tissue.  At 12 to 14 days post evisceration, the labeling loses its definite cellular contour and is observed as if dispersed within the ECM.  This suggests that the molecules recognized by the antibodies are added to the extracellular matrix of the intestinal primordia during regeneration.  The cells were also detected aligned along the mesentery during regeneration, and large numbers of cells are observed during the second week of regeneration, suggesting that cell migration from the mesentery is occurring.  Ongoing experiments are targeted toward identifying the molecules recognized by the antibodies and their possible role in ECM remodeling.  The results from these experiments can lead us to a better understanding of the cellular and ECM roles tat are important to regeneration processes.

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SEDA MIRÓ, JASMINE; Fontán Navarro, Ludalis Z.; Pérez Santos, Noemí J.; UPR-ARECIBO, Department of Biology; Méndez, Abel, Prof., UPR-ARECIBO, Department of Physics and Chemistry

Planetary Microbial Ecology: Microbial Growth Kinetics as Function of the Physical State of the Environment

There are many physical, chemical and biological variables that affect microbial growth. In particular, the environment temperature has a strong effect on microbial growth rates while hydrostatic pressure has almost no effect. However, there is almost no data of how microorganisms respond to both low-temperature and low-pressure environments. Here we report experiments and techniques to measure the growth kinetics of bacteria as function of both temperature and pressure. A new experimental method, using laser-based optical density of batch cultures, was devised to easily and continuously measure the specific growth rate of bacteria under constant temperature and hydrostatic pressure. Our experiments were calibrated and validated by measuring the generation time of E. coli at various temperatures between 20 ^OC to 50 ^OC under standard pressure. Other test experiments showed no effect on the growth rate of E. coli down to one-third standard pressure. Our current experiments try to measure the specific growth rate of E. coli and other bacteria at lower temperatures and pressures. The experiments will be used to define some physical limits of microbial life with respect to the environment and to understand microbial growth in cold low-pressure environments. This type of work is relevant to identify favorable physical environments for exogenous or indigenous life and to quantify backward and forward contamination risks between planetary bodies such as Earth and Mars.

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SERRANO, YENDI, PUCPR, Sciences College; Ruiz, Abigael; Matta, Jaime, Prof., Ponce School of Medicine, Pharmacology & Toxicology Department

Antineoplastic Properties of Frankincense R Essential Oil Against Human MCF-7 Breast Cancer Cells

Breast Cancer is the most frequently diagnosed non-skin cancer among women in the United States. It is the most common female malignancy and a major cause of death in middle-aged women. It has been demonstrated that Essential Oils have a unique ability to penetrate cell membranes and diffuse throughout the blood and tissues. In this study the potential antineoplastic effects of Frankincense Essential Oil will be examined. Frankincense Essential Oil constitutes 75% monoterpenes, compound that have shown chemopreventive and chemotherapeutic activity in mammary tumor models. Monoterpenes represent a new class of breast cancer therapeutic agents. We would like to demonstrate Frankincense Essential Oil as a chemotherapeutic agent against MCF-7, an aggressive human breast cancer cell line. It has been demonstrated that this specific cancer cell line is sensitive to monoterpenes. Because of a current hypothesis in scientific research that establishes that in addition to monoterpenes, the whole Essential Oil mixtures have multi-action processes, cells will be treated with various concentrations of whole Frankincense Essential Oil. Dose-response curves will be established. A hemocytometer will be used in order to measure a decrease in cell division. Cell chromatin will be stain in order to determine morphological changes; apoptosis will be detected by immunofluorescence.

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SILVESTRY, MARIENA, UPR-MAYAGÜEZ, Arts and Sciences, Department of Biology; Severinov, Konstantin; Minhakin, Leonid, Department of Genetics, Rutgers The State University of New Jersey

GreA-like Factor, Studying the Transcription Factor

RNA polymerase (RNAP) is the enzyme involved in the transcription of RNA from a DNA template. E. coli RNAP is the best studied of its class. This process is regulated by different transcription factors, which are mostly proteins. It is known that the Gre A factor facilitates transcript cleavage in stalled complexes and restart of elongation. Our goal was to clone and overexpress the GreA-like factor from Thermus aquaticus. Initially we have constructed a plasmid library of T.a. genome fragments enriched with the greA-like gene sequence. We cleaved the total DNA with StuI restriction enzyme, purified DNA fraction containing greA-like fragmentfrom the gel, and ligated the fragment into vector pT7Blue. To perform primary and secondary screening of the library we used internal part of greA-like gene cloned previously. The positive library clones were found and the sequence of greA-like gene was determined. To overexpress the corresponding protein we desined special primers containing NdeI and EcoRI sites, amplified the gene and cloned into expression vector pET28 cleaved with NdeI-EcoRI. Protein SDS electrophoresis gel demonstrated the overexpresson of the protein with proper size.

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SOTO, ANÍBAL; Cajigas Rodríguez, Iván J.;  Soto Cardalda, Aníbal;  García Crespo, Katia; Pérez Guzmán, Luis J.; Arroyo Cruzado, Gerardo; Candelas, Graciela C., Prof., UPR-RÍO PIEDRAS, Natural Sciences  

A Novel Approach for the Molecular Cloning of Proline tRNA Genes From the Orb-Web Spider Nephila clavipes

Time sequence studies monitoring of  elicited fibroin synthesis in the large ampullate glands of the orb-web spider Nephila clavipes have revealed a series of discrete tissue and time specific waves of prelude macromolecular syntheses.  During one of them, tRNAs are selectively accumulated through differential gene expressions.  We are currently analyzing the selective accumulation of the tRNAs, cognate to fibroin’s preponderant aminoacids, (glycine, alanine and proline) at the level of gene expressions. Work on alanine and glycine tRNAs are either published or in progress, so we are curently focusing on proline tRNA in its selective upgrading. The cloning of the genes encoding proline tRNAs is therefore, critical to the understanding of  the transcriptional controls underlying the differential accumulation in fibroin production.  Screening of proline tRNA genes within the spider genome has been performed using via probing with a synthetic oligonucleotide designed on a highly conserved sequence of these genes from different organisms.   A novel PCR-based cloning strategy, TOPO Cloning (Shuman, 1994) allows the amplification of the putative gene and their cloning from high molecular weight DNA as template.  Using this strategy a 500bp DNA fragment corresponding to  the assumed  proline tRNA genes has been amplified and cloned.  Sequence analyses of this fragment are currently under scrutiny for identification through alignment.

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SOTO RIVERA, JACKELINE; Uscian, John, UPR-MAYAGÜEZ, Biology

Purification and Characterization of Trypsin From Intestinal Tissues of the Silk Snapper, Lutjanus vivanus

The abundance of digestive proteases in fish intestines will vary with diet.  On the idea that the relative abundance of trypsin may therefore function as an environmental indicator, trypsin from the silk snapper, Lutjanus vivanus, was examined.  Digestive tracts were obtained from fishes caught by commercial anglers of Puerto Real, PR.  Intestinal tissues were selected and homogenized.  The homogenate was subsequently treated via centrifugation and ammonium sulfate fractionation procedures.  Highest activity was detected in the 50% ammonium sulfate fraction.  An attempt to purify the enzyme from this 50% ammonium sulfate fraction will be made using a Benzamidine "Hi-Trap" column (Amersham-Biosciences), an affinity column.

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SOTO-PANTOJA, DAVID, UPR-MAYAGÜEZ; Lizardi, Paul, Yale University School of Medicine

Escherichia coli In vitro Transcription Translation System

The search for a cure has considerably progressed during the last years, but still one of the things that we know is that the earlier the detection is made, the patient would response more efficiently to treatment. The purpose of this research is to create an in vitro Transcription/Translation system to stabilize mRNA by developing a Vector that protects protein from degradation of exonucleases, in order to get more protein to detect and identify mutations in single cells using MALDI-TOF to visualize for mutations. This investigation concentrates in the design and ligation of the system. The system is composed of two main parts a PCR product and a Vector. The PCR product is made of K-ras exon 1 primers , arginines, a termination sequence and an adelynation sequence. The vector has the initiation codon, Shine-Dalgarno sequence a 5¡|stem-loop, a promoter sequence, a 3¡| stem loop and a mini hairpin. Each of the sequences were compared with the Escherichia coli codon usage for optimun performance in transcription and translation. DNA length was verified for its efficiency. This phase of the research was completed after circle ligation of the Vector and the PCR product by using ligase H and incubating overnight heating at 55 ^OC.

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VALENCIA-RIVERA PATRICIA; Martínez-Cruzado, Juan C., Prof., UPR-MAYAGÜEZ, Arts and Sciences, Biology Department

Contemporary Haplogroup A Mitochondrial DNA Reveals Pre-Colombian Migrations to Puerto Rico

About 55% of the mitochondrial DNAs of Native American origin in Puerto Rico have been classified as belonging to haplogroup A.  Approximately 75% of the haplogroup A mtDNAs in the Americas lack a Hae III site at the nucleotide position 16,517. In our study, 120 samples of haplogroup A mtDNAs have been analized for the presence or absence of this site. The results obtained indicate that 82.5% of haplogroup A mtDNAs lack the 16,517 Hae III site while 17.5% of haplogroup A mtDNAs posses the site. This scenario is more consistent with a common origin for most of the Puerto Rican haplogroup A mtDNAs. To test this, all mtDNAs from each group are being tested for a set of diagnostic restriction sites that may identify close relatives in the Americas. This has been hindered by the fact that samples have presented the same restriction sites as those mtDNAs founders of the New World that are present in North, Central, and South America. Identification of possible haplogroup A migrations to Puerto Rico have been studied by Median network analysis of DNA sequences from two hypervariable regions (HV-I and HV-II). This has revealed two haplotype clusters that represent at least two haplogroup A migrations to the Island; one much older than the other. The average number of sites HV-I differing from any sequence to the root in the older cluster suggests that the first migration ocurred shortly before the disappearance of the land bridge that connected Cuba with the Yucatan Peninsula.

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VÁZQUEZ YARED, PUCPR, Biology; Matta, Jaime, Ponce School of Medicine

Detection of Apoptosis and Morphological Changes in UV Irradiated Human Skin Fibroblasts

UV radiation is a potent creator of oxygen free radicals. Today there are limited studies explaining the mechanism by wish they cause damage to humans, specifically to human skin fibroblasts. Having in mind the hypothesis that acute exposure to Puerto Rico UV light levels is a strong factor in oxidative damage to the skin and that it may later lead to apoptosis and necrosis we decided to test environmental levels of UV on human skin fibroblast to see the results. Environmental levels of UV were measured in a two year period and used to calibrate the Oriel solar simulator to Puerto Rico UV light levels specification. Cells were irradiated at different doses equivalent to different time periods of exposure that a regular person would endure in a day to day basis. Apoptosis and necrosis was measured using an R&D Apo Kit a commercial apoptosis detection kit. Also morphological changes were documented using digital microscopy. We found that acute exposure to the equivalent of of 1.5 hours of environmental levels of UV can cause necrosis in fibroblast cells. These results show that even a small exposure of the environmental levels of UV light can cause substantial oxidative damage to our skin and thus increase our changes of contracting more serious skin diseases.

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VÁZQUEZ, RANDY; Asencio, Carmen, Prof., PUCPR, Departamento de Biología              

Efectos de Petiveria alliacea L. y Plantago  Mayor L. en el Crecimiento de Células Cancerosas

Las plantas han demostrado poseer numerosas propiedades medicinales contra diferentes enfermedades.  Petiveria alliacea L. es una planta herbácea que crece silvestre en tierras tropicales de Centro y Sur América, El Caribe y África.  La raíz de la planta se emplea como antiespasmódico en casos de calambres, inflamaciones de la vejiga, coyunturas, contracciones nerviosas, etc.  Plantago mayor L. también crece silvestre en toda clase de terrenos llanos yermos y cultivados.  Esta planta se ha usado medicinalmente por décadas.  Es conocido mayormente por sus propiedades de sanar heridas.  El propósito de este estudio es determinar sí la P. alliacea y el P. mayor causa inhibición en el crecimiento de células cancerosas.  Para este estudio, primero se cultivan las células de CHO o células de ovario de hámster chino en unos “culture flasks”.  Luego se le descarta el medio de cultivo y se le añade 3 mL de extractos de P. alliacea y P. mayor en unas concentraciones de 5, 10, y 15 M y se exponen a las células por un periodo 2, 4 y 6 horas para cada concentración.  Luego se medirá el por ciento de absorbancia de cada muestra usando un  espectrofotómetro.  En este experimento se espera que halla una diferencia entre el por ciento de absorbancia de las células expuestas a los extractos de P. alliacea y P. mayor y las que no las están, (grupo control), dando indicios de inhibición  de las mismas.

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VÁZQUEZ-ACEVEDO REBECA; Ríos-Velazquez,Carlos, UPR-MAYAGÜEZ, Arts and Science

Isolation and Characterization of Specific Bacteriophages for the Photosynthetic Bacteria Rodobacter sphaeroides From Samples of Soil and Water of the Southwest Area of Puerto Rico

The bacterium Rhodobacter sphaeroides belongs to the |Á-subdivision of the proteobacteria (purple non-sulfur bacteria).  This group of gram-negative bacteria are among the most metabolically diverse organisms known, being capable of growing in a wide variety of growth conditions and being used as model organism to understand photosynthesis and cytochrome structure and function.  To date, there are not many genetics tools for the specific manipulation of this group of bacteria.  The main goal of this research consists in the isolation and characterization of bacteriophages for the photosynthetic bacteria Rhodobacter sphaeroides from soil and water samples from different parts of southwest region of Puerto Rico.  The strains used in this study included R. sphaeroides 2.4.1, 630, and 7001, this last one being a restriction mutant of the 630 strain.  The double layered cultured technique was used for the isolation and enumeration of the phages obtained.   From seven of the water samples that where analyzed, we isolated phages for the strains 7001 and 630 of R. sphaeroides.  This bacteriophages where mostly found for the strain 7001 in Ponce, Mayagüez and Cabo Rojo. The phages isolated from this study, will be characterize genetically and morphologically by genetic engineering tools and by Scanning Electron Microscopy.

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VEGA, MARÍA; Santiago, Angélica; Fernández, Denny S., Prof., UPR-HUMACAO, Ciencias Naturales, Departamento de Biología

El Efecto de la Luz y la Disponibilidad de Nitrógeno en la Densidad Estomática de Tres Especies de Árboles Tropicales

Los efectos de las condiciones hídricas y la intensidad de luz en la densidad de los estomas han sido ampliamente estudiados en las plantas, especialmente en aquellos ambientes donde los niveles de estos factores presentan una limitación al crecimiento.  Es menos conocido el posible efecto del nivel de fertilización en la distribución de estomas en la hoja. En este trabajo nos preguntamos como se afectará la densidad de estomas debido al efecto combinado de tres niveles de luz (45 %, 10 % y 2 % de la radiación solar diaria) y tres niveles de disponibilidad de nitrógeno (0, 0.007 y 0.14 g N/l/mes) en tres especies de árboles (Guarea guidonia, G. ramiflora y Manilkara bidentata) de un bosque muy húmedo tropical (Sierra de Luquillo).  Al cabo de un año de tratamiento se tomaron impresiones de la superficie foliar, utilizando pintura de esmalte, y se montaron en laminillas de microscopía.  Estimamos la densidad de estomas mediante contaje directo en campos de 0.5 mm de diámetro.   La especie Guarea guidonia presentó una densidad  de estomas significativamente menor  que las otras dos especies de árboles. Un análisis de varianza indicó un efecto significativo de los niveles de radiación en la densidad de estomas, la cual aumenta a niveles mas elevados de radiación. Por el contrario, la disponibilidad de nitrógeno no mostró un efecto significativo en la densidad de estomas.  Aunque el nitrógeno es un elemento esencial en el desarollo del aparato fotosintético, no parece jugar un papel importante en la distribución de las células guardianas en la superficie foliar.

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VEGA HERNÁNDEZ, MÓNICA; Martínez Cruzado, Juan C., Prof., UPR-MAYAGÜEZ, Art & Sciences

Estudio Sobre Taza Mutacional en la Población Viequense

Este proyecto intenta utilizar la hipermutabilidad de la región hipervariable del DNA mitocondrial para explorar la posibilidad de que la alegada contaminación ambiental a la que está expuesta la población de la isla de Vieques esté causando mutaciones somáticas o germinales.  Se presume que la neutralidad selectiva de esta región permitirá la detección de mutaciones que de lo contrario serían eliminadas rápidamente por la selección natural.   Un fragmento de 837 pb de la región hipervabiable fue amplificado con la reacción de la polimerasa en cadena en 19 muestras viequenses, incluyendo personas relacionadas por su línea materna.  La secuencia de un total de 671 pb del fragmento está siendo determinada.  Las secuencias permitirán detectar cualquier mutación que haya ocurrido en esta región entre las generaciones.  Como producto secundario de este proyecto, se obtendrán secuencias que darán información sobre el origen continental de los viequenses en contraposición a los habitantes de Isla Grande.

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VIDAL GONZÁLEZ, IVAN GABRIEL, PUCPR, Biology Department; Horton, Teresa H. Dept. of Neurobiology and Physicology and the Center for Sleep and Circadian Biology; Northwestern University Turek, F.W. Dept. of Neurobiology and Physicology and the Center for Sleep and Circadian Biology; Northwestern University          

Effect of Age-Dependent Changes in Circadian Rhythms on Photoperiodic Time Measurements in Golden Hamsters

Our laboratory conducts studies examining the relationship between aging, circadian rhythms, (CR) and seasonal reproductive cycles (SRC) in golden hamsters.  An internal clock responsible of regulating CR is located the suprachiasmatic nucleus (SCN) of the brain.  The SCN can drive rhythms in other structures, such as in the pineal gland (PG). The PG is responsible of producing a photo-sensitive hormone called melatonin.  In mammals that reproduce seasonally, melatonin is thought to provide information about the length of night and thereby control SRC. Deficits in locomotor activity and melatonin concentration have been observed in aging hamsters.  Despite the age-related decline in melatonin secretion. aging golden hamsters show reproductive cycles.  Our current study aims to examine how the information about seasonal day length is conveyed in hamsters with reduced melatonin levels.  Wheel-activity and melatonin levels will be monitored in young, middle-age and old golden hamsters.  Within the different age groups there will be an equal number of animals that will have wheel versus animals with no-wheel. Melatonin will be measured in blood samples taken every half-hour throughout the dark period.  We will examine and test for small phase shifts in the timing of both melatonin concentration and locomotor activity, which may influence the neuroendocrine responses to photoperiod.  In addition we can have a better idea of differences in phase shift between age groups.  Differences between age groups in locomotor activity and melatonin rhythm will lead us to better understand the mechanism of sleep disorders and sleep deficits in the elderly people.

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VIERA VERA, JORGE; Martínez, Juan C., Prof., UPR-MAYAGÜEZ, Department of Biology

Los Orígenes del DNA Mitocondrial Africano en Puerto Rico

Estamos caracterizando el DNA mitocondrial (mtDNA) de origen africano que hemos identificado en Puerto Rico para conocer mejor la procedencia geográfica de la herencia africana que hemos recibido los puertorriqueños por vía maternal.  La caracterización se hace a base de la identificación de haplogrupos por medio de análisis de sitios diagnósticos de restricción a un grupo de muestras representativo de la población puertorriqueña.  De un total de 803 muestras puertorriqueñas, 213 fueron identificadas como provenientes del África que queda al sur del desierto de Sahara.  Pruebas subsiguientes nos ayudan a caracterizar estos mtDNAs en los haplogrupos específicos de África:  Q, R, S, L1a1, L1a2, L1b1, L1b2, L2a, L2b, L2c, L2d y L3*.  Los resultados sugieren que todos los haplogrupos de África se encuentran en Puerto Rico.  Además, la data sugiere que la mayoría de los africanos que llegaron a Puerto Rico provenían de África occidental.  Esto se debe a que la frecuencia en Puerto Rico de haplogrupos que son exclusivos de África occidental es similar a la de esa región geográfica.

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VILLALBA-RAMOS, NADYA YANUSKA; Martínez-Cruzado, Juan C., Prof., UPR-MAYAGÜEZ, Arts and Sciences, Department of Biology

Identifying the Most Common Y-Chromosome Haplogroups in Puerto Rico

The genetic contribution to the Puerto Rican gene pool of the ethnic groups that populated the Island of Puerto Rico is not well understood. The Y chromosome helps us compare the different population contributions through the paternal lineage. The non-recombining region of the Y chromosome contains polymorphisms that have become useful tools in studies of population migration history. Series of polymorphisms define haplogroups. Because haplogroups are exclusive of or predominant in certain geographical regions the identification of the haplogroup to which a Y-chromosome belongs is a good start in the quest of identifying its continental origin. Haplogroup identification of Y-chromosomes is achieved through PCR-amplification and RFLP analysis of diagnostic biallelic loci. In a collaborative effort, ten samples collected of men from various parts of the Island have been analyzed for Y-chromosome haplogroup identification. The data reflects a trend towards the 1C haplogroup which is predominant in Spain. Nevertheless, the 1C haplogroup can also be found in approximately one-third of Native Americans. Future studies involving microsatellites and the alphoid heteroduplex polymorphic system will help clarify the data obtained from biallelic markers in the present investigation.

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VILLANUEVA, ENEIDA; Planas, José, Prof., UPR-AGUADILLA, Biology Department

Intranasal Pairwise Competition Among Penicillin-Sensitive and Non-susceptible Streptococcus pneumoniae isolates

The overuse of antibiotics has led to the emergence of resistant strains of bacteria. Bacteria acquire resistance in different ways, such as incorporating foreign DNA into their chromosomes by transformation. Often, plasmid a nd chromosomally conferred resistance causes a loss in the fitness of the bacteria, even though exceptions are known. We created penicillin-intermediate variants of two penicillin-sensitive Streptococcus pneumoniae strains by a combination of mutat ion and transformation of penicillin-binding protein genes. Intranasal colonization was initiated in infant rats with inocula containing a mix of a parental strain and a non-susceptible variant of that strain. We estimated the fitness cost of resistance a mong these variants by measuring the changes in the ratio of non-susceptible:sensitive strains during pairwise competition. Although, resistance was achieved to an intermediate level, some of the non-susceptible strains showed no detectable fitness cost, while other variants did have a cost.

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