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Díaz, Angel D.; Vivoni, Alberto*, Departamento de Biología, Química y Ciencias del Ambiente, UIA-San Germán
Evaluación de métodos computacionales en el estudio de sistemas de porfirinas
Durante décadas se ha tratado de entender el comportamiento del complejo de Fe-porfirina que se encuentra en los grupos heme de las proteínas mioglobina y hemoglobina. Este complejo tiene afinidad tanto para el elemento primordial para la vida, oxigeno, como también para compuestos tóxicos como monóxido de carbono. En este trabajo, evaluamos métodos computacionales con diferentes sistemas de porfirinas para determinar el mejor método de análisis. Se optimizaron las estructuras con los métodos computacionales ab initio, semi-empíricos y de mecánica molecular del programa CAChe. Se analizaron las estructuras libres y solvatadas de complejos de porfirina enlazada a oxígeno, monóxido de carbono e imidazole. Se compararon los resultados con estructuras obtenidas del banco de proteínas del NCBI. Los parámetros que se usaron en la comparación fueron longitudes de enlaces de Fe-porfirina y Fe-O, Fe-CO y Fe-imidazole. Para la molécula libre, el método de AM1 fue el que mejor reprodujo las longitudes de los enlaces Fe-profirina, Fe-O2 y Fe-CO. Este método fue también el que mejor reprodujo el tamaño del “core” de la porfirina. Los métodos MM2 y MM3 fueron los que mejor reprodujeron la distancia Fe-imidazole. También se analizaron los métodos de Mecánica Molecular con sistemas de porfirina en una simulación acuosa, por medio de una gota de agua que el programa CAChe permite crear, dando distancia cercanas a las experimentales tales como Fe-O 1.906 Å (experimental 1.846). Se calcularon las vibraciones IR de las estructuras antes mencionadas, pero sólo se obtuvo resultados con el método de AM1.
Rodríguez, Valerie; Vivoni, Alberto*, Department of Biology, Chemistry and Environmental Sciences, UIA-San Germán
AM1 calculations of chemical and physical properties of gemfibrozil and derivatives
Gemfibrozil, 5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid is a lipid-regulating agent, which decreases serum triglycerides as well as very low-density lipoprotein cholesterol, and increases high-density lipoprotein cholesterol. In a previous work, the biological activities of gemfibrozil and the quiral analogues of 5-(2,5-dimethylphenoxy)-2-methylpentanoic acid and 5-(2,5-dimethylphenoxy)-2-ethylpentanoic acid were studied. The purpose of the present work is to analyze the biological activity of these drugs through quantum mechanical calculations. For this work, we used the AM1 method of the MOPAC package of the CAChe Software. The properties studied were: energy of optimized geometry, partial charges, nucleophilic susceptibility, dipole moment, solvation energy, solvent accessible surface, and LUMO density. Previous studies show that gemfibrozil undergoes oxidation of the ring methyl group to successively form hydroxymethyl and carboxyl metabolites. The calculation results of the present work show that the methylic cation at the ortho position is more stable than the one at the meta position. Consequently, it is likely that the oxidation of the compound occurs at ortho position. Furthermore, the calculations show that changes of the substituent group on the pentanoic acid chain do not affect the properties of the benzylic carbocation, but that they do affect the properties of dipole moment and solvent accessible surface. These properties, however, are not affected by the stereoquemistry of the molecule. From these results, it can be inferred that the differences in the biological activities of the stereochemically different drugs is due to steric hindrance in the interaction of the substituent groups with the enzyme that propitiates the reaction.
Rodríguez Rodríguez, Yainitza; González, Wigberto; López, Gina, Biology Department, UPR-Mayagüez; Rivera, Lidia, Crop Protection, UPR-Mayaguez; Fernández, José, Industrial Biotechnology, UPR-Mayagüez; Diaz, Emilio*, Chemistry Department, UPR-Mayagüez
Induction of oxidative enzyme activities in P. cinnamomi with hydrogen peroxide
Phytophthora cinnamomi has been catalogued as a seriously destructive pathogen for avocado trees (Persea americana), in addition to other plant species in Puerto Rico and around the world, causing mayor economic loss. The avocado tree produces hydrogen peroxide as defense against the fungus, among other things, but P. cinnamomi still causes rotten roots. It may be that the avocado plant causes oxidative stress on the organism when it produces hydrogen peroxide and the organism responds with oxidative enzymes such as catalase and glutathione peroxidase, which then act as scavengers of reactive oxygen species that can degrade and destroy organelles, cells and tissues. It is believed that there is a relationship between the defense mechanism of the plant, the level of oxidative enzymes in Phytophthora, and its virulence towards the plant. That would suggest the enzymes may be induced by oxidative stress on the fungus, as a defense mechanism. The organism was grown in Lima Bean broth, and different concentrations of hydrogen peroxide were added, after 24 hours of incubation. The cellular extracts were examined with enzymatic essays for catalase and glutathione peroxidase. The results will be presented.
Varela-Agront, Gladys M., Faculty of Arts and Sciences, Department of Biology, UPR-Mayagüez; Rios, Robert*, Department of Chemistry, UPR-Mayagüez
Synthesis of peripheral benzodiazepine receptor (PBR) ligands
The physiological role of the PBR sites is still under investigation, but is thought that they are related to cell growth and proliferation. PBR’s are known to be concentrated in solid malignant tumor tissue. We wanted to examine the effect of varying the oxidation state of the diazepine ring, including the three possible methylated derivatives to better understand the structural requirement of these ligands to the receptor. The synthesis and characterization of various methylated derivatives of 7-Chloro-5-phenyl-1,4-benzodiazepine-2-one will be reported. Also preliminary data on affinities of these derivatives to the PBR will also be presented.
Segarra, Aidmari*UPR-Humacao, Departament of Biology;
MECOBI: Un Curso Experimental de Integración de la Biología, la Matematíca y la Computación a Nivel Subgraduado
MECOBI (Métodos Computacionales Aplicados a Sistemas Biológicos) es un curso experimental multidisciplinario, que fue ofrecido durante el primer semestre del 2002-2003 en la Universidad de Puerto Rico en Humacao. En el mismo, se combinaron temas de biología, matemáticas, y computación con especial énfasis en el área de Biología de Poblaciones. En este curso participaron estudiantes de las concentraciones de biología y matemáticas computacionales. A los estudiantes se le presentaron conceptos tales como: poblaciones no-estructuradas, ecuaciones de diferencias lineales y no-lineales, poblaciones estructuradas, matrices, competencia y depredador–presa, ecuaciones diferenciales, y modelos espaciales. El curso consistió de conferencias, y prácticas de laboratorio computacional y la evaluación se basó en asignaciones y un trabajo de investigación final. El mismo fue ofrecido por dos profesores simultáneamente: uno de los cuáles aportó sus conocimientos en el área de la biología y el otro en el área de matemáticas computacionales. Durante el semestre se utilizaron varias aplicaciones y lenguajes de programación tales como Mathematica, Populus y Star Logo. En una encuesta realizada al finalizar el curso los estudiantes señalaron que el curso cumplió exitosamente con los objetivos de integrar las tres disciplinas. Además indicaron su interés en tomar otros cursos multidisciplinarios, así como recomendarlo a otros estudiantes. Se presentarán ejemplos de ejercicios de laboratorio y proyectos realizados.
Cartagena, María*UPR-Humacao, Department of Biology; Ortiz, Jomar, Calcaño, Wanda, Claudio, Gladys, Cintrón, Isabel Fuentes, Francisco
Presence of Prokaryotes from the Bacteria Domain in Tropical Hypersaline Salterns Ponds
Thalassohaline solar salterns are considered habitats with a low species diversity. In order to assess the actual genetic diversity of prokaryotes populations in salterns ponds, finger-printing analyses of 16S rDNA genes were conducted. An analysis of the genotypic diversity in saltern pond’s waters and in Larsen’s broth cultures, by amplification of the 16S rDNA genes, showed the presence of both Bacteria and Archea. Until recently, Archea were identified as the only microbial inhabitants of hypersaline habitats. This work presents the second scientific report of Bacteria in hypersaline waters. Our results suggest that variability within the extreme halophilic prokaryotes in our tropical environment is greater than what is generally expected.
Estrada, Luis D.*UPR-Humacao, Microbiology; Méndez Cruz, D.,University of Puerto Rico-Humacao, Visscher, P.,T., University of Connecticut-Groton, Nieves-Méndez, D. University of Puerto Rico-Humacao, Hernández-Cruz, C., University of Puerto Rico-Humacao, Casillas-Martínez, L. University of Puerto Rico-Humacao
Characterization of Extreme Halophilic Bacteria Isolated from Crystallizer Ponds
Recent discoveries have shown that extreme halophilic bacteria play an important role in hypersaline ecosystems such as the crystallizer ponds from solar salterns. However, studies regarding the in situ activities and diversity of such novel bacteria are limited. Consequently, we have studied the bacterial diversity and physicochemical conditions of several crystallizer ponds with salinity levels up to 630 practical salinity units (PSU). Further analysis of the ponds indicated a large concentration of commonly limiting nutrients such as nitrogen and a high chemical oxygen demand. Using microelectrodes we also determined oxygen penetration in the water column up to 3mm depth. Such eutrophic environment harbors an abundant bacterial diversity. To enrich for such bacteria we used several standard media such as Larsen, Marine Agar and natural brine water supplemented with extracts of Rodospirilium salinarum. Eleven uncharacterized bacteria were cultured using this natural media. Although most isolates were Gram negative bacteria form the Vibrio genus, analysis of their fatty acids indicate a low similarity index to previously identified bacteria. Three of the isolates were extreme halophiles as they require more than 2.0 M NaCl for optimal growth. We also studied the changes on fatty acids of one of these extreme halophiles (CR-11) as the salinity of the medium was increased. A ten fold raise in the salinity of the growth medium induced a 20% increase in the total content of unsaturated fatty acids of CR 11. 18:3 was the dominant unsaturated fatty acid produced under salt stress. Our next step is to identify this bacteria using phylogenetic analysis to determine if novel extreme halophiles have been discovered.
Hernández-Cruz, Luz Raquel*UPR-Humacao, Departamento de Biología; Aponte-Rodríguez, José A. Departamento de Matemáticas, Universidad de Puerto Rico en Humacao
Análisis Poblacional de Dermochelys coriacea (Tinglar)
Utilizando los datos recopilados por el programa de patrullaje de tortugas del proyecto Sea Grant en Humacao durante los años 1997-2000, analizamos el comportamiento poblacional del Tinglar, Dermochelys coriacea. Por medio del uso del programa Mathematica, calculamos las matrices de proyección, el análisis de sensibilidad y el parámetro lamda para sus diferentes etapas, las cuales definimos como: Huevos, Neonatos, Juveniles y Adultos. Dermochelys coreacea disminuirá significativamente con el pasar del tiempo hasta lograr un equilibrio donde se encontrará en una línea muy fina entre la extinción y la sobrevivencia. El ciclo de vida oceánico de Dermochelys coreacea limita la exactitud de su estudio poblacional debido al desconocimiento exacto del número de individuos en las etapas juveniles y adultas. Este proyecto al igual que otros análisis poblacionales basa sus resultados en una serie de supuestos, los cuales deben ser descifrados para poder realizar análisis poblacionales con resultados más significativos.
Malavé, Josué*UPR-Mayagüez, Department of Biology; Aponte, Aidali, Villanueva, Eneida
Isolation and characterization of bioluminescent bacteria from marine environments in Puerto Rico
As the phenomenon of bioluminescence increases its interested observers, new possible applications such as a better gene reporter that are being used for the improvement of molecular biology. These species of bacteria are known to emit a blue green light for communication purposes provoked by the enzyme luciferase regulated by the lux gene operon. This research intends to isolate bioluminescent bacteria from marine habitats and characterize it molecularly for future applications. With a collection of pure isolated bioluminescent bacteria we accomplished the microbiological characterization and a preliminary molecular analysis. On going experiments are in progress in order to isolate the lux region to compare the lux genes of the isolates.
Martínez, Ramón*UPR-Mayagüez, Industrial Biotechnology Program;
Preliminary determination of microbial flora in several soils in Puerto Rico by using molecular analysis and metagenomic libraries generation.
Physiological and chemical factor are limitating the discovery of new organism in diverse ecosystems. It is know that only 0.1% of microorganisms are cultivable in laboratory but there are a 99% that remain uncultivable. Accessing the genome of the microbial flora in (metagenome), and their analysis by using Molecular techniques such as 16S rDNA, will allow the understanding of the real soil population and diversity, and will permit the isolation of new products of industrial, and clinical importance. The project seeks to develop environmental libraries from soil samples in several forest and crops in Puerto Rico, and determine the microbial population. DNA was isolated from soil samples, and the Domain specific primers were used to amplify the rDNA. Genetic engineering techniques were used to generate and evaluate the environmental libraries. Successful genomic preps, 16S and 18S rDNA amplifications, and their restriction analysis from the soils are presented, and the possible generation of a high molecular weight environmental libraries are also shown.
Ocasio, Juan*UPR-Mayagüez, Department of Biology; Acosta, Jaime, Guzmán, Angélica
Study of the Aquatic Macro invertebrates in Canalized and Non Canalized Areas of Quebrada de Oro at Mayaguez
The study was performed at Quebrada de Oro, a small stream across the University of Puerto Rico at Mayagüez Campus. The main purpose of the study was to collect and identify the insects found at the stream and uses this information as indicators of the quality of the water. Two areas were sample; a canalize and the non-canalized area of the stream. A total of thirteen insects families were found, eleven were found at the canalize site and nine were found at the non-canalized site. The insect’s fauna consisted mostly of pollution-tolerant midge larvae, indicating poor to fair water quality. Midges (Diptera) were more abundant at the canalize site. This group can tolerate more sediment and needs less oxygen to survive. Members of the order Diptera, or true flies, are especially good biondicators of water quality because they occupy the full spectrum of habitats and condition. Mayflies (Ephemeroptera) were more abundant at the non-canalize site. This group is very sensitive to the water conditions and less tolerant, their abundance indicating improve water quality in insects found at the stream and use this information as indicators of the quality of the water.
Ortiz, Collazo, Yamarie* Pontifical Catholic University of Puerto Rico, Dept of Biology; De Jesús Martínez, Caly A,. López Moa, Mildredenid, León Zayas, José A., Molina Colón, Sandra
Reforestación con Especies Nativas de Bosque Seco
Se estudio el crecimiento de 244 plántulas de especies nativas de Bosque Seco en dos lugares: Bosque Seco de Guánica y Sierra Bermeja en Puerto Rico, en dos parcelas en cada lugar, con 61 plántulas en cada una, bajo dos tratamientos: una parcela con sombra ( producida por el árbol de Leucaena leucocephala) y otra parcela sin sombra. Se midió el crecimiento alcanzado mensualmente y se irrigó las plántulas bisemanalmente. En general, se esperaba que el mayor crecimiento fuera alcanzado en las dos parcelas con sombra debido a que el árbol de Leucaena además de proveer sombra, enriquece el suelo con nitrógeno y otros nutrientes. Se encontró que la diferencia en el crecimiento entre las plántulas con sombra y las plántulas sin sombra no era significativo, mientras que si hubo una diferencia significativa en el crecimiento de las plántulas por lugar. Las plantas en el Bosque Seco de Guánica crecieron mejor que las plantas en Sierra Bermeja. La diferencia fue estimada en siete centímetros de altura. Esta diferencia puede deberse a que ya que las plantas son nativas de Bosque Seco su crecimiento es mejor en este habitad para el cual ya están adaptadas, otro factor importante también pudo haber sido el viento, la composición del terreno así como el factor competencia. Durante este año se buscará correlacionar el crecimiento relativo de las plantas con la precipitación ajustada.
Padilla-Crespo,Elizabeth*UPR-Mayagüez, Industrial Biotechnology; Rios-Velázquez, Carlos, Hazen,Terry, Center for Environmental Biotechnology, DOE Lawrence Berkeley National Laboratory
Molecular Analysis of the Microbial Community Structure in Aerobic and Anaerobic Landfill Bioreactors
Landfill management is becoming one of society’s greatest problems. Bioremediation approaches represent a solution to treat these sites, but in order to design the most optimal strategy, a better understanding of the microbial communities involved in detoxification and stabilization is needed. In this study we analyzed the microbial community present in different landfill bioreactor simulations by molecular techniques. Genomic DNA from the bacterial communities of leachate and gravel samples from the bioreactors was extracted, and the total 16S rRNA’s were amplified by Polymerase Chain Reaction (PCR) using universal bacterial primers. Terminal restriction fragment polymorphism (T-RFLP) was used to analyze the community structure, and individual 16S rRNA from the bioreactors were cloned and analyzed by T-RFLP and sequencing. The presence of TCE and PCE dechlorinating bacteria Dehalococcoides was also tested in the bioreactors by PCR. T-RFLP results showed a highly diverse communities in all bioreactors, with the anaerobic bioreactors giving the highest number groups. The anaerobic bioreactor communities were more diverse in the leachate than the gravel. An amplification product using Dehalococcoides specific primers was obtained from samples taken at the Yolo County Landfill. Clone libraries from the anaerobic bioreactors were generated. T-RFLP of selected clones showed that they have dominant peaks in the community, and sequencing analysis suggests that they belong to two different phylogenetic groups of unculturable bacteria.
Sánchez, Elia Enid*UPR-Río Piedras, Department of Biology; Toranzos, Gary A.
Characterization of Microbial Water Quality in Recreational Waters from Puerto Rico
Anthropogenic sources of microbial polluted waters can increase the probability of diseases, but no formal study has been conducted to quantify this in the Caribbean island of Puerto Rico. Five public beaches located mainly on the north coast of the island were sampled from January to August, 2002. Four samples were taken (two at chest depth and two at ankle depth) with a 200 ft of separation in the beach. Samples were taken in the morning (9:00 am), at noon (12:00pm), afternoon (3:00pm) and in the evening (6:00pm) for two consecutive days during the weekends. Physico-chemical characteristics of water were analyzed in each sample. The quantity of swimmers at every hour in the beach was also recorded. Samples were analyzed for microbial concentrations by Membrane Filtration (MF), Colilert and Enterolert methods.
Mean total coliforms and termotolerant bacteria for all beaches sampled with MF was 70.0 CFU/100mL and 18.17 CFU/100mL respectively (N=160). Mean MPN values for Colilert and Enterolert in a 100 mL were and 2418.3 and 228.44 respectively (N=96). Simple linear regression model to relate bacterial concentrations with physical parameters of water showed a statistically significant association between enteroccoccus bacteria and total dissolved solids in the water (p=0.009). One-way analysis of variance (ANOVA) showed a significant difference (p=0.000) in total coliforms concentrations within methods and within beaches (p=0.000). No significant relationship was found between the quantity of swimmers and bacterial concentrations in any of the two methods of analysis. Bacterial indicators of fecal pollution in beaches from Puerto Rico can reach high levels and can be hazardous to human health.
A prospective epidemiological study will be useful to investigate the morbidity of bathers and it's relationship with bacterial indicators of fecal contamination in these tropical waters.
Almodóvar, Wanda*UPR-Mayagüez, Department of Biology, Martínez-Cruzado, Juan C.
Contribución caucásica e indígena en el genoma puertorriqueño a través de la línea paterna
El propósito de este estudio es determinar la contribución por vía paternal de los grupos caucásicos 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, 29 muestras de DNA de estudiantes de la Universidad de Puerto Rico-Mayagüez, procedentes de distintas áreas de la isla. En un estudio previo, dichas muestras demostraron tener los genotipos DYS257A, DYS199C, SRY10831G y SRY2627C. Cromosomas Y con tales genotipos componen el 68% de los cromosomas Y en España y el 25% de los amerindios. En este estudio, dichas muestras fueron analizadas para el locus DYS194 que puede indicar si el cromosoma Y pertenece al haplogrupo 1L (europeo) o al 1C (indígena). Los resultados al presente son que el 14% tiene ascendencia indígena 1C mientras que un 66% procede del haplogrupo 1L (europeo); 20% de las muestras aún se encuentran bajo estudio. Por otra parte, 3 muestras pertenecientes al haplogrupo 1B, el cual tiene origenes africanos y europeos, fueron analizadas con respecto a un polimorfismo en el locus DYS188 resultando tener procedencia europea. Las muestras pertenecientes a los haplogrupos 1C y 1L están siendo analizadas en términos de sus microsatélites, DYS19, DYS389, DYS390, DYS391, DYS392 y DYS393, para mayor especificidad de los resultados. Hasta el momento, dentro del 20% de las muestras analizadas para DYS389 se ha notado una diferencia entre las de origen indígena 1C y europeo 1L. Próximas investigaciones serán dirigidas al análisis de la secuencia de nucleótidos de microsatélites.
Arce, Julio A.*UPR-Río Piedras, Department of Biology File-Emperador, Sharon
Morphological Parameters for Determinig the Sex of Marisa Cornuarietis
The large ampullate snail Marisa cornuarietis was introduced into Puerto Rico from South America. In the 1960s it was cultured and spread over the lakes and streams of the island as a biological competitor for the snail intermediate host of Schistosoma mansoni (bilharzia). More recently this large ampullate snail has been reported to be very sensitive sentinel for pollution with estrogenic chemicals. For these studies it is important to have a noninvasive method to differentiate the sexes but in the ampullariidae the sexes are separate but very similar and only certain subtle external differences allow us to guess the sex of an intact individual. The present study was designed to verify and quantitate the report of Demain and Ibrahim (1970) in which they reported that the sexes could be differentiated from the shape of the aperture. (i.e. the ratio of the height vs. the width of the shell aperture in males is close to <1.1 and the females have a ratio >1.2) This study extended those parameters by using Sigma-scan software to determine the area of the aperture. The method of evaluation was verified by dissecting each snail that died to determine its sex. In conclusion, the apertures of the two sexes are slightly different and various quantitative means of determining this reinforce each other but there remain some individuals where this method provides ambiguous results.
*The support of AMP during the course of this study is gratefully acknowledged.
Armaiz, Guillermo*UPR-CAYEY, Department of Biology; Kesterson, Robert, Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School Gardner, Wendy, Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School
CNS obesity pathway identification of melanocortin regulated genes by differential display
Dominant mutant alleles of agouti (e.g. Ay) direct over-expression of agouti signaling protein (ASP) and induce several phenotypic changes in the mouse including complete yellowing of the fur, increased linear growth, hyperinsulinemia, male-specific hyperglycemia, hyperphagia, and adult-onset obesity. These latter phenotypes are due to ASP inhibiting the actions of the fourth melanocortin receptor (MC4-R), which is expressed in the brain. Of the five known Gs-coupled ( cAMP) melanocortin receptors (MC-R), MC3-R and MC4-R are the primary mediators of the Central Nervous System (CNS) actions of melanocortin peptides. Although the expression of MC3-R is restricted mainly to the hypothalamus, both MC3-R and MC4-R are widely expressed throughout the brain. In order to discover downstream mediators of MC4-R action, we isolated novel cDNA fragments that are selectively turned on or off in response to melanocortin signaling using differential display methodology. This PCR-based technique can detect differentially expressed mRNAs, which is a powerful tool for studying differentiation, cell cycle, carcinogenesis, inductive events, and other biological phenomena that involve changes in gene expression. We treated mice neuroblastoma Neuro2A cells, a neuronal cell line known to express MC4-R, with melanotan II (MTII), a melanocortin receptor agonist that will stimulate MC4R, and compared the expression profile of mRNAs to control untreated cells. We have isolated 3 products by PCR that have been purified by the Gene Clean III method, and then sub-cloned into a plasmid vector for screening and sequencing. Validation of true differential expression of cloned cDNAs will be made by ongoing Reverse Northern Blot experiments. Final confirmation will be done using Northern blot analysis of RNA derived from control and hormone treated cells, whereas in vivo expression will be examined by in situ hybridization analysis of obese Ay and lean C57 littermates
Baerga, Rebecca*, Pontifical Catholic University of Puerto Rico; Sesti, Federico, Ph.D. Dept. of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School
Investigation of the molecular mechanism by which T8A-MiRP1 causes susceptibility to sulfamethoxazole
Long QT Syndrome (LQTS) is a cardiac disease characterized by a prolonged QT interval in the ECG. Common factors for acquired LQTs include drugs, metabolic abnormalities and gender. One of the primary factors for the onset of LQTs reside in malfunction of cardiac ion channels. These proteins generate electrical impulses by allowing exchange of ions across the cell membrane. IKr is an important repolarizing potassium current in the human ventricle and is produced by a channel formed by the pore-forming subunit HERG and the beta subunit MiRP1. A single-nucleotide polymorphism in MiRP1, T8A, was identified in patients affected by drug induced LQTS. When MiRP1 mutants are transiently coexpressed with HERG in CHO cells, they are inhibited by the antibiotic sulfamethoxazole (SMX), while channels formed with wild type MiRP1 are not. The natural mutation T8A occurs in the first consensus sequence for a N-glycosylation site and thus disrupts the ability to covalently attach sugar groups whose absence might facilitate drug accessibility to the site. To understand the molecular mechanism that determines susceptibility to SMX, we assessed drug blockage with HERG channels alone in the whole cell configuration of the patch clamp. We found that homomeric channels are inhibited like channels formed with T8A. This suggests that HERG provides a binding site for SMX and that MiRP1 protects its accessibility from SMX molecules. These results have important implications for the design of better drugs and may lead to a better understanding of the molecular mechanisms causing cardiac arrhythmia.
Bonilla, Margarita*UPR-Cayey, Department of Biology; Zheng Zhou, Ph.D. Department of Developmental Biology, Baylor College of Medicine
Genetics Characterization of New Engulfment Mutants
Apoptosis or programmed cell death is a process that occurs in multicellular organisms throughout life. The apoptosis of specific cells is closely linked to the removal of apoptotic bodies or cell corpses. The removal of the dying cells before the integrity of the cell membrane is lost prevents the release of potentially harmful contents, thus protects the neighboring cells and the entire organisms. Engulfment also is involved in the regulation of cell-cell communication, cell-cell interaction, and cytoskeletal reorganization. Recent genetic studies in C. elegans have identified 7 genes in this pathway. One of these genes is ced-1, which has been shown to encode a transmembrane protein in phagocytic cells. The aim of this study is to determine if the mutants have been isolated in the Zhou lab from EMS mutagenesis are new alleles of ced-1 or define new ced genes. I performed complementation tests for 23 of these mutants with CB3203 [ced-1 (e1735)]. Alleles of ced-1 will fail to complement and thus poses unengulfed cell copses in the heterozygous embryo. From the 23 mutant strains all the F2 embryos showed many persistent cell corpses, suggesting that all these mutants are new alleles of ced-1. I also participated in an on going genetic screen and isolated a cell corpse engulfment mutant, B60, which is yet to be characterized.
Burgos, Ricardo*UPR-Mayagüez, Department of Biology; Santos-Flores,Carlos, Ríos-Velázquez,Carlos
T7 Phage Display Technology for the Development of cyanotoxin Biomarkers
The areas of genomics and proteomics have become important for the development of new research tools in order to answer important biological questions. The area of functional genomics is been used to understand and to study interactions between proteins and other important molecules. The phage display technology is based on the surface expression of the peptide sequences fused with phage capsid protein and can be used to study proteins in a structural and functional level. The main focus of this project is to use phage display technology to develop biomarkers for toxin detection. T7 phages displaying peptides from different human cDNA libraries, were exposed to membranes and cytosolic fractions prepared from cultures of toxin-producing cyanobacteria. After three rounds of biopanning, a series of putative toxin specific phages displaying peptides were isolated, and the cDNA amplified by PCR. Ongoing studies are in process to sequence the candidates cDNA for further analysis using Nucleic acids- protein data bases.
Cabello, Lourdes*Pontifical Catholic University of Puerto Rico, Department of Biology; Vega, Nina Biology, Román, Jorge , Robles, Karina, Rodríguez, Jesusalyn Secondary Education, Pontifical Catholic University of Puerto Rico
Biología reproductiva de Zamia pumila L.
Zamia pumila L. (Cycadophyta: Zamiaceae) es una planta nativa de las Antillas Mayores. Fue declarada erradicada de Puerto Rico en 1998 debido al usa de terrenos para desarrollo urbano. Se encontro una población de esta planta en las montañas cársicas de Juana Díaz. El propósito de este estudio es determinar el ciclo de vida de Z. pumila en Puerto Rico. Se colectaron datos climatológicos y biológicos dos veces por mes durante el año 2002, tales como: temperatura, humedad relativa, pH del suelo, número de conos/planta, sexo, largo y ancho de los conos. Se determinó el número de semillas por megaesporófila y se determinó la razón de semillas y abortos. Los resultados indican que las plantas masculinas producen unpromedio de cuatro conos y las femeninas producen un promedio de dos conos por planta. La producción de semillas es baja; se determinó un 52% de abortos. Los conos masculinos tiene una duración de alrededor de siete meses. Los posibles polinizadores podrían ser coleópteros de las familias Scolytidae y Lathridae, cuyos adultos fueron encontrados en algunos conos.
Cabrera, Arelys*UPR-Río Piedras, Department of Biology; García-Arrarás, José E. Inhibition of fibronectin interaction by RGD injections delays intestinal regeneration
Fibronectin, a glycoprotein found in the extracellular matrix, carries out functions associated to the, adhesion, migration and growth of cells. Fibronectin has a well-known sequence identified as RGD (Arg-Gly-Asp), that is necessary for the recognition of integrins in the cellular surface and, therefore, to maintain a communication between components of the extracellular matrix and the cytoskeleton. The sea cucumber, Holothuria glaberrima, is an invertebrate that has the capacity to regenerate its viscera following evisceration. Previous studies have demonstrated that during regeneration of the intestine a remodeling of the extracellular matrix occurs. Cellular migration is one of the processes that occur in the formation of the new organ. To study a possible role of fibronectin in cellular migration during intestinal regeneration, sea cucumbers were treated with peptides that compete with the RGD sequence. The peptides used were GRGDTP (Gly-Arg-Gly-Asp-Thr-Pro), RGDS (Arg-Gly-Asp-Ser) and RGD (Arg-Gly-Asp) in concentrations between 0.8-10 mg/ml. Control groups were injected with a saline solution or with a control peptide that does not compete with the RGD sequence. The regenerated structures were analyzed by immunohistochemistry for the presence of collagen and muscle cells. In addition the number of cells in several tissues was quantified. The results show a delay in regeneration in animals injected with RGD as can be seen by decrease formation of muscle and reduced cellular migration. Our results suggest that fibronectin has an important function in the intestinal regeneration of echinoderms. Funded by SCORE, NSF, RCMI, AMP and the UPR
Capella , Omar*UPR-Mayagüez, Department of Biology; Deliz, Beverly, Biology Department-UPR Aguadilla, Diaz, Gloria E., Physiology Department, School of Medicine, Hernandez, Pedro J., Physiology Department, School of Medicine Vargar, Noel, Biology Department, UPR Aguadilla, Segarra, Anabel, Physiology Department, School of Medicine
Estrogen and mu opiates: modulator of cocaine-induced locomotor sensitization in female rats
Estrogen is responsible for many of the gender differences observed in addictive behavior, particularly in the locomotor response to cocaine. The mechanism by which this occurs is unknown. In this study we investigated if estrogen modulates the locomotor response to cocaine via the ì-opioid receptor system. Adult female rats were ovariectomized, half received implants with estradiol benzoate (OVX-EB), the other half received empty implants (OVX). After a 7 day recovery period, rats were divided into the following groups: saline injected (OVX-SAL, OVX-EB-SAL), naloxonazine injected (OVX-NLX, OVX-EB-NLX), cocaine injected (OVX-COC, OVX-EB-COC) and naloxonazine and cocaine injected (OVX-NLX-COC, OVX-EB-NLX-COC). Animals were injected for 5 consecutive days, the dosage of cocaine and of naloxonazine injected was 15 mg/kg i.p. To insure blockade of opioid receptors, naloxonazine was injected 12-20 hours prior to behavioral testing. Cocaine-induced locomotor activity (horizontal, stereotyped and vertical activity) was measured on days 1, 3 and 5. Animals were then allowed a drug free period of two days and on day 8 the rats were challenged with the same dose of cocaine and locomotor activity recorded. Rats treated with estrogen showed a significant increase in the locomotor response to cocaine, indicating that only OVX-EB rats became sensitized to cocaine. Treatment with naloxonazine, a ì-opioid antagonist, blocked locomotor sensitization to cocaine in EB treated females. OVX rats do not show any significant difference neither ì-opioid receptor blocked nor saline control in the locomotor response to chronic cocaine administration. Curiously, consecutive ì-opioid receptor blockade in OVX rats increased the locomotor response to an acute dose of cocaine. These results suggest that naloxonazine could be used as an effective pharmacotherapy in females to decrease the motivational component of cocaine addictive behavior.
Collado, Enixy*UPR-Humacao, Department of Biology; Sastre,M.P, Muller,R., Morales, S., Rivera, P. , Veléz,Y.
DNA Damage in Mantle Cells of the Caribbean Mussel Brachiodontes Exutus: A Comet Assay Evaluation
The mussel Brachiodontes exustus (Mollusca: Mytilidae) lives in the intertidal zone of the Caribbean and is often observed attached to rocks, mangrove roots and piling. Although much smaller, this species can be considered an ecological equivalent of Mytilus spp., which is often associated with bioaccumulation studies but is not found in the Caribbean. The objective of this work is to assess B. exustus as an indicator of environmental stress in Puerto Rico using the single cell gel electrophoresis/comet assay. This sensitive technique is used to detect single strand breaks in DNA of single cells. Ambient levels of DNA damage in B.exustus mantle cells are described as well as those under culture conditions. A preliminary experiment was performed exposing mantle cells for 15 minutes to 0, 10, 25,50 and100 micromolar hydrogen peroxide concentrations in order to test the sensitivity of the comet assay. As expected DNA damage increased along with hydrogen peroxide concentrations. Another experiment was performed exposing organism in aquaria to 0,20,100 and 200 ppb copper concentrations (as copper chloride) for 24 and 96 hours. DNA damage levels increase along with copper concentrations both at 24 and 96hours. Field samples from Ferry, Rampa, Vietnam and Isla de Cabras in San Juan Bay were analyze to contrast with Fajardo area with no significance difference in DNA damage but, achieve inside the San Juan Bay. Spermatozoids were found in few experiments at various samples in the San Juan Bay showing small “comets”. This study indicates the comet assay can be used effectively in B. exustus mantle cells to assess DNA damage in Caribbean waters.
Comenencia, Eydith*UPR-Cayey, Department of Biology; Chiesa, Ricardo
CREM-1 Expression in the Rat Amygdala and its Possible Role as a Negative Modulator of CREB-dependant Gene Expression During the Acquisition of Emotional Memories
The synaptic plasticity theory states that learning and memory processes involve synaptic changes. Memory consolidation involves a series of molecular events requiring transcriptional gene regulation, which eventually ends in long-term changes at the synapse. Western Blot analysis showed a decrease in the levels of cAMP Responsive Element Modulator Protein (CREM), 1 hour after conditioning. These results, combined with gene expression studies in the amygdala using cDNA array technology, led to the proposal of a model for the molecular events involved in the acquisition and consolidation of the aversive memory of CTA and of the modulation of emotions generated during emotional learning. The model proposes that ERK 1/2 inactivation induces a decrease in the levels of CREM-1, a negative modulator of CREB, this, in turn, induces an increase in CREB mediated transcription, resulting in an increase in the levels of the transcription factors Nor-1 and Fra-1 . We found that the levels of CREM-1 decrease 1 to 6 hours after the CTA training. We postulate that this decrease in CREM-1 allows an increase in CREB-mediated transcription, which initiates gene regulatory networks responsible for the synaptic changes involved in the acquisition and consolidation of emotional memories in the amygdala. The molecular events modulating the generation of a strong emotional memory, such as the one in CTA, might also be involved in other learning experiences or psychoemotional disorders. Most of these psychoemotional disorders involve associative relation between events, very similar to the associative learning in CTA.
Cosme-Blanco, Wilfredo*UPR-MAYAGUEZ, Department of Biology; Denson,Lee A., Pediatrics,Yale University Held, Matthew Pediatrics,Yale University
Regulation of Growth Hormone Receptor Expression by Inflammatory Cytokines
Background:Children with Extrahepatic Biliary Atresia (EHBA) experience growth failure and muscle wasting which is associated with an acquired growth hormone (GH) resistance. Hepatic expression of the GH receptor (GHR) is reduced in these children. Rodents also exhibit growth failure after bile duct ligation (BDL), however the factors which regulate GHR abundance are not known. Anabolic effects of GH are dependent upon activation of STAT5 and IGF-I expression. Cytokines including TNF and IL-6 are up regulated in EHBA and after BDL. Our prior studies reported that TNF down regulates the GHR, via alterations in Sp1/Sp3 DNA binding. We hypothesized that TNF inhibits GH signaling after BDL by down regulating GHR abundance. Methods: Wild type (WT) or TNF Receptor I (TNFRI) null mice received sham surgery or bile duct ligation (BDL). Control mice were fed ad lib or pair fed with BDL mice. 7 days after BDL, mice were injected with PBS or GH, and liver and muscle were harvested. H4IIE rat hepatoma cells were treated with GH ± cytokines or bile acids and protein was isolated. STAT5, GHR, Sp1 and Sp3 abundance were determined by immunoblot (IB). IGF-I expression was determined by real time PCR. TNF and IL-6 abundance were determined by ELISA. Results: STAT5 activation by GH was inhibited in liver and muscle of BDL mice. Pair fed mice exhibited normal STAT5 activation. IGF-I expression was also reduced in BDL mice; this was associated with up regulation of TNF and IL-6. GHR abundance was reduced in BDL mice. However, Sp1/Sp3 abundance was preserved. Treatment of H4IIE cells with TNF prevented STAT5 activation and reduced GHR abundance. However, GHR abundance was also reduced after BDL in TNFR I null mice. Conclusion: Mice with obstructive cholestasis due to BDL are GH resistant in liver and muscle; this is associated with down regulation of the GHR. However, this does not appear to require intact TNFR I signaling. Therapies which restore GHR abundance in this setting may improve growth and body composition of children with EHBA.
Díaz Muñoz, Samuel L.*UPR-Maygüez, Department of Biology; Martínez Cruzado, Juan C.
The Contribution of Ancestral Amerindian Populations to the Genetic Makeup of Puerto Rican Men
The human Y chromosome possesses a large non-recombinant area and is passed from males to their male offspring, without changes, generation after generation. The presence of slow evolving binary markers in the DNA sequence, single nucleotide polymorphisms or SNP’s, that occurred at different evolutionary moments, provides information about the migration of human populations, which enables an assessment of the contribution of ancestral continental groups to the genetic makeup of modern day populations. Our objective is to use SNP’s in conjunction with additional DNA markers to elucidate the contribution of Amerindian groups to the genetic makeup of modern Puerto Rican men.
We will collect buccal cells from 30 Puerto Rican men, from different municipalities of the island, in order to obtain DNA samples. We will use RFLP (Restriction Fragment Length Polymorphism) techniques to analyze certain SNP’s and the faster evolving microsatellites, so as to find what proportion of the samples exhibits an Amerindian haplogroup and haplotype. The first step will be the analysis of the 92R7 locus, which reveals if the sample does not belong to the 1C haplogroup. Past studies of the 1C haplogroup have revealed that 68% of the samples in this haplogroup are of European origin and the remaining 32% has Amerindian ancestry. Therefore, after said analysis we will conduct analysis of the DYS194 locus and, if necessary, various microsatellite markers in order to reach a conclusion of the continental origin of the samples.
Fernández, José R.*UPR-Mayagüez, Industrial Biotechnology; Zito, Christina, Department of Pharmacology, Yale University-School of Medicine, Bennett, Anton M.,Department of Pharmacology, Yale University-School of Medicine
Regulation of Insulin-Like Growth Factor Receptor (IGF-1R) Signaling by the SH2 Domain Containing Protein Tyrosine PHOSPHATASE 2 (SHP-2) Ii Cell Growth
SHP-2 is a ubiquitously expressed protein tyrosine-specific phosphatase that contains SH2 domains at its NH2 terminus and a catalytic domain at its carboxyl terminus. SHP-2 has been shown to be an important factor in signaling pathways initiated through receptor tyrosine kinases (RTK) for insulin growth factors (IGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and nerve growth factor. The catalytic activity of SHP-2 is required for activation of the Ras/Raf/Erk pathway downstream of several growth factor receptor and cytokines. The direct targets of tyrosyl dephosphorylation by SHP-2 that regulate the Ras/Raf/Erk pathway, however, remain to be identified. Catalytically inactivated mutants of SHP-2 were used to demonstrate Erk and Akt regulation signaling downstream of IGF-1R. A mutation within Asp425 to Ala425 of SHP-2 “wild-type” renders it catalytically inactive and SHP-2-substrate complexes can be formed and detected by anti-phosphotyrosine immunoblotting. Our studies demonstrate that the SHP-2 substrate-trapping mutant interacts with a potential substrate of 90 kDa. The data suggest that SHP-2 is a major regulator of Erk and Akt signaling downstream of IGF-1R. Our data also suggest a role for Protein Tyrosine Phosphatase 2 in IGF-mediated human cell survival. Drugs targeting the catalytic activity of SHP-2 might be useful in combination with chemotherapy for the treatment of cancer and the genetic autosomal-dominant condition of Noonan Syndrome.
Flores Pérez, Iehsus*UPR-Mayagüez, Department of Biology; Rivera Santos, Mishelle , Uscian, John M.
Purification and Kinetic Characterization of Trypsin from Selected
Trypsin is a proteolytic enzyme that is secreted into the lumen of virtually all animals possessing a gut. In order to better understand the underlying biochemistry of protein digestion/nutrition in the silk snapper, Lutjanus vivanus, we used standard chromatographic and spectrophotometric procedures to purify and kinetically characterize trypsin from intestinal and pyloric caecal tissues of this species. The enzyme was found to have a molecular weight of approximately 25 kDa. It was optimally active at a pH range of 6 - 8. The enzyme activity could be abolished with a trypsin-specific inhibitor. In addition, the silk snapper trypsin activity varied with temperature, displaying highest catalytic rate at 50 oC. The data are considered with respect to their possible application as a tool in helping to assess silk snapper trophic environment.
Fourquet, Jessica* Pontifical Catholic University of Puerto Rico, Dept of Biology; Flores Caldera, Idhaliz Microbiology, Ponce School of Medicine
Genetics of Endometriosis: The Role of the Estrogen Receptor Gene
Endometriosis is defined as the presence of endometrial tissue (gland and stroma) outside of the uterine cavity, especially in the peritoneum, ovaries, fallopian tubes and the cul the sac. This condition has been associated with several candidate genes, including the estrogen receptor (ER) gene. The ER gene is found in chromosome 6 and has been associated with hormonal independence during the luteal phase and to alter biologic activities. The objective of this study is to investigate the role of the ER polymorphisms in endometriosis, and to conduct genetic linkage in chromosome 6, where ER is located, using DNA from affected families. This study is based on the observation that variable number of tandem repeats of thymidines and adenines (VNTRs) are associated with endometriosis. This research involved 118 volunteers of the following study groups: a) sporadic cases (n=53): women who have been diagnosed with endometriosis by surgery and who do not have family history, b) family cases (n=32): patients with one or more relative with endometriosis, c) female controls (n=22): healthy women and d) male controls (n=11): a group used to determine the mutation rate in our population. After genomic DNA extraction from blood lymphocytes, a region upstream from the ER gene and containing the VNTR was amplified by PCR. PCR amplification was verified by agarose gel electrophoresis prior to the analysis of the polymorphisms by polyacrylamide gel electrophoresis (PAGE). The sizes of the different bands visualized by PAGE were determined using the Multi-Analyst software. Bands were categorized according to sizes in small, medium, large and extra-large VNTR’s. The majority of the patients had medium-sized VNTR’s. This observation contrasts with the results observed in a Greek population where endometriosis was associated with having small-sized VNTR’s. Statistical analysis of our data showed that, among those individuals with small VNTR’s, there was a significant difference between sporadic cases and controls (p<0.051). No other associations with VNTR size were found.
González Avilés, Gladys D.*UPR-Mayagüez, Arts and Sciences, Department of Biology; Jon P. Woods Medical Microbiology, University of Wisconsin-Madison
Inhibition of Iron Acquisition in Histoplasma capsulatum
Histoplasma capsulatum is a thermally dimorphic, pathogenic fungus that infects humans and other mammals. The saprobic mold form lives in rich soil particularly in association with bird and bat guano. Human infection occurs through the inhalation of conidia or mycelial fragments. Once in the lungs at 37C, the fungus converts into the yeast form and infects pulmonary macrophages. Although most infections in immunocompetent hosts are self-limited, histoplasmosis poses greater risks for severe pulmonary or systemic disease in immunocompromised individuals. Iron limitation has been shown to be a specific host mechanism of H. capsulatum control. Our laboratory recently showed that the fungus expresses ferric reductive activities that may enable it to obtain this essential nutrient in iron-limited conditions. We tested whether exposing H. capsulatum to the oxidizing agent (KSO3)2NO that inhibits the conversion of Fe+3 to Fe+2 could inhibit its growth. We also examined protein expression profiles in different iron-limited cultures using SDS pages. The results from this experiment are that (KSO3)2NO inhibit the growth of H. capsulatum and the inhibition is quantitative dependent. Iron limited culture expressed proteins that differ from the optimal condition cultures. We think this proteins can help to the survival of H. capsulatum . This work should increase our understanding of H. capsulatum iron acquisition and these proteins can be the targets for the development of treatments for histoplasmosis.
González Laboy Millie Lee*UPR-Humacao, Department of Biology; Casillas-Martínez, Lilliam, Visscher, Pieter, Department of Marine Sciences, University of Connecticut-Groton CT. Hernández-Cruz, Carmen, Biology Department, University of Puerto Rico – Humacao
Microbial diversity analysis of the beige and black layer of a cyanobacterial mat from Cabo Rojo salterns
Physicochemical conditions are the most important factors that influence the presence of the different microbial communities within a cyanobacterial microbial mat. We have studied the physicochemical conditions that prevail during the rainy season at the Cabo Rojo salterns and how such conditions select for the microbial communities present in their cyanobacterial mats. After dissecting the bottom layers of the Cabo Rojo mats we characterize the most abundant microorganisms in each layer using Transmission Electron Microscopy (TEM). Two main layers were studied, one exhibit a beige coloration and the other was black. On the beige layer a series of bacterial phototrophs including green filaments of Chloroflexus were abundant. Filamentous cyanobacteria that resemble Microcoleus and Oscillatoria species were also found in large numbers. On the black layer of the mat a series of anoxygenic bacteria were visualized. Interestingly in this black layer we also found presence of unidentified spirochets with their characteristic axial filaments. Such spirochets were always found occupying disintegrating cyanobacterial sheaths. Bacterial with spiral forms were previously reported to be present in this ecosystems but there specific role is not yet known. In the future we plan to use molecular methods (i.e.16SrDNA sequencing analysis) to identify the most important microorganisms per layer. We also plan to enrich for this spirochets by supplementing our growth media with Rifampicin as spirochets are known to be resistant to this antibiotic.
Hernández-Alvarado, Nelmary*UPR-Mayagüez, Industrial Biotechnology; López-Garriga, Juan, Department of Chenistry
Expression and purification of hemoglobin I from Lucina pectinata
Hemoglobin I (HbI) from Lucina pectinata, a tropical clam, has the unusual ability to bind in hydrogen sulfide. HbI cDNA was amplified and cloned into an expression vector that contains a fusion tag of six histidine residues; vector was transformed in E. coli BL21 Star. The expression of HbI was optimal expressed when induced for 5 hours with 1.0M isopropyl-b-thiogalactoside (IPTG). Heme group was synthesized and insert by the presence of D-aminolevulinic acid (D-ALA), a precursor in the heme biosynthetic pathway, in the medium. Western Blot using an antibody against the histidine tag detected the presence of the recombinant protein. HbI was purified by ionic exchange chromatography using diethyl aminoethyl-cellulose (DEAE-cellulose) and size exclusion through fast performance liquid chromatography (FPLC). UV-Vis spectrum of the recombinant protein showed a Soret band at 420 nm, the same of OxyHbI under natural condition. However, to optimized the percent of purification, further experiments will be performed such ammonium sulfate precipitate and metal affinity chromatography.
Iglesias-Torres, Emanuel*UPR-Mayagüez, Department of Biology; Maldonado-Ramirez, Sandra
Mycelial fungi associated to disease fungi in three orchid species
In Puerto Rico, few studies have attempted to characterize the mycobiota associated to diseases in orchids. In this reasearch, fungal elements associated to diseased tissue in three orchid species were isolated. Samples from leaves, flowers and stems were collected and analyzed. Portions of each tissue were surface sterilized and placed in Petri dishes containing Potato Dextrose Agar as a culture media. After the incubation period fungal colonies were isolated and sub-cultured. Pure cultured were characterized by macroscopic characteristics. Nigrospora sphaerica and Culvularia lunata were recovered from lesions in Phanaelopsis lelolai. Nigrospora sphaerica was also isolated from an orchid variety named "Sweet Sugar". From Dendrobium var "yoko" fungal colonies of Alternaria alternata were recovered.
Leiva-Nieves, Jexsenia*UPR-Mayagüez, Department of Biology; Quiñones-Camacho, Brendaliz, Maldonado-Ramirez, Sandra L.
Preliminary studies of fungal endophytes in Thalassia testudinum
Sea grass prairies are one of the most common coastal ecosystems in Puerto Rico. One of the most common sea grass in these prairies is Thalassia testudinum. Prairies of T. testudinum are very abundant in shallow and peaceful zones of the sub-litoral with soft substrates. Few studies have attempt to recover and identify the superficial microbiota and pathogens associated with T. testudinum, but no previous research has attempted to identify fungal endophytes. During this research, plants of T. testudinum were collected from two different coastal areas in Lajas and Cabo Rojo. Portions of healthy tissue were surface sterilized and placed in Petri dishes contaning a specially designed culture medium. After incubation, fungal colonies were isolated and sub-cultured. Both mycelial and yeast-like fungi were recovered. Mycelial fungi includes species of Penicillium, Aspergillus, and Mucor. Yeast-like fungi will require further biochemical analysis to corroborate preliminary identification.
Lozada Villaseñor, Carla*UPR-Mayagüez, Department of Biology;Maldonado, Sandra
Airborne fungi in Cabo Rojo Area
Bioaerosols have been found in many occupational environments. This study was undertaken to examine the quantity of fungi present on the atmosphere of Cabo Rojo Area. The study was made to show how temperature and humidity usually affect airbone concentrations on the atmosphere. Knowledge of fungi in a given city or region is important for the ecological diagnosis and specific treatment of allergic manifestations induced by inhaled allergens. In order to diagnose the presence of fungi, several qualitative and quantitative techniques are used. This study of fungal air spores where collected using Petri dishes. A total of two samples were obtained, then isolated and classified by genera. The results revealed prevalence of Aspergillus, Curvularia, Penicillium, Cladosporium, Peyronellaea and Bipolaris.
Maisonet Colón, Jorge*UPR-Río Piedras, Department of Biology; Dr.Fernando Renaud , Carlos Ballester
Detección de dímeros entre receptores opioides mu y delta
En esta investigación se busca probar la existencia de un dímero entre los receptores opioides mu y delta en macrófagos peritoneales de ratón. La existencia de este dímero fue sugerida por resultados farmacológicos encontrados en investigaciones previas en donde se observó ligamiento cooperativo para agonistas mu y delta. Para esto se está trabajando en la detección de los receptores mu y delta por medio de Western blots utilizando anticuerpos específicos para receptores mu y delta.
Para realizar esta investigación se aíslan macrófagos peritoneales de ratón después de que éstos son inyectados con tioglicolato(agente inflamador) y en un término de cinco días son sacrificados. Con los macrófagos se lleva a cabo cultivo celular. Luego utilizando técnicas de homogenización, por medio de las cuales se rompen las células y se centrifuga varias veces la muestra hasta obtener finalmente fracciones de membranas. Se utilizan las técnicas de Lowry para saber qué concentración de proteínas se tiene. Luego comienza el proceso de inmunodetección con anticuerpos específicos para receptores mu y delta. Las proteínas son sometidas a Western Blot, donde el primer paso es hacer una electroforesis en la cual usualmente se corren g de proteínas por carril a través de una gel de poliacrilamida. Una vez finalizada la electroforesis se procede con la transferencia de las proteínas hacia una membrana. Luego de varios intentos para lograr la optimización de la transferencia se determinó que aumentando el voltaje de 10 a 20 voltios y el tiempo de la transferencia de 12 a 20 horas se obtiene una transferencia efectiva. Al terminar la transferencia se tiñe con ponceau la membrana y con comassie blue la gel para corroborar si la transferencia ha sido efectiva, y si esto es así se procede a la incubación de la membrana con los anticuerpos. Este proceso requirió que se optimizara la cantidad de anticuerpo que debe ser utilizada para detectar la banda de interés sin que salga “background” en la autoradiografía. La dilución que mejor resultado ha dado hasta el momento es con el anticuerpo primario 1: 2500 y con el anticuerpo secundario 1:1000. Es en esta parte del experimento que ahora se esta utilizando “blocking peptide” para bloquear los receptores que estamos estudiando y hacer que desaparezca la banda que los representa en la autoradiografía. De esta manera se puede tener certeza de que la banda que se observa representa los receptores opioides de interés en es en esta investigación. Hay que señalar que antes de realizar los experimentos con macrófagos peritoneales de ratón, se estuvieron realizando experimentos similares con células de cerebro de ratón. La razón para esto es que ya se tenía conocimiento de la existencia de los receptores mu y delta en ese tipo de célula, servirían como control para poner a prueba las técnicas.
Próximamente se realizará este experimento utilizando un ligando covalente en la incubación y se espera que aparezca una banda que reaccione con ambos tipos de anticuerpos.
Las alternativas que tenemos para realizar esta investigación son:
1) Utilizar un ligando covalente en la incubación y determinar si aparece una banda que reacciona con ambos tipos de anticuerpos.
2)Otra alternativa para detectar estos dímeros sería determinar si los receptores mu y delta se co-localizan utilizando un microscopio confocal. Si existen dímeros mu/delta, su localización por medio de inmunofluorescencia debe coincidir.
La importancia de este trabajo es que si estos receptores funcionan como dímeros, este hecho se debe tomar en consideración al diseñar drogas que interaccionen con estos receptores.
Martínez Daviana*UPR-Rio Piedras, Department of Biology; González, Carlos I. Characterization of DSE's in S.cerevisiae
There are certain regulatory events that can control gene expression. At the post-transcriptional level, mRNA turnover plays a key role in determining the final amount of an encoded protein. The nonsense mediated mRNA decay (NMD) pathway is a very well conserved surveillance mechanism that promotes rapid degradation of mRNA’s harboring premature termination codons. This pathway is a good example of how mRNA turnover and translation are related. In addition to the premature termination codon, several downstream sequence elements (DSE’s) are required to activate the NMD pathway. The main goal of our investigation is to identify and characterize potential DSE’s in the 3’ UTR of the CYC1 mRNA. Our results will help to further characterize these DSE’s and elucidate the trans-acting factors that interact with these sequences in order to activate degradation via the NMD pathway.
Narvaez Carla Rubi*UPR-Mayagüez, Department of Biology; Rodríguez, Lolita, Souto, Fernando
Tratamiento de litiasis urinaria con plantas medicinales: Cultivo in-vitro y variación hormonal de la Rubia tinctorum sp.
La raíz de la planta herbácea Rubia tictorum proveniente de Europa fue utilizada por muchos años como remedio oficial para tratar la piedra del riñón. Esta planta presenta ciertos compuestos hidroxi-antraquinonicos (HAQ, derivados de Alizarina) y glucósidos en su raíz madura de dos a tres años de edad. Estos son responsables aparentemente de su acción antilitica. Sin embargo la utilización de su raíz se ha prohibido ya que a su vez posee ciertos componentes tóxicos, como la Lucidina, los cuales en cantidades grandes pueden ocasionar la muerte. La posibilidad de cultivar in-vitro las raíces de esta planta bajo condiciones que disminuyan la acumulación de Lucidina nos propone establecer un nuevo sistema in-vitro. La planta ha mostrado características propias de una planta madura, por tanto se han realizado ciertas variaciones hormonales de Auxinas y Citocianinas para la proliferación su raíz. Se crearon dos matrices especificas para evaluar y re-evaluar el rango optimo en producción de raíz, alargamiento de los meristemos apicales y proliferación de vástagos. Durante estos meses se ha trabajado detalladamente con la herbácea a la cual se le ha encontrado y re-evaluado su estado optimo al ser propagada in-vitro por vía asexual. Las cantidades de hormonas utilizadas han sido ppm con un rango de 0-1ppm en ambos reguladores. Esta planta ha demostrado una gran inclinación en cuanto al rango que envuelve los 0.876ppm de IAA y 0 Kin, observamos un aumento significativo en alargamiento del vastago y aumento de raiz. Por otro lado obtuvimos en un rango de 0Kin/0.8ppm IAA con caracteristicas similares al anterior de alargamiento meristematico. Hubo un rango especial de 0.2ppmKin/0.8ppmIAA donde no presentó nada de alargamiento apical, pero sí crecimiento del sistema radicular. Este es uno de los propositos de esta investigacion, la obtención de suficiente raíz para posteriormente recolectar las raíces in-vitro, comparar los componentes de su raiz, llevarlas a secado, peso y molienda, para poder realizar las decocciones de la planta in-vitro. De esta forma la compararemos con la de campo y trataremos de determinar todos los compuestos hidroxi-antraquinonicos, glucosidicos y más aún los tóxicos, para poder aislarlos, especialmente los tóxicos y lograr así nuestros objetivos medicinales.
Núñez Santana, Félix* Pontifical Catholic University of Puerto Rico, Department of Biology; Stone, Marielle K., Neurobiology and Phisiology, Northwestern University, Levine, Jon E. Neurobiology and Phisiology, Northwestern University Turek, Fred W., Neurobiology and Phisiology, Northwestern University Horton, T