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Alicea, Ricardo; del Llano, Ana M. Ph.D. Department of Biology, UPR-Humacao - Muñiz, Rebecca. Department of Biology, UPR-Aguadilla - Narvaez, María. Department of Biology, UPR-Rio Piedras - Galarza, Danny. Department of Biology UPR-Ponce - Lavergne, Julio Ph.D. Department of Microbiology and Medical Zoology, UPR-Medical Sciences Campus, Rio Piedras.
Early and late apoptosis-related events in an HIV-1 chronically infected cell line treated with nitric oxide or dexamethasone.
Cells undergoing apoptosis exhibit recognizable membrane, mitochondrial and nuclear changes which can be measured in order to assess the degree of damage exerted by death-inducing agents such as nitric oxide and dexamethasone. Since apoptosis plays an important role in the pathogenesis of AIDS disease, we have used in our experiments an HIV-1 chronically infected cell line (J1.1) treated with two well known death-inducing agents, nitric oxide donated by SIN-1 and dexamethasone as in previous experiments this particular cell line has be found to be relatively resistant to the effects of death-inducing agents, namely, anti-Fas and TNF. Both the J1.1 cells and the Jurkat parental cell line were exposed to either agents or left as untreated controls, in 72 hours kinetic study. At the 24, 48 and 72 hour time point the following parameters were measured by flow cytometry: cell cycle progression and occurrence of apoptosis, exposure of phosphatidylserine at the outer membrane, mitochondrial membrane potential and integrity. Our result indicate that both cell lines are susceptible to dexamethasone and nitric oxide, with dramatic effects at all parameters measured. Of particular interest were the effects on the different phases of the cell cycle and the fact that all the observed damage were present by the first 24 hours of the kinetic study. Our results suggest that although the J1.1 HIV-1 infected cell line in relatively resistant to other death-inducing agents, it is vulnerable to the potent effects of nitric oxide or dexamethasone.
Arce, Julio; File, Sharon. Biology, UPR Rio Piedras.
Infección con trematodos Thiara spp. en Puerto Rico
Dos caracoles asiáticos Thiara granifera y Thiara (Melanoides) tuberculata fueron introducidos en Puerto Rico y reportados por primera vez 1954 (Harry & Aldrisch 1958) y (Abbot) respectivamente. Estos moluscos exóticos son los huéspedes intermediarios para un grupo de trematodos parasíticos de importancia médica en Asia y Sureste de Asia. En el hemisferio occidental han servido de control biológico sobre Biomphalaria glabrata el huésped intermediario de Schistosoma mansoni desplazando este completamente. Hemos detectado un trematodo juvenil en estos caracoles que no ha sido reportado previamente en Puerto Rico. Estos han mostrado en el laboratorio metacercarias enquistadas en las agallas de los peces de agua dulce. Se ha encontrado el parásito en menos del 1% de los caracoles examinados. Creemos que este trematodo heterófido es Centrovestus sp. El mismo que ha sido reportado matando peces nativos en peligro de extinción en Texas y un pez importado en México. Las etapas de cercaria y metacercaria han sido descritas y comparadas con otros reportes de parásitos en este hemisferio. Los intentos para completar el ciclo de vida y confirmar la identidad de esta especie están en progreso y serán reportados próximamente.
Arenas Alicea, Agnes V.; Esteban, Ernesto. Department of Physics, UPR Humacao.
Exact solutions for a cell population kinetic model
We develop a carcinogenesis cell population kinetic model valid for high-LET HZE radiation. A unique feature of this model is to consider n possible mutations before a normal cell becomes a cancer cell. We present exact solutions describing the normal, injured, mutant, killed, and cancerous cell populations at any time t, and for a given number n of mutations. Explicit analytical solutions for the cases of n=1, n=2 will be presented and discussed.
Cabrera Piquer, Fernando; Ríos Velazquez, Carlos; Ramos, Gloria. Microbiology, UPR Mayagüez.
Culturing the unculturable: Searching for strategies to develope culture media for traditional non-cultured microorganisms.
Due to the fact that 99% of the organisms form soil cannot be cultured, and that limiting tools are available for culturing the soil micro flora, new strategies need to be developed in order to unravel new soil groups of microbes with potential medical and industrial applications. Our research seeks to develop culture media, and culturing strategies to "culture the unculturable", for isolation of microorganisms considered non-cultured, based on the notion that some non-cultured microorganisms should grow in media that are provided with chemical components that resemble their habitat. Different soil samples will be used to prepare modified culture media (MCM). The different MCM will be used to isolate the microbial biota from the soil, and the new cultivable candidates will be detected by comparing the MCM with the microbes isolated from general enriched media. The students will use from general microbiological techniques, to genetic engineering approaches, for isolation and characterization of the MCM isolates.
Capella-Miranda, Omar; Nicholl, Suzanne M.; Roztocil, Elisa; Fegley, Allison J.; Davies, Marc G. Department of Surgery, University of Rochester.
Sphingosine-1-phosphate induced vascular smooth muscle cell
The development of restenosis involves an integrative program of cell proliferation, migration and extracellular migration. The bioactive sphingolipid metabolite, sphingosine-1-phosphate (S-1-P), has been implicated in cell migration by regulating G-protein dependent signaling pathways such as the MAP Kinase pathway. Recent studies suggest that S-1-P may also function as an integrator linking growth factor receptors to cell migration. We hypothesize that S-1-P stimulates smooth muscle cell migration through transactivation of receptor tyrosine kinases of the Epidermal Growth Receptor Factor (EGFR), Insulin-like Growth Factor Receptor (IGF-1R) and Platelet-Derived Growth Factor Receptor (PDGFR). Rat pulmonary artery smooth muscle cells were either pre-treated with/or without specific inhibitors of EGFR (AG1478), IGFR (AG1024) and PDGFR (AG1295) and stimulated with S-1-P (0.1µM) over a period of 30 minutes. Linear wound assay was employed as an assay of migration and protein was isolated for Western blot analysis. AG1024 (IC50: 10 M) and AG1295 (IC50: 100M) dose dependently inhibited S-1-P induced cell migration. EGFR inhibition had no effect. S-1-P results in a time dependent activation of ERK1/2 and p38MAPK both of which have been shown to be involved in S-1-P mediated cell migration. Both AG1024 and AG1295 significantly inhibited the S-1-P induced activation of ERK1/2 and p38 MAPK. EGFR inhibition had no effect. These data suggest that the S-1-P MAPK activation and subsequent migration involves the transactivation of two receptor tyrosine kinases, IGFR and PDGFR.
Cardona, Vanessa; Cruz, Nelly; Vega, Lissette; Rios-Velasquez, Carlos. Biologia, UPR Mayagüez.
Isolation and characterization of heavy metal resistance microorganism with potential bioremediative capability
Geomicrobiology is a new interdisciplinary field that involves the specialized study of microbiology in geologic environment. Besides understanding the role of microbes in processes such as mineral degradation, organic matter mineralization, and biogeochemical cycles, the presence of these microbes as part of the indigenous population of contaminated areas can be used as a tool for in situ treatment for the removal of heavy metals for the contaminated sites. The main purposes of this research are: isolate, identify and characterize the microbes that may tolerate high concentration of metals, and to test the potential of the isolated metal resistant geomicrobes for bioremediation, biotransformation, and biodegradation of heavy metals. Also, to test their capability as bioindicators to detect contaminated areas. Soil samples were collected in the west side of Puerto Rico. A blue-pigmented colony that had a metallic copper-colored sheen was isolated. The microorganism was molecular and biochemically characterized. We have found this unknown organism to be very similar to Vogesella indigofera, a microorganism used as a bioindicator for chromium contaminated areas.
Casiano, Madalis. Chemistry Department, Pontifical Catholic University of Puerto Rico - Rivera, Elizabeth; Abreu, Silkha; Rios-Bedoya, Carlos; Flores, Idhaliz. Microbiology, Ponce School of Medicine
Endometriosis is associated with a mutated progesterone receptor
Endometriosis is a gynecological disorder characterized by severe menstrual pain, painful intercourse and infertility. Endometriosis has been associated with polymorphisms in candidate genes, including the progesterone receptor gene (PR). We investigated the association of a polymorphism consisting of a 306 base pair insertion in the PR gene (PROGINS) in endometriosis patients of Puerto Rican origin. Wild type (T1) and mutant (T2) alleles were identified by PCR amplification of the region of interest and agarose gel electrophoresis. We observed significant differences in the frequencies of the PROGINS genotypes (T2/T2 + T1/T2) between women with endometriosis (30%) and controls (13%) [OR=2.9, CI=1.2-7.1; p<.05]. In addition, the frequency of the mutant allele T2 was higher in patients (17%) than controls (6%) (OR=2.9, CI=1.2-6.7, p<.05). In addition, we observed that sporadic patients were more likely to have the mutant genotypes T2/T2 + T1/T2 than familial patients (36% vs. 18%); however, this difference was not significant. The differences in the frequencies of PR genotypes between the two study groups were significant only when sporadic patients were compared to female controls (p<.05). Our findings are in accord with a previous study that found an increased frequency of the PROGINS polymorphism in a white population of endometriosis patients. In conclusion, these data supports a role for deficient PR function in the pathophysiology of endometriosis. This study is possible thanks to support from grants NIH-RCMI #5G12RR0305-19, NIH-RCMI #2G12RR003050-19, NIH #5T34GMO7732-23 and NIH RLK NRSA GM07732
Colón, Enid. Industrial Biotechnology Program, UPR Mayagüez – Rivera, Michelle; Rios Velázquez, Carlos. Department of Biology, UPR Mayagüez
Isolation and characterization of novel antimicrobial agents, and antimicrobial encoding genes from diverse soils in Puerto Rico.
Soil is a diverse medium in which we can find a variety of microorganisms. Resent research has demonstrated that only .01% of the total soil microbial diversity can be cultivated only by traditional microbiological techniques. In order to access the uncultivable microbiota present in soil we can use functional genomic techniques, which include the generation of soil metagenomic libraries. Genomic DNA extraction from soil samples has been done for further molecular characterization. By serial dilutions techniques, crowded plate, disk diffusion method and radial inhibition assays, we have identified several Antimicrobial Producing Agents (APA). The APA candidates are mainly non-spore forming gram-positive rods and have shown specific inhibition only to bacteria, especially Staphylococcus aureus. The genomic DNA of the candidates has been extracted, and will be used for molecular analysis by PCR, RFLP and sequencing specifically of the APA candidates rDNA. Screening of a developed soil metagenomic library is in progress to detect the presence of antimicrobial encoding genes.
Colon - Luna, Felicita. Department of Chemistry, Pontifical Catholic University of Puerto Rico - Mercado-Cruz, Joanna; Santiago-Cortes, Alma L. Department of Biology, Pontifical Catholic University of Puerto Rico.
HLA-DRB1 genotyping using PCR and automated DNA sequencing
Type 1 Diabetes is a chronic autoimmune disease that results from a T lymphocyte-dependent, selective destruction of the insulin-producing pancreatic β-cells and subsequent irreversible insulin deficiency. The human leukocyte antigen (HLA) region on chromosome 6p 21 contains the major locus of Type 1 diabetes. This locus has been associated with the development of some autoimmune diseases like Juvenile Chronic Arthritis and Rheumatoid Arthritis. Genetic susceptibility to Type 1 diabetes is affected by a combination of HLA-DQ and DRB1 alleles. Polymerase chain reaction (PCR) and Automated DNA Sequencing will be used to identify the DRB1 alleles present in thirty Puerto Rican families with at least one diabetic child. HLA-DRB allele frequencies will be determined and associated with susceptibility or resistance to Type 1 diabetes. Genomic DNA has been amplified for twenty individuals and are pending to be sequenced. Results of the automated DNA sequencing will be presented orally at the Junior Technical Meeting. This investigation was supported by NIH RLK NRSA GM07732 from the Pontificia Universidad Católica de Puerto Rico, MARC U*STAR Honor Program.
Colón Santos, Eric M.; Erick M; Menéndez Berríos, Nayda I.; Hidalgo, Marie B.; Alvarez, Jedisse M.; Nieves Xaymara; Torres Lilith. Natural Science Department, UPR-Aguadilla.
Aislación de las proteínas en infusión de hojas de Boussingoltia basselloides “Planta de Insulina”
Boussingoltia basselloides, es una planta oriunda de la región del Ecuador, la cual es utilizada tradicionalmente para el tratamiento de la diabetes. La misma es conocida popularmente como “planta de insulina”, ya que se le atribuye la función de regular los niveles de glucosa en la sangre. El objetivo de nuestra investigación es aislar proteínas a partir de una infusión de hojas de la planta. Para aislar las proteínas utilizamos dos muestras a las cuales sometimos a pruebas en la infusión. Luego ambas se concentraron utilizando dos métodos: diálisis y filtración. Posteriormente se llevo a cabo una digestión utilizando las enzimas proteinasa K y tripsina. Todos los resultados de estas técnicas fueron analizados por electroforesis en gel de poliacrilamida. Los resultados de esta electroforesis reflejaron la presencia de proteínas en la infusión. Sin embargo, el gel presentó una mezcla de proteínas las cuales están en proceso de ser identificadas.
Comenencia Ortiz, Eydith J. Biology Department, University of Puerto Rico at Cayey - Copper, Michael. Center for Molecular Neuroscience, Vanderbilt University.
Characterization of Hegehog signaling in the adult mouse brain
The Hedgehog genes encode for a family of secreted signaling molecules called Desert, Indian and Sonic hedgehog. Sonic hedgehog is the best characterized for its involvement in embryonic development, tumor formation and stem cell regulation. Although roles for Hedgehog signaling relating to embryogenesis, tumorigenesis, and stem cell regulation have been studied the most extensively, adult roles for Hedgehog signaling remain unknown. Interestingly, previous studies have found that components of the Hedgehog signal transduction cascade are expressed within many structures of the adult brain. As part of an ongoing effort to explore roles for Hedgehog signaling in the adult brain, we utilized a line of transgenic mice expressing a reporter gene for Hedgehog pathway. In these transgenic mice, the Patched gene was replaced by homologous recombination with the Lac Z gene. As the Lac Z gene disrupts the targeted Patched allele, it reports Patched transcription. These mice were used to characterize the ß-Galactosidase staining pattern of Patched, and therefore hedgehog expression in the adult brain. ß-Galactosidase staining pattern expressed hedgehog activity in many structures of the adult brain. Among these regions are the Basal Ganglia, Subventricular zone and Hippocampal region and the Suprachiasmatic nucleus, suggesting the possible involvement of hedgehog signaling in the control of movement, adult neurogenesis, and in circadian rhythms respectively. These findings could be part of the foundation for future exploration of roles of hedgehog signaling in the adult brain.
Cotto Rios, Xiomaris. Department of Biology, University of Puerto Rico at Cayey - Castillo, Adrew; Justice, Monica. Department of Molecular and Human Genetics, Baylor College of Medicine.
Mapping of transgene insertion site of Pim-1/ Eg5 transgenic mice
Proviral insertions in the mouse genome have previously shown to result in misexpression of neighboring genes. Using the technique viral insertion site amplification (VISA), two genes Hex and Eg5 were both found to be up regulated in tumors obtained from AKXD recombinant inbred strain of mice. As it was seen that Eg5 was upregulated in AKXD tumor samples, transgenic mice were created to reproduce the expression profiles in the hematopoietic tissues. One of the transgenic lines currently displays a phenotype at 12-16 weeks of age, at which they become ill and usually succumb to their illness within a few days. Due to the randomness of transgene insertion into the mouse genome upon pronuclear injection, it is not known where the Eg5 transgene is inserted in the mouse genome and if it is disrupting additional coding sequences which may be the cause of the phenotype seen in this line. To identify flanking sequences we created a sub-genomic library using a derivative of the lambda phage as a cloning vector, with which we would localize the insertion site of our Eg5 transgene. We digested genomic DNA for creation of our library, performed a southern analysis of gel slices from our digested genomic DNA, prepared the phagemid, infected bacteria with phage particles, and made colony lifts. We screened our sub-genomic library using a radiolabeled probe specific for the 3’ end of our transgene and our primary screen identified multiple positive plaques. Results are part of the foundation of an ongoing project, and additional screens are currently being done to verify positives plaques.
Cruz, Noel; Santiago, Alma. Department of Chemistry, Pontifical Catholic University of Puerto Rico.
Class II - HLA polymorphism of Taino Indians
It has become increasingly common to use ancient DNA (aDNA) to investigate population origins and evolution. A number of published studies have demonstrated that it is possible to extract endogenous DNA from archeological remains and amplify specific targets by the polymerase chain reaction (PCR). The HLA system is very useful in such anthropological studies because it is a polymorphic system with large differences in the allele frequencies between populations. aDNA from bone samples belonging to extinct Taino Indians from Puerto Rico will be extracted. Class II-HLA alleles will be determined using PCR, automated DNA sequencing, sequence-specific primers and probe hybridization. Statistical Analysis of allelic frequencies of the class II loci will be determined. The results obtained will be compared with other Amerindian populations already published.
Cruz, Nelly M. Department of Biology, University of Puerto Rico, Mayagüez, Puerto Rico - Maddock, Janine R.; Fuentes, Jennifer L.; Cantor, Robert H. University of Michigan, Ann Arbor, MI.
Cloning and expression of a yeast GTP-binding protein
The eukaryotic ribosome is a large ribonucleoprotein particle that is assembled from a small 40S and a large 60S subunit. Many non-ribosomal proteins involved in ribosome biogenesis have been identified, but are functionally uncharacterized. One of these proteins, Nog1p, has recently been found to be involved in 60S ribosome biogenesis. Nog1p is essential for Saccharomyces cerevisiae viability and is localized to nucleus and nucleolus. Nog1p harbors a GTP binding domain and is highly conserved in Archaea and eukaryotes. This study’s main purpose is to functionally characterize Nog1p. In order to investigate the biochemical properties of wild type and mutant Nog1p it is necessary to purify soluble and active Nog1 protein. We have found that the majority of full-length Nog1p expressed from Escherichia coli is insoluble. The purpose of this experiment was to isolate a form of the Nog1 protein that can be characterized biochemically. To do this, we created five different Nog1p truncations that contain the GTP-binding domain but lack either the amino and/or carboxyl terminal domain(s). Our aim was to determine whether these variants of Nog1p are soluble when purified from E. coli. Thus far, we have cloned the NOG1 truncations into a shuttle vector (pCR®2.1-TOPO®). We also cloned these NOG1 truncations into pET-42a(+), a plasmid that will allow for expression of the truncated Nog1 protein in E. coli. Next, these constructs will be used to overexpress and purify Nog1p. The biochemical characterization of Nog1p and complementary in vivo studies will contribute to a better understanding of the biological significance of Nog1p and its role in ribosome assembly.
Cuevas, Marielly. Department of Biology, PUCPR - Morales, Mara; Santiago, Cariluz; Appleyard, Caroline. Department of Physiology, PSM.
Specific phosphodiesterase inhibition can ameliorate chronic intestinal inflammation
The aim of these studies was to examine the therapeutic action of phosphodiesterase (PDE) inhibition in an animal model of chronic intestinal inflammation. We compared the effects of a PDE specific inhibitor, rolipram, and a non-specific inhibitor, pentoxifylline. METHODS: Inflammation was induced by the intracolonic administration of 30mg trinitrobenzenesulfonic acid (TNBS) in 50% ethanol. This was reactivated 6 weeks later by the intravenous administration of TNBS (5 mg/kg in saline) to produce chronic colitis. Control animals were reactivated with saline alone. Animals received pentoxifylline (PTX; 50mg/kg) or rolipram (ROL; 4mg/kg) treatment during the reactivation period. After sacrifice the colons were removed, scored for damage, and samples were taken for the measurement of myeloperoxidase. Relative gene specific RT-PCR was performed to analyze TNF expression. Segments of colon were stripped of the outer muscle layers and mounted in Ussing chambers for continuous measurement of short circuit current (Isc). RESULTS: Experimental animals had significantly higher levels of TNF mRNA (p<0.05) and colonic damage (p<0.01) than controls. Treatment with ROL, but not PTX significantly attenuated both the colonic damage and increased TNF expression. ACh caused a concentration-dependent increase in Isc in all groups which was depressed in the reactivated animals. ROL treatment could ameliorate, but not reverse this decreased responsiveness. CONCLUSIONS: Specific PDE inhibition can reduce the inflammatory parameters associated with chronic colitis. Supported in part by NIH MBRS SO6GM08239 & RLK NRSA 5T34GM07732.
De Jesús Jiménez, Doriann; Rivera, Evasomary; Martínez, Daviana; González Vargas, Carlos I. Biology, University of Puerto Rico, Río Piedras Campus.
An in vitro system using HeLa cytoplasmic extracts to study adenosine/uridine-rich element-mediated regulation of the VEGF transcript
RNAs transcribed from several genes implicated in cardiovascular diseases are regulated at the post-transcriptional level. These include the β-adrenergic receptors and the vascular endothelial growth factor (VEGF). These transcripts harbor Adenosine/Uridine-rich elements (AREs) in their 3’ untranslated region (3’UTR) which play a role in regulating their stability and translation. Inappropriate control of the abundance of these transcripts leads to disease. The mRNA destabilizing regulatory functions of these AREs are thought to be mediated by ARE-binding factors (ARE-BPs). Our laboratory is currently investigating the effects of the VEGF ARE on mRNA decay using an in vitro HeLa cell-based mRNA turnover system. We are using this system to reproduce regulated aspects of the VEGF ARE-mediated mRNA decay. We are also studying the effects of the cap structure and the poly (A) tail in the kinetics of the mRNA turnover of the VEGF constructs. This in vitro mRNA decay system will also be used to investigate the effects of various ARE-BPs on the stability of the VEGF mRNA. Initial data suggest an increase in mRNA turnover in VEGF constructs harboring ARE sequences. The elucidation by which VEGF AREs controls the expression of its mRNA eventually can provide a powerful tool for regulating gene expression and altering disease. Supported by a grant from NHLBI (KO1 HL-04355)
Diaz Maldonado, Naomi. Chemistry, University of Puerto Rico Mayagüez - Thornton, Tina; Taparowsky, Elizabeth. Biological Sciences, Purdue Univesity
Identification of BATF target genes
To define the role of a transcription factor, the specific genes that it regulates must be determined. We are studying the BATF protein, a member of the AP-1 family of transcription factors that is expressed in hematopoietic cells including M1 mouse myeloid leukemia cells. Work by the Taparowsky lab has shown that over expression of BATF can reduce cell division and cellular transformation of oncoproteins in vitro by dimerizing with the Jun family members (JunB, c-Jun and Jun D). The mechanism by which BATF regulates cell proliferation is still unknown. In order to elucidate this mechanism, it is crucial to investigate when, how, and at which sites the BATF protein associates with DNA. The way that our lab is trying to achieve this goal is by chromatin immunoprecipitation (ChIP) which will enable us to study the association of BATF with DNA in vivo. Our goal within this project was to optimize all the conditions needed to accomplish the ChIP assay. This included the optimization of the DNA shearing, testing primers for AP-1 sites likely to be regulated by BATF, and finding conditions for the immunoprecipitation of BATF. The optimization of the DNA shearing was made by sonication and verified by gel electrophoresis. Time points ranged from 30seconds to 6x30seconds. The best approximation to 500bp was obtained in the 2x30seconds. Sonication conditions were repeated to ensure reproducibility of the DNA shearing. Primers were designed to flank the AP-1 sites within the promoter of several genes known to be or suspected to be regulated by AP-1. These include c-Jun, IL-15 and BATF. Each primer pair was tested on mouse genomic DNA in a gradient PCR experiment. Primers were tested on the optimized sheared M1 DNA in which we had specific amplification of the Ap-1 sites. In order to test the affinity of the BATF antibody (á-BATF), M1 cells were induced with LIF (leukemia inhibitory factor) which induces the expression of BATF, prior to the IP. By a western blot of the IP of BATF we showed that á-BATF was highly specific and also confirmed that BATF gene is upregulated in response to IL-6/LIF. In conclusion, future work will be done to perform the ChIP for BATF, now that all the conditions needed have been optimized.
Fernandez, José R. Department of Industrial Biotechnology, University of Puerto Rico-Mayagüez Campus - Zhang, Lei; Bennett, Anton M.. Department of Pharmacology, Yale University-School of Medicine.
Generation and characterization of Noonan Syndrome mutations in the protein tyrosine phosphatase SHP-2
Noonan Syndrome (NS) is a common autosomal dominant disorder characterized by unusual facial features, short stature, skeletal anomalies, and defects of the heart, the most common of which are pulmonary valve dysphasia and hypertrophic cardiomyopathy. Incidence is said to be 1 in 1000-2500 births. Noonan syndrome was mapped to chromosome 12q in the PTPN11, a gene encoding the nonreceptor protein tyrosine phosphatase 2 (SHP-2). Several mutations have been found in the N-SH2 and PTP domains of SHP-2 in NS patients, which give the suggestion that mutations in PTPN11 are the cause of NS. SHP-2 is a key component of several signal transduction pathways that control protein developmental processes, particularly cardiac semilunar valvulogenesis. Binding of the NH2-terminal SH2 domain (N-SH2) of SHP-2 with its cognate phosphotyrosyl peptide is necessary and sufficient to activate the PTP. Noonan Syndrome mutations in SHP-2 (PTPN11) were generated in the N-SH2 and PTP domain and their expression verified in HEK 293 cells. The data reveals that NS mutants, as anticipated, produce constitutively elevated levels of SHP-2 phosphatase activity as compared to wild-type SHP-2. The rank order of these NS mutants indicate that the N-SH2 mutation E76A>> D61A> N308D>E69K. The variance in SHP-2’s phosphatase activity amongst these NS mutations, suggest the possibility that mechanisms other than excessive SHP-2 phosphatase activity are solely responsible for NS pathogenesis. Future work will utilize these mutants to establish a causal link between NS and aberrant SHP-2 catalytic activity.
Fontanez-Nuin, Darah. Pontifical Catholic University of Puerto Rico - Porter, James. Ponce School of Medicine.
Presynaptic A1 adenosine receptors inhibit thalamic stimulation of inhibitory neurons in the mouse somatosensory cortex
The cortical processing of sensory information relayed by the thalamus is important for our perception of the world. To determine if presynaptic adenosine receptors modulate the thalamic excitation of cortical inhibitory neurons we used a thalamocortical slice preparation and patch clamp electrophysiology. The ventrobasal thalamus was stimulated and excitatory postsynaptic currents (EPSCs) were recorded in layer IV inhibitory neurons in the somatosensory “barrel” cortex of mice. Inhibitory neurons were identified based on their pattern of action potential discharges in response to injected current and their morphology. Bath application of 10 µM adenosine reversibly reduced thalamocortical EPSCs in inhibitory neurons by 44 ± 5%. The inhibition was dose-dependent as 100 µM adenosine reduced the EPSCs by 84 ± 7%. The inhibition produced by 100 µM adenosine was reversed by 8-cycloentyl-1,3-dimethylxanthine (CPT), a selective A1 adenosine receptor antagonist, indicating that the inhibition was mediated by A1 adenosine receptors. Consistent with a presynaptic inhibition of glutamate release, adenosine did not affect the input resistance of the neurons, but did increase the paired pulse ratio and the coefficient of variation of the EPSCs. We conclude that presynaptic A1 adenosine receptors modulate thalamocortical stimulation of inhibition within the somatosensory cortex. Supported by S06 GM08239 and NIH NRSA CM07732 from PCUPR, MARC U STAR Honor Program.
Garcia La Torre, Edwin; Gonzalez, Carlos I. Deparment of Biology, UPR Rio Piedras.
Recognition of the marker protein Hrp1p by Upf2p is required for activation of nonsense-mediated mRNA decay
In eukaryotes, a nonsense codon in a given transcript will trigger its rapid decay by the nonsense-mediated mRNA decay (NMD) pathway. In yeast, a surveillance complex is thought to assemble at the translation termination event and search 3’ for a marker to determine whether the termination codon is premature. In yeast this marker can consist of Hrp1p bound to a downstream element (DSE). Hrp1p interacts with Upf1p, a known component of the surveillance complex. We show here that activation of mRNA decay also requires an interaction between Upf2p and Hrp1p. Importantly, binding of Upf2p to Hrp1p is modulated by the Upf2p/RNA complex and by the phosphorylation status of Upf2p. Our results indicate that the surveillance complex undergoes a remodeling process upon recognition of the Hrp1p/DSE marker to trigger decapping of the mRNA. We propose that the translation initiation factor Mof1/Suip may be involved in this remodeling step.
Gonzalez, Francis. Chemistry Department, University of Puerto Rico-Mayagüez Campus - Stuart, Jeremy; Yuan, Zhi-Min. Cancer Cell Biology Department, Harvard School of Public Health.
Investigation of the interaction between Nap 1 BP and c-Abl
c-Abl is a protein kinase that can localize in the nucleus as well as the cytoplasm, performing different functions according to its cellular location. Many functions of nuclear c-Abl have been described, but the cytoplasmic activities of c-Abl remain poorly understood. Previous mass spectrometry analysis of c-Abl stable lines indicated that a protein which binds c-Abl is Nap 1 BP, a cytoplasmic protein whose function has not yet been established. The interaction between these two proteins was verified and strong phosphorylation of Nap 1 BP mediated by c-Abl was observed in vivo. An in vitro kinase assay showed that the region containing amino acids 150-382 in Nap 1 BP is phosphorylated by c-Abl. An in vitro binding assay was also performed and showed that the region containing the SH3 domain in Nap 1 BP, namely residues 382-453, was the fragment that bound c-Abl. This finding suggests an additional level of interaction between Nap 1 BP and c-Abl besides the previous report stating that Nap 1 BP bound to c-Abl's SH3 domain in vitro. These findings may lead to the identification of the possible significance of the Nap 1 BP/c-Abl interaction and provide the basis for elucidating any cellular effects attributed to the functions of cytoplasmic c-Abl.
Irizarry, Nydia. Department of Biology, UPR Mayagüez Campus - Lopéz Garriga, Juan. Department of Chemistry, UPR Mayagüez Campus - Nadathur, Govind. Department of Marine Science, UPR Mayagüez Campus - Ríos Velázquez, Carlos. Department of Biology, UPR Mayagüez Campus - Rosado, William. Department of Marine Science, UPR Mayagüez Campus - Almodóvar Rivera, José R. Department of Biology, UPR Mayagüez Campus.
Pursing the hydrogen sulfide symbiotic bacterias present Lucina pectinata
Lucina pectinata inhabits in the sulfide rich coastal sediments of Puerto Rico and houses a chemoautotrophic symbiont bacteria, which uses hydrogen sulfide and carbon monoxide to produce sugars from the clam. Despite this, little is known about the nature and characteristics of these bacterias. In an effort to identify the bacterias, Scanning Electron Microscopy (SEM) and Denaturing Gradient Gel Eletrophoresis (DGGE) were used. The samples were prepared to observe SEM from 1µm to 10µm resolution and measurement were performed in longitudinal sections of gill tissue showing the presence of bacteriocytes and microvilli. Similarly, SEM also helps to identify the presence of bacterias living inside L.pectinata in the apparent symbiotic relationship. At the same time, DGGE was used to determinate from DNA samples, different patterns from cultivate and non-cultivate bacterias.
Itara, Luis Omar; Ayala, Limaris; Carrión, Luán; Marcano, Miguel; Rodriguez-Toro, Wanda; Dávila, Iván. Biology, UPR-Humacao.
Study and chraracterization of microbial flora of Macrobrachium faustinum
Macrobrachium faustinum is one of the most common species of prawns present in the streams and rivers of Puerto Rico. In the eastern and northern areas of Puerto Rico, this prawn is used as food. At the moment no study has been found mentioning the microbial flora associated to this prawn. Study the microbial flora and the possible presence of foodborne pathogen, such as Listeria monocyogenes is the main goal of this project. Prawns were collected and two different species samples (Atya sp. and Macrobrachium sp.) were processed to conduct a standard plate count. Diluted samples were plated in PCA for total count, MOX and PALCAM for Listeria, Potato Dextrose Agar for fungi and Brilliant Green Broth for coliforms. Preliminary results show a variety of organisms and the possibility of Listeria sp. Currently we are working on the characterization of the isolates.
Mcfaline Figueroa, Jose R. Química, UPR-Mayagüez - Hastie, Nicholas D.; Ijpenberg, Annemieke. Comparative and Developmental Biology Group, Human Genetics Unit Medical Research Council, Edinburgh, United Kingdom.
Quantification of glomeruli in adult mouse kidneys using Optical Projection Tomography (OPT)
During development, the Wilms’ tumour suppressor 1 gene (Wt1) is mainly expressed in the kidneys, the gonads and in the mesothelial lining of the main organs. In adult mice, the gene continues to be expressed in the podocytes lining the glomerular basement membrane of the kidneys. This expression pattern is faithfully reproduced in the yWT470lacZ mouse reporter line1, expressing the lacZ transgene under the control of 470kb of the human WT1 locus. In order to compare different models of kidney disease, we are interested in developing methods allowing us to quantify changes in kidney structure/function. We therefore decided to apply the newly developed technique of optical projection tomography2 to study glomerular distribution within the kidney. To this end, yWT470lacZ glomeruli were visualized either by direct staining with X-gal, or by using an anti-lacZ antibody. Following scanning, the glomerular distrubution within the tissues was 3-dimensionally reconstructed. The number of glomeruli was approximated from the thus generated quantitative data by using a combination of thresholding and size-limitation, and was estimated at 6600 per adult kidney. This roughly corresponds to two-thirds of the expected number of glomeruli as reported by Takemoto3 et al. Although there clearly is a need to optimize the experimental conditions, notably the penetration of the tissue with the stain/anti-body, these first results are very promising.
Medina, Sahira; Betancourt; Carlos. Biology Department, University of Puerto Rico, Mayagüez.
Aquatic Hyphomycetes from the Maricao River, Puerto Rico
The aquatic hyphomycetes, a group of fungi responsible for the degradation of organic material in sources of fresh water, were studied in the Maricao River in order to determine the biodiversity. Spores were obtained from the river’s foam, transferred to WhirlPak bags and stained with Lactophenol Cotton Blue. Samples obtained were viewed with a Microscope. The following species were found: Anguillospora crassa, Brachiosphaera tropicalis, Campylospora chaetocladia, Clavariopsis aquatica, Diplocladiella apendiculata, Helicomyces spp., Triscelophorus monosporus, and three unknown species.
Mercado, Benjamín; Arbelo, José; Hernández, Edwin. Department of Biology, UPR-Arecibo - Ramos Maiella. Physic and Chemistry, UPR-Arecibo - González Jessica. Biology, UPR-Arecibo.
Identification of isolated from the rhizosphere of water hyacinth from the Caño Tiburones wetland
Rhizobacteria were isolated from the aquatic plant water hyacinth (Eichornia crassipes). Identification of these bacteria was performed by gas chromatography (GC) coupled to the MIDI database system using a quantitative method based on a statistical analysis of the fatty acid content in these microorganism. The following bacteria were identified as: Bacillus-pumilus-GC subgroup B, Bacillus subtillis, Aeromonasichthiosma A/hydrophila, Aeromonas-trota/enteropelogenes, Aeromonas-veronii GC subgroup B (biogroup sobria), Pseudomonas-mendosina/straminea, Pseudomonas-stutzeri ( P. perfectomarina ), Chryseonomas-luteola ( Pseudomonas VE1), Flavimonas-oryzihabitans ( Pseudomona VE2 ) Pseudomonas-pseudoalcaligenes, Pseudomona-mendosina/straminea, Pseudomonas-alcaligenes, Aeromonas-ichthiosma A/hydrophila, Klebsiella-pneumoniac-pneumoniae-GC-subgroup B, Bacillus megaterium GC-subgroup A, Chryseobacterium-indologenes (Flavobacterium), Salmonella-cholaesuis-houtenae, Bacillus subtilis, Paenibacillus alvei GC-subgroup B, Brevundimonas-vesicularis ( Pseudomonas vesiculares), Kluyvera-cryocrecens-GC-subgroup B and Paenibacillus alvei GC-subgroup A. Ongoing projects include tolerance and uptake studies with these bacteria in culture media with heavy metals and elements of environmental concern.
Morales, Sulimar. Natural Sciences, UPR-Aguadilla – Tesfaye, Mengiste. Department of Botany and Plant Pathology, Purdue University.
Genetics control of Arabidopsis resistance to Botrytis cinerea
Botrytis cinerea is an important plant pathogenic fungus with a wide host range. Botrytis causes the gray mold disease in a wide range of crop plants under different production conditions. It causes significant economic loses in Agriculture. The principal means of disease management is the application of chemical fungicides. However, pollute the environment and are toxic to animals and humans. Genetic resistance provides environmentally safe and sustainable strategy to protect plants against plant pathogens. The molecular genetics of plant interactions with Botrytis are being investigated to determine plant resistance mechanisms. We are using Arabidopsis thaliana as a model to understand the Biology of Botrytis-plant interactions. I participated in a research project that is designed to identify plant genes that protect plants from Botrytis infection. Previously genes which are induced during Botrytis infection were identified in the lab. Plants that carry mutations of these Botrytis induced genes are used to determine their role in Botrytis resistance. My goal was to isolate T-DNA insertion mutants in the selected genes and analyze the effects of these mutations on plant resistance to Botrytis infection. DNA was extracted from these plants and used as a template for Polymerase Chain Reaction (PCR). PCR was conducted using pairs of primers designed from the gene of interest and the T-DNA insert. The resulting PCR products were resolved on an agarose gel. Using this approach I have identified homozygous mutants in two genes of interest. These mutant plants will be studied for their level of disease resistance.
Morell, Gloriner; Maldonado, Sandra. Biology, UPR-Mayagüez.
Muestreador aereo portable utilizado para determinar la incidencia de esporas pigmentadas en la baja atmósfera del área oeste de Puerto Rico
Para esta investigación se utilizó un globo de helio tratado, para muestrear la baja atmósfera en la finca Alzamora del Recinto Universitario de Mayagüez. Se recuperaron esporas de hongos a una altura de aproximadamente 50 metros. El muestreador de esporas fue especialmente diseñado para esta investigación, la cual se concentró principalmente en la recuperación de esporas pigmentadas colectadas en Agar con Rosa de Bengala. Luego del periodo de incubación las esporas fueron transferidas a un medio de cultivo, donde crecieron por siete días, para ser caracterizadas macroscópicamente y microscópicamente. El género más frecuente fue Curvularia. También se recuperaron esporas viables de Cladosporium, Nigrospora, Alternaria y Penicillium.
Negron, Veronica. Biology Department, UPR Humacao - Muller, Rafael J. Physics Department, UPR Humacao - Sastre, Miguel; SanJuan, Mariola. Biology Department, UPR Humacao.
The coupling of a astronomical CCD camera with a brighly microscope
In order to obtain one better quality of image we have combined the use of an astronomical camera CCD with a microscope composed of light. With this combination we can obtain clearer images, to store the information of a concise form and until being able to measure the microorganisms accurately. We have developed these techniques and we have including the use of fluorecens and programs of manipulation of images. Our results have been helpful for other investigations since the professors have been able to know their subjects better study. We continued developing more techniques with the purpose of being able to obtain an improved image every day that helps us to know plus the microorganisms.
Nieves Laspina, Aníbal F. Department of Chemistry, Pontifical Catholic University of Puerto Rico - Suárez, Edu; Montealegre, Federico. Department of Microbiology, Ponce School of Medicine.
Molecular identification of pathogenic mycobacteria species from indoors environmental samples of asthmatic patients homes in Ponce, Puerto Rico.
Rapidly growing mycobacteria are a complex group of environmental organisms that cause human diseases. Recent studies have suggest a role of the gram-positive mycobacteria tuberculosis in allergic diseases such as atopic diseases and asthma. Different positions have been presented, producing a controversy in this field. A preliminary study (data not published) suggested the unexpected presence of mycobacteriaceae family members in pooled dust samples collected from mattresses of asthmatic patients in Ponce, Puerto Rico. In this study, we propose to search individual dust samples for the presence of pathogenic strains of Mycobacteria ( M. smegmatis, M. intracellulare, M. tuberculosis, M. bovis and M. avium). The identification of these species in indoor dust samples, may imply a role of these agents in the development and/or sensitization of allergic patients symptoms. This project may also contribute to understand if we are underestimating the importance of monitoring indoor environmental exposures to these pathological agents. An early detection and accurate identification of pathogenic Mycobacteria will help both to apply measurements to eliminate or avoid further exposure and to initiate the appropiate treatment earlier.
Ocasio Torres, Yomayra; Esteban Avila, Ernesto P. Departamento de Fisica y Electrónica, Universidad de Puerto Rico-Humacao - Arenas Alicea, Agnes V. Departamento de Biologia, Universidad de Puerto Rico-Humacao.
A radiation-induced carcinogenesis model for space radiation
We develop a mathematical carcinogenesis model with 3n+8 parameters characterizing different biological processes arising when a stem cell population is continuously irradiated with high-LET HZE radiation. This multi-step model is mathematically described by a set of n+4 first order linear differential equations, where n is the number of possible mutations before a normal cell becomes a cancerous cell. At any time t, we present exact closed form solutions describing different irradiated stem cell populations for any given n mutations. The cases for n=1 and n=2 mutations are explicitly written down. Two applications are carried out. First, we show that the two free parameters α and β in target theory, are in fact related to one and two-killing, respectively. Second, we solve the proposed model were is more plausible to occur, i.e. in deep space, where Earth's magnetic shield and atmosphere can not longer shield us from galactic cosmic rays and special particle events. In particular, by using MARIE data we present plot survival curves in a Martian environment.
Oliveras, Yerai. Department of Chemistry. University of Puerto Rico, Mayagüez - Rivera-Bermudez, Moises A.; Masana, Monica I.; Dubocovich, Margarita L. Department of Molecular Pharmacology and Biological Chemistry and Drug Discovery Program, Feinberg School of Medicine, Northwestern University.
Characterization of melatonin receptor expression and function in immortalized rat SCN cells (SCN2.4)
In the mammalian SCN, the pineal hormone melatonin (MLT) inhibits neuronal firing rate through activation of MT1 MLT receptors and phase shifts circadian rhythms through activation of MT2 MLT receptors (Dubocovich et al., Front. Biosci. 8:d1093, 2003). Immortalized rat SCN2.2 cells are used as an in vitro model to study the mechanisms of circadian rhythm regulation. This cell line is composed of flat polygonal glial-like cells and small round neuronal-like cells (6-10 mm) cells. MT1 and MT2 melatonin receptor mRNA is expressed only in small-round cells with neuronal-like morphology, comprising ~10 % of the total cell population. Here we examined the morphological phenotypes of four clonal immortalized rat SCN cell lines. The percentage of neuronal-like cells were: 18.3 ± 2.2 % (n=3) in SCN2.1; 18.1 ± 2.4 % (n=4) in SCN2.3; 28.9 ± 4.9 % (n=3) in SCN2.4; 24.4 ± 4.2 % (n=3) in SCN3.1. The neuronal specific marker MAP-2 was expressed in 20.2 ± 0.4 % (n=4) of SCN2.4 cells, representing ~82 % of cells with neuronal morphology. MAP-2 expressed in only 50-65% of cells with neuronal morphology in the other clonal cell lines. The SCN2.4 clonal cells were further studied for expression and signaling of MLT receptors. SCN2.4 cells expressed MT1 and MT2 MLT receptors mRNA as detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and MLT receptor proteins as assessed by specific 2-[125-I]-iodomelatonin (300 pM) binding (0.56 ± 0.14 fmol/mg protein, n=7). In addition, MLT (10 nM, 10 min) stimulated protein kinase C (PKC) activity in SCN2.4 cells suggesting expression of functional MLT receptors. We conclude that the SCN2.4 cells provide a new in vitro model with higher number of neuronal cells to study MLT signaling pathway(s) involved in the regulation of circadian rhythms. Supported by USPHS grant MH 52685 to MLD, MH 52685 Minority Supplement to MARB, SROP FASEB MARC Program and ASPET SURF Program to YOS
Pagán, Beatriz. Biology, Pontifical Catholic University of Puerto Rico - Santiago, Cariluz; Appleyard, Caroline. Physiology, Ponce School of Medicine.
P53, K-Ras and APC mutations in a dimethylhydrazine “reactivated” rat model of colitis
Inflammatory Bowel Diseases (IBD) including Crohn’s disease and Ulcerative colitis, are disorders of the gastrointestinal tract that are of unknown etiology. Patients with this disease have a higher risk for developing colorectal cancer. Aim: Design and characterize an IBD-cancer rat model to investigate molecular and histological correlations, then establish a profile of cancer related gene mutations of p53, k-ras and APC during the transition of inflammation to dysplasia. Methods: Four groups of animals will be used: Prolonged Reactivated Colitis: a single intracolonic dose of trinitrobenzenesulfonic acid (TNBS) will be followed six weeks later by “reactivation” with intravenous TNBS via a tail vein for three days. TNBS will be administered twice a week thereafter. Reactivated-DMH: For induction of cancer the rats will receive a subcutaneous injection of 1,2-dimethylhydrazine (DMH) once a week following the reactivation. Control-DMH: Control rats will be induced then reactivated using sterile saline, and receive DMH; Normal: normal control rats will receive no treatment. Rats from each group will be sacrificed at 5, 10 and 15 weeks after the first reactivation or injection of DMH. Segments of the colon will be analyzed by macroscopic analysis, molecular analysis and histology. Expected outcomes: It is expected that this research will develop an animal model in which the pathophysiology and initial phases of colon cancer can be investigated. These experiments will also help to improve the early detection of colorectal cancer in patients with long standing ulcerative colitis. Supported in part by NIH RLK NRSA GM07732 and NIH MBRS SO6GM08239.
Reyes-Ortiz, Vimalier. Arts and Sciences (Industrial Biotechnology), UPR Mayagüez - Martinez-Cruzado, Juan C. Arts and Sciences (Biology), UPR Mayagüez.
Identification of native groups on Dominican population using mitochondrial DNA
I am identifying Amerindian Mitochondrial DNA (mtDNA) haplogroups in the Dominican Republic. The goal is describing the Amerindian maternal ancestry phylogeography in this country. Mitochondrial DNA will be extracted from hair roots and submitted to RFLP analysis. Selected mtDNA fragments will be amplified by PCR and tested for the possible presence of restriction sites that are haplogroup-specific. In that way, the haplogroup of each mtDNA will be determined. Because haplogroup are continent-specific, determining the identification of a haplogroup also identifies its continental origin. Techniques to be used are PCR amplification, restriction digestion, gel electrophoresis and photo identification.
Rivera, Zelieann. Department of Biology, University of Puerto Rico at Mayagüez - Bourguet, Shannon M.; Christian, Patricia J.; Hoyer, Patricia B. Department of Physiology, University of Arizona at Tucson.
Role of the aryl hydrocarbon receptor in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in C57BL/6 mice: involvement of caspase-3 activity
VCD has been shown to specifically destroy oocytes contained in primordial and primary follicles of rats and mice, therefore, causing follicle depletion and ultimately ovarian failure. Previous studies revealed that in primordial and primary follicles, VCD increased the activity of caspase-3 suggesting that caspase-3 might be involved in VCD-induced follicle loss. Furthermore, it has been observed that á-naphthoflavone (ANF), a know aryl hydrocarbon receptor (AhR) antagonist, had a protective effect against VCD-induced follicle loss in rats. However, the same experiment when conducted in mice determined that there was no such protective effect. The present study was designed to assess differences in caspase-3 activity in VCD and VCD/ANF-treated mice. Female C57BL/6 mice were given daily injections of either sesame oil (control), VCD, ANF or VCD+ANF (co-injected) for 15 days. Four hours after the final dose animals were sacrificed by CO2 inhalation and ovaries were excised. Pre-antral follicles were collected and isolated into two fractions, fraction 1 (primordial and primary) and fraction 2 (secondary). Protein concentration was measured and caspase-3 activity determined. Fraction 1 and 2 follicles collected from mice treated with VCD, ANF or VCD+ANF showed no significant difference in caspase-3 activity as compared to the control. After 15 days of daily dosing, VCD reduced primordial and primary follicles and ANF did not protect against follicle loss. Caspase-3 did not appear to play a major role in VCD-induced ovotoxicity in either fraction. This data suggest that, unlike rats, AhR and caspase-3 are not involved in VCD-induced follicle loss in C57BL/6 mice.
Rodriguez, Eillen. Biology Department, University of Puerto Rico at Cayey - Santos, Giovelly; Toranzos, Gary. Biology Department, University of Puerto Rico at Rio Piedras.
Detection of Aeromonas in treated and non treated drinking water of urban and rural areas of Puerto Rico
In 1998 EPA (Environmental Protection agency) publish a drinking water contaminant candidate list (CCL), on which they include contaminants that are know or anticipate to occur in water systems and which may require regulations. The American water work Association (AWWA) Microbial contaminant research committee selected about 11 microbes considered as potential disease causants. The genus Aeromonas was included on this list. The organisms that belong to this genus are facultative anaerobic, Gram-negative rods possessing polar flagella. Aeromonas are present on flowing freshwaters environments. Is know that some strains of Areomonas are pathogenic; this strains produce a toxin that can cause illness on humans. Until today is unclear which diseases are related to Aeromonas presence. There isn’t information about the presence of Aeromonas in drinking waters of Puerto Rico and/or the Caribbean. Due to the potential risk for our population is crucial to found evidence for the presence or absence of Aeromonas on our water supply systems; especially of pathogenic strains. Our principal goal is to detect Aeromonas on treated and non-treated drinking waters of different rural and urban area of Puerto Rico. To achieve our goal we are going to collect samples of drinking waters of different areas of Puerto Rico (e.g. ground waters, lakes, beaches, etc.) Those samples are going to be analyzed following the method 1605 for Detection of Aeromonas in finished drinking waters by membrane filtration using Ampicillin-Dextrin agar with Vancomycin (ADA-V) provided by the EPA.
Rodriguez, Yanitza; Betancourt-López, Carlos. Biology Department, UPR-Mayagüez.
Filamentous fungi in human respiratory system
During the rainy seasons in the tropics, nasal allergies are amongst the most common diseases. Most of these are caused by fungi. We studied fungi in nostril passages from August to October. Samples from different individuals were taken from each nostril using sterile vials. Mucous material from the vials were transferred to Petri plates with MEA and lactic acid and incubated at 37˚C. Permanent slides were prepared for fungal identification under microscope. Results suggest that during the months from August to October the most common allergens were Aspergillus Papulospora and yeasts.
Rodriguez, Laura; Betancourt, Carlos. Department of Biology, UPR-Mayagüez.
Aquatic Hyphomycetes of the Calvache Stream, Rincón, Puerto Rico
Aquatic Hyphomycetes (Deuteromycotina) are microorganisms that live in clean and very oxigenic fresh waters . Their distribution are universal with more diversity in tropical regions. In the Central mountain range of Puerto Rico, various studies have been elaborated in creeks, rivers and stream, however never in Calvache Stream. The purpose of this investigation is to prepare a mycological census of Aquatic Hyphomycetes in this stream. We used a plastic sterile spoon to extract foam suspended on the water, where conidias are highly concentrated. This foam was transfered to a plastic bag Whirl Pak and we mixed the foam with a dye called Lactofenol cotton blue to dye the conidia. Samples were taken to the laboratory and the foam was transfered to micropipettes with a dropper. The micropipettes were placed in a microcentrifuge to precipitate sediments and conidia found in the foam. Then the liquid from the micropipettes were extracted leaving only the precipitation. A drop of Lactofenol was added to the precipitate to create a solution and preserve the conidia. We took and placed a drop of the solution in a slide with a cover glass and were studied with a microscope using ocular 15x objective 60x. The genus found were: Alatospora (1 species), Aguillospora (3 species), Beltrania (3 species), Brachiophaera (1species), Campylospora (4 species), Clavariopsis (1 species), Flabellospora (1 species), Heliscus (1 species), Helicomyces (2 species), Isthmotricladia (1 species), Isthmolongispora (1 species), Jaculispora (1 species), Lemonniera (1 species), Phalangispora (1 species), Tricelophorus (1 species), Tricladium (1 species), Speiropsis (1 species), Varicosporium (1 species) and 7 unknowns.
Rodriguez-Torres, Sharon. Department of Biology, Pontifical Catholic University of Puerto Rico - Cruz-Pacheco, Noel. Department of Chemistry, Pontifical Catholic University of Puerto Rico - Santiago-Cortes, Alma L. Department of Biology, Pontifical Catholic University of Puerto Rico.
Genotyping of HLA- DQBI locus in Puerto Ricans with Type I Diabetes
Type I Diabetes (T1D) is an autoimmune disease that affects the beta-cells of the pancreas. The destruction of these cells leads to an insulin dependency in diabetic patients. Genetic and environmental factors are believed to influence the development of T1D. Human Leukocyte Antigen (HLA) class II genes have been identified to contribute to T1D susceptibility. The DQ region of the HLA class II locus has been linked as a high risk factor to the predisposition of T1D. The alleles identified to confer risk are: DQB1*0201, DQB1*0302, DQB1*0501, DQB1*0502, DQB1*0604. The DQB1 locus of diabetic families will be molecularly typed using Polymerase Chain Reaction (PCR) and probe hybridization (Dynal RELI SSO HLA-DQBI Typing) techniques. Data analysis will allow the identification of diabetogenic markers in Puerto Ricans with T1D. Exon 2 of the DQB1 locus has been amplified for twenty seven samples. Hybridization results will be presented at the meeting. “This investigation was supported by NIH RLK NRSA GM07732 from the Pontifical Catholic University of Puerto Rico, MARC U * HONOR PROGRAM.”
Rosario Colon, Leonardo. Chemistry Dept, UPR Mayagüez - Perez Rivera, Alex A.; Galligan, James. Dept. of Pharmacology and Toxicology, Michigan State University.
Potential interaction between alpha-1 and alpha-2 adrenoceptors in murine mesenteric veins
The sympathetic nervous system (SNS), which its main effector is norepinephrine (NE), is an important contributor to the pethogenesis of hypertension and other cardiovascular diseases. Previous studies done in this lab have demonstrated that murine mesenteric veins are more sensitive than arteries to adrenergic stimulation by the alpha-1 and alpha-2 agonist NE. However, the selective alpha-2 agonists clonidine and UK 14,304 did not elicit a direct vasoconstriction in mesenteric vessels. Given this, we assessed the effect of the alpha-1 adrenoreceptor (AR) antagonist prazosin and the alpha-2 ARs antagonist yohimbine on NE-induced vasoconstriction in small veins of SHAM control and DOCA-salt hypertensive mice in an effort to determine the main role of alpha-2 ARs in vasoconstrictor responses to NE. Blood vessel diameter changes were measured in-vitro by computer-assisted video microscopy. Prazosin produced a concentration-dependent inhibition of NE-induced constrictions with no apparent difference seen between SHAM and DOCA-salt veins. Yohimbine produced a concentration-dependent inhibition of NE-induced constrictions with a bigger effect seen in DOCA-salt veins. Also, as determined by changes in EC50 values, yohimbine had the greater inhibitory effect on NE-induced constrictions in SHAM control and DOCA-salt veins. Even though the antagonists produced rightward shifts in the NE concentration-response curves, they did not affect maximum responses to the agonist. A potential interaction between alpha-1 and alpha-2 ARs could help explain the effectiveness of yohimbine (an alpha-2 AR antagonist) in antagonizing NE-induced constrictions in murine mesenteric veins with greater effect than prazosin (an alpha-1 AR antagonist) in the absence of a direct alpha-2 mediated vasoconstriction.
Soltero-Rivera, Ingrid M.; Martinez-Cruzado, Juan C. Department of Biology, University of Puerto Rico-Mayagüez.
Identificando ADNmt de origen indígena en la República Dominicana
El ADN mitocondrial (ADNmt) se hereda estrictamente de forma maternal no recombinante. Como no sufre cambios al menos que ocurra una mutación, su uso es muy importante en la caracterización de linajes ancestrales de las poblaciones humanas. Ello se ha logrado mediante los llamados haplogrupos. Un haplogrupo es un grupo de haplotipos que comparten una mutación que se hereda de un ancestro cercano que tiene en común. En la República Dominicana se tomaron muestras de cabello de diferentes personas no relacionadas genéticamente. El ADNmt fue extraído para cada muestra utilizando la solución Chelex. Cada muestra fue sometida a la técnica de “Restriction Fragment Length Polymorphisim” (RFLP) para así poder clasificarlas bajo los haplogrupos L (ancestro africano), A, B, C, D y X los cuales tienen ancestro indígena. Se encontró que los haplogrupos indígenas con mayor frecuencia fueron los haplogrupos C y D ya que ambos se obtuvieron un 6%. El haplogrupo L fue el de mayor frecuencia con un 23% no obstante, un 50% no perteneció a ningún de los haplogrupos estudiados lo que sugiere que pertenezca a otro ancestro africano diferente al haplogrupo L. Un 78% resultó tener orígenes africanas mientras que el restante 22% resultó con ancestros indígenas.
Sosa, Greychan; Alvarado, Darys.; Castro, Moraima; Jimenez, Joan; Loperena, Omayra; Ruiz, Johanna. Natural Sciences Department, University of Puerto Rico in Aguadilla.
HAART resistance mutation rates in a population of HIV+ Puerto Ricans
Human immunodeficiency virus (HIV) infection causes progressive immune deficiency by a decline in numbers of CD4+ T-cells. The human immunodeficiency virus-1 (HIV-1) has infected over 57 million and killed over 22 million individuals worldwide since the beginning of the epidemic. According to the Department of Health of Puerto Rico, as of June 2002, there have been 27,662 AIDS cases reported in Puerto Rico. Puerto Rico held the sixth highest incidence rate among the United States and its territories. To contain the viral infection, highly active antiretroviral therapy (HAART) is used. We determine the genetic diversity and prevalence of primary and secondary antiretroviral resistant genotypes in a population of HIV positive Puerto Ricans. The data was generated from the 2000 to 2003 using the TRUGENE™ HIV-1 Genotyping system at the Immunoretrovirus Research Laboratory at the Universidad Central del Caribe. We found mutations associated with resistance to Nucleoside reserve trascriptase inhibitors (NRTI’s), non-nucleoside reserve trascriptase inhibitors (NNRTI’s), reserve trascriptase inhibitors (RTI’s), and Protease Inhibitors (PIs) in the population. The continuous screening of mutations rates in the HIV virus will help researchers identify better treatment regimens and possible targets for treatment.
Torruella, Carmen; Ferrer, Iván. Natural Sciences Department, Inter American University, Bayamón Campus.
Characterization of the P. yoelii mdr1 gene (pymdr1)
Drug resistance in malaria remains a major health problem worldwide. In falciparum malaria, the most deadly form of the disease, the Plasmodium falciparum chloroquine resistant transporter (pfcrt) gene and multidrug resistance (pfmdr1) gene have been linked to the resistant phenotype. Mutations in the pfmdr1 gene have been implicated in resistance to chloroquine, mefloquine, quinine, halofantrine and artemisinin. Previous work from our laboratory showed amplification of the mdr1 orthologue in drug resistant lines of the rodent malaria parasites P. berghei and P. yoelii. The mdr1 genes from P. falciparum, P. chabaudi, P. berghei and P. yoelii were retrieved from the PlasmoDB database and the coding sequences were aligned. To ascertain if point mutations were involved with drug resistance in P. yoelii, the complete open reading frame of the mdr1 gene was amplified, cloned and sequenced in a collection of drug sensitive and resistant lines. The previously described resistant phenotype point mutations at positions 86, 184, 1034, 1042, and 1246 of the pfmdr1 were not detected in our collection. Analysis by RT-PCR confirmed the expression of the mdr1 gene in intraerythrocytic and in hepatic stages of P. yoelii.
Vega Alvarado, Lissette. Biology Department, University of Puerto Rico, Mayagüez Campus - Lackland, Dr. Daniel T. Biometry and Epidemiology, Medical University of South Carolina - Vega Alvarado, Lissette. Biology, University of Puerto Rico, Mayagüez Campus.
The potential impact of the growing Hispanic population on diabetes and cardiovascular health: a descriptive assessment
Diabetes is a disease that shows high levels of sugar in the blood. One of the major problems related with diabetes are cardiovascular diseases, such as: heart attacks, strokes, hypertension, and others. The major objective is to determine how many Hispanics with diabetes have a cardiovascular disease (CVD). Medline, CDC and US Census Bureau were used to gather information about cardiovascular diseases among Hispanics with diabetes and the characteristics of this population. This study showed that White women with diabetes have a CVD rate of 55.7, 65.2 in Blacks and 56.6 in Hispanics. The rate of cardiovascular disease in women with diabetes during 1997-1999 was twice that of women without diabetes. Some characteristics of the Hispanic population influence health in different ways. Lack of health insurance directly affe