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R Contreras

Publications and source records attributed to R Contreras.

At least 19 recordsLinked to original sources
PubMed1998

[Polymerase chain reaction for rapid detection and serotyping of dengue virus in clinical samples].

This study describes the benefits of using reverse transcriptase polymerase chain reaction (RT-PCR) for the rapid detection and typing of dengue virus in clinical samples. Twenty-seven serum specimens from patients with dengue fever and dengue hemorrhagic fever in Colombia, Nicaragua, and Panama were directly subjected to RT-PCR for the detection of dengue virus. The resulting double-stranded DNA product was typed by a second round of PCR amplification (nested PCR) with type-specific primers, viral culture/indirect immunofluorescence (IIF), and enzyme-linked electroimmunoassay for IgM anti-dengue antibodies. The amplified virus genome was detected and typed within 8 hours. Nested RT-PCR, using viral culture and IIF as the gold standard, showed 100% sensitivity; 78% specificity; 69% positive predictive value, and 100% negative predictive value. It is noteworthy that two of the specimens whose results were positive with nested RT-PCR and negative with viral culture showed specific IgM antibodies. The results of the RT-PCR were in close agreement with those obtained through viral culture. This suggests PCR can greatly facilitate the rapid and early diagnosis of dengue infection.

Colombia↗
PubMed1997

In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharides--evidence for N-acetylglucosaminyltransferase-I-accepting glycans from Trichoderma reesei.

To investigate the potential of filamentous fungi to synthesize N-glycans that are convertible to a mammalian type, in vitro glycosylation assays were performed. Recombinant human N-acetylglucosaminyltransferase I, human beta-1,4-galactosyltransferase and rat alpha-2,6-sialyltransferase were successively used to mimic part of the mammalian glycosylation synthesis pathway. High-mannose carbohydrates on Trichoderma reesei cellobiohydrolase I were converted to a hybrid mammalian-type structure. Successful modification varied markedly with the strain of T. reesei used to produce cellobiohydrolase I. In vitro pretreatment of fungal glycoproteins with Aspergillus saitoi alpha-1,2-mannosidase improved subsequent hybrid formation. It was, however, not possible to trim all fungal oligosaccharides to an acceptor substrate for mammalian glycosyltransferases. With T. reesei RUTC 30, capping glucose residues and phosphate groups were shown to be responsible for this lack of trimming. N-glycan processing in T. reesei apparently involves different steps, including alpha-1,2-mannosidase trimmings, and thus resembles the first mammalian glycosylation processes. The alpha-1,2-mannosidase trimming steps can be exploited for further in vitro and/or in vivo synthesis of complex oligosaccharides on (heterologous) glycoproteins from filamentous fungi.

Acetylglucosamine↗
PubMed1997

Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase.

The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-mating-factor signal sequence. Cultures in shake flasks provided expression levels of approximately 2.5-3 mg/l. Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity. Although P. pastoris-secreted products are believed to carry shorter N-linked carbohydrate side chains than glycoproteins of S. cerevisiae, secreted neuraminidase was hyperglycosylated, with N-glycans of the high-mannose type containing up to 30-40 mannose residues. N-glycans were phosphorylated and only partially sensitive to alpha-mannosidase treatment. Balb/c mice immunized three times with 2 microg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse-adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection. The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.

Animals↗
PubMed1997

Structural characterization of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei RUTC 30.

We have characterized the primary structures of the predominant N-linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chromatographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N-linked core. In the bulk of the N-glycans, the alpha-1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The alpha-1,6 arm shows a remarkable heterogeneity: in addition to alpha-1,2-Man and alpha-1,6-Man, the presence of a terminal mannose alpha-1,6-phosphodiester was observed. This latter substituent has not been characterized before on mannosidase-processed N-glycan and its function and synthesis pathway are entirely unknown. The predominant N-glycans on cellobiohydrolase I can be represented as follows: GlcMan8GlcNAc2, GlcMan7GlcNAc2, Man7GlcNAc2, ManPGlcMan7GlcNAc2, GlcMan5GlcNAc2 and Man5GlcNAc2.

Carbohydrate Sequence↗
PubMed1997

NMR evidence for a novel asparagine-linked oligosaccharide on cellobiohydrolase I from Trichoderma reesei RUTC 30.

The primary structure of a novel phosphate-containing oligosaccharide, isolated from T. reesei cellobiohydrolase I, was determined by NMR techniques. The new compound has the same structure as GlcMan7GlcNAc2, but it is extended by one alpha-mannopyranosyl unit (Man-P) through a phosphate link. Three different heteronuclear (31P-1H) NMR techniques were used to prove that the phosphate links the glycosidic site of Man-P with C-6 of unit Man-B. The presence of mannoses linked through a phosphate diester resembles glycosyl synthesis in yeast.

Asparagine↗
PubMed1997

Diabetes and insulin-induced stimulation of L-arginine transport and nitric oxide synthesis in rabbit isolated gastric glands.

1. The properties of L-arginine transport have been characterized and correlated with cGMP production (index of nitric oxide (NO)) in whole gastric glands isolated from non-diabetic and alloxan-diabetic rabbits. 2. In non-diabetic and diabetic glands, transport of L-arginine was stereoselective, Na+ and pH independent and inhibited by other cationic amino acids. L-Arginine transport was slightly inhibited by L-leucine and L-phenylalanine, but unaffected by other neutral amino acids. 3. Diabetes enhanced the Vmax for saturable L-arginine transport from 10.7 +/- 1.0 to 17.7 +/- 0.5 pmol (mg protein)-1 s-1, with negligible changes in K(m). 4. Accumulation of the membrane potential-sensitive probe tetra[3H]phenylphosphonium (TPP+) was increased 2-fold in diabetic compared with non-diabetic gastric glands, suggesting a membrane hyperpolarization. 5. Basal intracellular cGMP levels were elevated 2-fold in diabetic gastric glands, and in non-diabetic glands histamine, vasoactive intestinal peptide, and bradykinin increased cGMP levels. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (100 microM) abolished basal cGMP accumulation. 6. Addition of extracellular L-arginine induced a concentration-dependent increase in cGMP levels in gastric glands isolated from non-diabetic rabbits, but had no effect on elevated cGMP levels in diabetic glands. 7. Insulin induced a rapid (5 min) concentration-dependent increase in cGMP levels in non-diabetic gastric glands, but reduced elevated cGMP levels in diabetic gastric glands. 8. The present study has identified a specific transport system for L-arginine in gastric glands which resembles the classical system y+. Our findings also provide the first direct evidence that diabetes increases the basal activity of system y+ and NO synthase in gastric glands. The differential modulation of L-arginine transport by insulin and L-arginine identified in non-diabetic and diabetic glands, may be of importance in protecting the gastric mucosa from injuries associated with diabetes.

Animals↗
PubMed1997

Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei.

The Aspergillus niger and Trichoderma reesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70-80% identity to the SAR1 protein. Complementation of S. cerevisiae sar1 and sec12 mutants by expression vectors carrying the A. niger sarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. niger sarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger.

Alleles↗
PubMed1996

A20, an inhibitor of cell death, self-associates by its zinc finger domain.

A20 is a primary response gene which is induced after monocyte adherence or cytokine stimulation of a variety of cells. The A20 protein belongs to a novel class of Cys2/Cys2 zinc finger proteins, and has been characterized as an inhibitor of both apoptotic and necrotic cell death. In order to clarify its molecular mechanism of action, we used the yeast-based two-hybrid system to screen for A20-associated proteins. Here we report that A20 is able to self-associate, and demonstrate that the latter interaction is mediated by its zinc finger domain.

Base Sequence↗
PubMed1996

Fostering the health of communities: a unifying mission for the University of New Mexico Health Sciences Center.

Fostering the health of communities can serve as a unifying mission of the academic health center (AHC), which can set the AHC apart from other health providers in the community. To achieve this mission, the University of New Mexico's AHC is increasingly focusing education, research, and service upon the identified health and service needs of communities in its state. Since major health problems in our society have social, behavioral, and economic roots, New Mexico's AHC has tapped into the broad expertise of its different components as well as that of its state and community partners to adequately address health problems in the community. Its hospitals offer financing and management resources, its colleges offer innovative approaches to community-based education, and the state department of health offers expertise in health policy development. To adequately respond to the complexity of community health needs, the different colleges and departments at New Mexico's AHC are increasingly merging into integrated governance units. Measures of community outreach success include evidence of strengthened community development, increased health care access, and improved indices of community health. New Mexico's AHC formed an interdisciplinary rural outreach task force, which has demonstrated its ability to form partnerships with state and local agencies and to mobilize institutional resources in education, research, and service from the AHC's different departments, colleges, and hospitals to respond promptly to unique community health needs. Evidence shows that such an integrated, coordinated AHC intervention can generate strong and lasting AHC-community alliances, improve the quality and economic viability of community health systems, and enhance the financial resources of the AHC.

Academic Medical Centers↗
PubMed1995

The effect of in utero administration of buthionine sulfoximine on rat development.

Glutathione (GSH) is a tripeptide that is thought to be an essential cell component playing an important role as a cellular antioxidant and scavenger of free radicals. GSH depletion has been shown to render cells more sensitive to various insults. GSH has a protective effect. GSH levels can be decreased by inhibition of its synthesis with buthionine sulfoximine (BSO), which inhibits gamma-glutamylcysteine synthetase. Several studies have shown that treatment with BSO enhances the toxicity of some drugs and radiation. A previous study indicated that the effects of BSO on the developing embryo were short lived and did not persist to birth. In the above-mentioned study, mothers were treated with BSO only on days 10 and 11 of gestation. The objective of the present study was to determine the effects of BSO administration on GSH depletion throughout pregnancy on the developing rat. Timed pregnant Sprague-Dawley rats were placed on a liquid BioServ diet containing BSO starting on day 1 of pregnancy. The mothers received a daily dose of BSO ranging from 2 to 6 mmol/kg/24 h. The mothers were maintained on the diet until gestation day 21 when they were anesthetized with sodium pentobarbital and the pups delivered by Cesarean section. GSH levels were measured in brain and liver, and various parameters relating to development were assessed. A dose-response curve showed that a maximum depletion (86%) of GSH in the mother's liver was produced by the 6 mmol/kg dose of BSO. However, no change was seen in brain GSH levels of the mothers.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Drug-Induced↗
PubMed1995

Effects of selected synthetic compounds on growth of Mucor rouxii.

The in vitro activity of four synthetic compounds was tested on fungal cells from Mucor rouxii. The compounds included phenylenediamine, two phenolamines, and quinone. At the concentrations tested (10(-2)-10(-4)M) the compounds exhibited antifungal activity, with the exception of quinone. On the basis of their effects on spore germination, and development of hyphae, phenylenediamine was the more active. The mechanism of action of the compounds is still unknown, but hyphae show morphological alterations and disturb the distribution of calcofluor in the cell wall. This suggests variations in the genesis of the cell wall.

Aniline Compounds↗
PubMed1995

[Hematological serological valves, and organ weight in adult Sprague-Dawley rats].

Sprague-Dawley rats are frequently used in experiments and their normal hematological, serologic and anatomical parameters are not easily available. The aim of this study was to measure these parameters in adult Sprague-Dawley rats. Twenty-four rats (12 males) whose weight ranged between 200 and 300 g, raised at the Instituto de Salud Pública in controlled environmental conditions and fed with pellets were studied. After a 24 h fast, animals were anesthetized with Droperidol and Ketamine, and after obtaining a cardiac blood sample, sacrificed by exsanguination. Most organs were withdrawn and weighed. Mean values for blood glucose, calcium, amylase, total proteins, cholesterol, AST, ALT, bilirubin, alkaline phosphatases, electrolytes and complete blood count were determined. Heart, lung, pancreas, kidney and testicle mean weight was also calculated. Male and female rats had significant differences in packed red cell volume, white blood cell count and hemoglobin.

Animals↗
PubMed1994

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Base Composition↗
PubMed1994

Sequence analysis and expression studies of a gene encoding a novel serine + alanine-rich protein in Trichoderma harzianum.

The promoters of constitutive genes have proven themselves to be useful parts of expression vectors for genetic engineering in different organisms. Based on a differential screening of an induced cDNA library, we have used a new approach to isolate constitutively expressed genes which can be used for this purpose. Following this strategy, we have isolated constitutively expressed cDNA clones of the mycoparasitic fungus Trichoderma harzianum. One of these cDNA clones corresponds to a gene (cob4) that encodes a novel serine + alanine-rich protein. The predicted amino acid (aa) sequence of this protein (171 aa, deduced M(r) 15,564, pI 12.49) displays a motif that is also found in structural proteins of different species and in a yeast thermoinducible protein. Northern (RNA) blot analysis has demonstrated that cob4 is expressed during growth when glucose or cell walls of a phytopathogenic fungus are provided as carbon source. Hybridizations of T. harzianum genomic DNA indicate that this gene most probably belongs to a multigene family.

Alanine↗
PubMed1994

Human interferon-beta, expressed in Saccharomyces cerevisiae, is predominantly directed to the vacuoles. Influence of modified co-expression of secretion factors and chaperones.

Expression of the human interferon-beta (hIFN-beta) gene was found to be very toxic for Saccharomyces cerevisiae. An integrative expression cassette, containing the hIFN-beta gene under control of the inducible galactokinase (GAL1) promoter in combination with the alpha-factor prepro-secretion signal, was used to study the secretion process in more detail. Specific differences were found between a vacuolar proteinase--mutant and a normal laboratory yeast strain. Cell organelle fractionation, carried out with the recombinant C13-ABYS66 strain, revealed that 99% of the hIFN-beta remained intracellular and that the majority was associated with the vacuolar fraction. The secretion efficiency in the latter strain was investigated by overexpressing chaperone molecules (HSP70 and BiP) and homologous secretion factors (SEC1 and SEC18). Only the presence of HSP70 resulted in a 5-fold increase in secreted hIFN-beta.

Adenosine Triphosphatases↗
PubMed1994

Phosphoribosylpyrophosphate synthetase (PRS): a new gene family in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains at least four PRS genes, all of which have been cloned and sequenced. Each of the four derived amino acid sequences have more than 60% similarity to the corresponding polypeptides of man, rat, Escherichia coli and Salmonella typhimurium. The PRS1 gene maps on chromosome XI, PRS2 on chromosome V, PRS3 on chromosome VIII and PRS4 on chromosome II. One member of this gene family, PRS1, contains a region of non-homology (NHR) shown by cDNA cloning and sequencing not to be an intron. The results presented here suggest that the presence of this NHR is not detrimental to the function of the gene. To date the possibility of protein splicing can be neither proven nor disputed.

Amino Acid Sequence↗