Biomedical subjects

I Gomes

Publications and source records attributed to I Gomes.

At least 19 recordsLinked to original sources

PubMed2003

Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization.

Five strains of the extreme thermophilic Rhodothermus marinus were screened for the production of amylolytic and pullulytic activities. The culture medium for the selected strain, R. marinus ITI 990, was optimized using central composite designs for enhanced enzyme production. The optimized medium containing 1.5 gl(-1) of maltose and 8.3 gl(-1) of yeast extract yielded amylase, pullulanase and alpha-glucosidase activities of 45, 33 and 2.1 nkatml(-1), respectively. Among the various carbon sources tested, maltose was most effective for the formation of these enzymes, followed by soluble maize starch, glycogen and pullulan. The crude amylase and pullulanase showed maximum activities at pH 6.5-7.0, and 85 and 80 degrees C, respectively. At 85 degrees C amylase and pullulanase had half lives of 3 h and 30 min, respectively.

Amylases↗

PubMed2001

A novel nuclear protein, 5qNCA (LOC51780) is a candidate for the myeloid leukemia tumor suppressor gene on chromosome 5 band q31.

Interstitial deletion or loss of chromosome 5, del(5q) or -5, is a frequent finding in myeloid leukemias and myelodysplasias, suggesting the presence of a tumor suppressor gene within the deleted region. In our search for this gene, we identified a candidate, 5qNCA (LOC51780), which lies within a consistently-deleted segment of 5q31. 5qNCA expresses a 7.2-kb transcript with a 5286-bp open reading frame which is present at high levels in heart, skeletal muscle, kidney, placenta, and liver as well as CD34+ cells and AML cell lines. 5qNCA encodes a 191-kD nuclear protein which contains a highly-conserved C-terminus containing a zinc finger with the unique spacing Cys-X2-Cys-X7-His-X2-Cys-X2-Cys-X4-Cys-X2-Cys and a jmjC domain, which is often found in proteins that regulate chromatin remodeling. Expression of 5qNCA in a del(5q) cell line results in suppression of clonogenic growth. Preliminary sequence results in AML and MDS samples and cell lines has revealed a possible mutation in the KG-1 cell line resulting in a THR to ALA substitution that has not been found in over 100 normal alleles to date. We propose 5qNCA is a good candidate for the del(5q) tumor suppressor gene based on its predicted function and growth suppressive activities, and suggest that further mutational and functional study of this interesting gene is warranted.

Acute Disease↗

PubMed2001

Highly abundant genes in the transcriptosome of human and baboon CD34 antigen-positive bone marrow cells.

Nonhuman primates are useful large animal model systems for the in vivo study of hematopoietic stem cell biology. To better understand the degree of similarity of the hematopoietic systems between humans and baboons, and to explore the relevance of such studies in nonhuman primates to humans, this study was designed to compare the global gene expression profile of bone marrow CD34(+) cells isolated from these 2 species. Human complementary DNA (cDNA) filter arrays containing 25 920 human cDNAs were surveyed for this purpose. The expression pattern and relative gene abundance of the 2 RNA sources were similar, with a correlation coefficient of 0.87. A total of 15 970 of these cDNAs were expressed in human CD34(+) cells, of which the majority (96%) varied less than 3-fold in their relative level of expression between human and baboon. Reverse transcriptase-polymerase chain reaction analysis of selected genes confirmed that expression was comparable between the 2 species. No species-restricted transcripts have been identified, further reinforcing the high degree of similarity between the 2 populations. A subset of 1554 cDNAs, which are expressed at levels 100-fold and greater than background, is described, which includes 959 expressed sequence tags and uncharacterized cDNAs, and 595 named genes, including many that are clearly involved in hematopoiesis. The cDNAs reported here represent a selection of some of the most highly abundant genes in hematopoietic cells and provide a starting point to develop a profile of the transcriptosome of CD34(+) cells.

Animals↗

PubMed2001

Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation.

G-protein-coupled receptors (GPCRs) have recently joined the list of cell surface receptors that dimerize. Dimerization has been shown to alter the ligand-binding, signaling, and trafficking properties of these receptors. Recent studies have shown that GPCRs heterodimerize with closely related members, resulting in the modulation of their function. In this study, we have attempted to determine whether members of GPCR superfamilies that couple to different families of G-proteins can associate and form oligomers. We chose the beta2 adrenergic receptor that couples to stimulatory G-proteins and delta & kappa opioid receptors that couple to inhibitory G-proteins. beta2 and delta receptors undergo robust agonist-mediated endocytosis, whereas kappa receptors do not. We find that when coexpressed, beta2 receptors can form heteromeric complexes with both delta and kappa receptors. This heterooligomerization does not significantly alter the ligand binding or coupling properties of the receptors. However, it affects the trafficking properties of the receptors. For example, we find that delta receptors, when coexpressed with beta2 receptors, undergo isoproterenol-mediated endocytosis. Conversely, beta2 receptors in these cells undergo etorphine-mediated endocytosis. However, beta2 receptors, when coexpressed with kappa receptors, undergo neither opioid- nor isoproterenol-mediated endocytosis. Moreover, these cells exhibit a substantial decrease in the isoproterenol-induced phosphorylation of mitogen-activated protein kinases. Taken together, these results provide direct evidence of heteromerization of GPCRs that couple to different types of G-proteins, which results in the modulation of receptor trafficking and signal transduction.

Adrenergic beta-2 Receptor Agonists↗

PubMed2001

G protein coupled receptor dimerization: implications in modulating receptor function.

Protein-protein interactions are involved in the regulation of a large number of biological processes. It is well established that a variety of cell surface receptors interact with each other to form dimers, and that this is essential for their activation. Although the existence of G protein coupled receptor dimers was predicted from early pharmacological and biochemical analysis, solid evidence supporting dimerization has come within the past few years following the cloning of G protein coupled receptor cDNAs. Using differential epitope tagging and selective immunoisolation of receptor complexes, dimerization of a number of G protein coupled receptors including members of the rhodopsin, secretin, and metabotropic glutamate receptor families have been reported. More recently fluorescence or bioluminescence resonance energy transfer techniques have been used to examine dimerization of these receptors in live cells. These studies have found that whereas in some cases there is an agonist induced increase in the level of dimers, in others there is a decrease or no change in dimer levels. Several recent studies have also reported the ability of related members of G protein coupled receptors to heterodimerize. These heterodimers exhibit distinct physical and functional properties. Examination of possible sites of interactions between receptors has implicated a role for extracellular, transmembrane and/or C-terminal region in dimerization. The functional consequences of dimerization, explored mainly using mutant receptors, have demonstrated a role in modulating agonist affinity, efficacy, and/or trafficking properties. Thus dimerization appears to be a universal phenomenon that provides an additional mechanism for modulation of receptor function as well as cross-talk between G protein coupled receptors.

Animals↗

PubMed2001

Linkage analysis of the 5q31-33 candidate region for asthma in 240 UK families.

Atopy and asthma are complex genetic diseases resulting from the interactions of a number of genetic and environmental factors. We had previously reported allelic association between the IL9 marker on chromosome 5q31-33 and atopy. In order to further investigate the role of susceptibility genes on 5q31-33 in the development of atopy and asthma we have studied 240 UK families comprising 131 families selected at random, 60 multiplex families with affected sib pairs, and 49 single proband nuclear families. Polymorphic markers on 5q31-33 were genotyped and both single and multipoint linkage analysis was undertaken using the BETA program. We have used both affection status and quantitative scores for atopy and asthma for phenotypic variables, combining data into scores for asthma and atopy. The strongest suggestion of linkage using multipoint analysis was centred around D5S410 with a maximum Lod of 1.946 at location 171.3 cM and a standard error of 3.3 for the asthma quantitative score. There was no evidence of linkage with atopy, the atopy quantitative score or total serum IgE.

Asthma↗

PubMed2000

Heterodimerization of mu and delta opioid receptors: A role in opiate synergy.

Opiate analgesics are widely used in the treatment of severe pain. Because of their importance in therapy, different strategies have been considered for making opiates more effective while curbing their liability to be abused. Although most opiates exert their analgesic effects primarily via mu opioid receptors, a number of studies have shown that delta receptor-selective drugs can enhance their potency. The molecular basis for these findings has not been elucidated previously. In the present study, we examined whether heterodimerization of mu and delta receptors could account for the cross-modulation previously observed between these two receptors. We find that co-expression of mu and delta receptors in heterologous cells followed by selective immunoprecipitation results in the isolation of mu-delta heterodimers. Treatment of these cells with extremely low doses of certain delta-selective ligands results in a significant increase in the binding of a mu receptor agonist. Similarly, treatment with mu-selective ligands results in a significant increase in the binding of a delta receptor agonist. This robust increase is also seen in SKNSH cells that endogenously express both mu and delta receptors. Furthermore, we find that a delta receptor antagonist enhances both the potency and efficacy of the mu receptor signaling; likewise a mu antagonist enhances the potency and efficacy of the delta receptor signaling. A combination of agonists (mu and delta receptor selective) also synergistically binds and potentiates signaling by activating the mu-delta heterodimer. Taken together, these studies show that heterodimers exhibit distinct ligand binding and signaling characteristics. These findings have important clinical ramifications and may provide new foundations for more effective therapies.

Analgesics, Opioid↗

PubMed2000

Optimisation of culture medium and conditions for alpha-l-Arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus.

The culture medium for Rhodothermus marinus was optimised on a shake-flask scale by using statistical factorial designs for enhanced production of a highly thermostable alpha-L-arabinofuranosidase (AFase). The medium containing 3.6 g/l birch wood xylan and 8.2 g/l yeast extract yielded a maximum of 110 nkat/ml AFase activity together with 125 nkat/ml xylanase and 65 nkat/ml beta-xylosidase activity. In addition, low levels of beta-mannanase (30 nkat/ml), alpha-galactosidase (0.2 nkat/ml), beta-galactosidase (0.3 nkat/ml), endoglucanase (5 nkat/ml) and beta-glucosidase (30 nkat/ml) were detected in the culture filtrate. Among the various carbon sources tested, birchwood xylan was most effective for the formation of AFase and xylanase activities, followed by oat spelt and beechwood xylans, and xylan-rich lignocelluoses (e.g., starch-free sugar beet pulp and wheat bran). Constitutive levels of enzyme activities were detected when the bacterium was grown on other polysaccharides and low-molecular-weight carbohydrates. A fermentation in a 5-l fermenter (3-l working volume) using the optimised medium yielded 60 nkat/ml AFase associated with 65 nkat/ml xylanase and 35 nkat/ml beta-xylosidase activities. The crude AFase displayed optimal activity between pH 5.5 and 7 and at 85 degrees C. It had half-lives of 8.3 h at 85 degrees C and 17 min at 90 degrees C. It showed high stability between pH 5 and 9 (24 h at 65 degrees C). The combined use of AFase-rich xylanase and mannanase from R. marinus in the prebleaching of softwood kraft pulp gave a brightness increase of 1.8% ISO. To our knowledge, this is the first report on the production of a high AFase activity by an extreme thermophilic bacterium and this enzyme is the most thermostable AFase reported so far.

Journal Article↗

PubMed2000

Opioid receptor endocytosis and activation of MAP kinase pathway.

Opioid receptors, members of the G-protein coupled receptor (GPCR) super family, bind to endogenous opioid peptides or opiate drugs and induce a wide variety of signal transduction processes by inhibiting adenylyl cyclase, modulating cation channels, and activating the mitogen-activated protein (MAP) kinases. Similar to other GPCRs, agonist binding causes rapid internalization and down-regulation of opioid receptors. The interdependence between receptor endocytosis and activation of MAP kinase pathway are increasingly being examined. We have examined these using ligands that exhibit differential extent of endocytosis as well as mutants of mu and delta opioid receptors that are unable to internalize. We find that ligands, including morphine, that do not induce receptor internalization are able to stimulate MAP kinase phosphorylation not only in heterologous cells but also in neuronal cell lines that express endogenous mu and delta receptors. Moreover, mutant receptors that fail to undergo agonist-mediated internalization are able to efficiently phosphorylate MAP kinases. Taken together, these data are consistent with the notion that the activation of MAP kinase pathway is an internalization independent phenomenon in the case of opioid receptors and that GPCR internalization and activation of MAP kinase are governed by complex regulatory mechanisms.

Amino Acid Sequence↗

PubMed2000

Simultaneous production of high activities of thermostable endoglucanase and beta-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus.

The culture-medium composition was optimised, on a shake-flask scale, for simultaneous production of high activities of endoglucanase and beta-glucosidase by Thermoascus aurantiacus using statistical factorial designs. The optimised medium containing 40.2 g l(-1) Solka Floc as the carbon source and 9 g l(-1) soymeal as the organic nitrogen source yielded 1130 nkat ml(-1) endoglucanase and 116 nkat ml(-1) beta-glucosidase activities after 264 h as shake cultures. In addition, good levels of beta-xylanase (3479 nkat ml(-1)) and low levels of filter-paper cellulase, beta-xylosidase, alpha-L-arabinofuranosidase, beta-mannanase, beta-mannosidase, alpha-galactosidase and beta-galactosidase were detected. Batch fermentation in a 5-1 laboratory fermentor using the optimised medium allowed the production of 940 nkat ml(-1) endoglucanase and 102 nkat ml(-1) beta-glucosidase in 192 h. Endoglucanase and beta-glucosidase showed optimum activity at pH 4.5 and pH 5, respectively, and they displayed optimum activity at 75 degrees C. Endoglucanase and beta-glucosidase showed good stability at pH values 4-8 and 4-7, respectively, after a prolonged incubation (48 h at 50 degrees C). Endoglucanase had half-lives of 98 h at 70 degrees C and 4.1 h at 75 degrees C, while beta-glucosidase had half-lives of 23.5 h at 70 degrees C and 1.7 h at 75 degrees C. Alkali-treated bagasse, steam-treated wheat straw, Solka floc and Sigmacell 50 were 66, 48.5, 33.5 and 14.4% hydrolysed by a crude enzyme complex of T. aurantiacus in 50 h.

Ascomycota↗

PubMed2000

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties.

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l(-1) xylan and 10.2 g l(-1) yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml(-1) after 168-h shake culture at 4 degrees C. In addition, very little endoglucanase, beta-mannanase, beta-xylosidase, beta-glucosidase, alpha-L-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10 degrees C in a 5-1 fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0-5.5 and good stability at pH 4-9 (21 h at 4 degrees C). Although the enzyme was maximally active at 45 degrees - 50 degrees C, it appeared very thermolabile, showing a half-life of 78 min at 35 degrees C. At 40 degrees - 50 degrees C, it lost 71%-95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast.

Antarctic Regions↗

PubMed2000

Expression of immediate early gene pip92 during anisomycin-induced cell death is mediated by the JNK- and p38-dependent activation of Elk1.

We report here that immediate early gene pip92 is expressed during anisomycin-induced cell death in fibroblast NIH3T3 cells. To determine the mechanism by which this occurs and to identify downstream signaling pathways, we investigated the induction of the pip92 promoter. The activation of pip92 by anisomycin is mediated by the activation of MAP kinases, such as JNK and p38 kinase, but not ERK. Deletion analysis of the pip92 promoter indicated that pip92 activation occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required for anisomycin-induced pip92 expression. Elk1, which binds to the Ets site, was phosphorylated by the JNK- and p38-dependent pathways and the phosphorylation of Elk1-GAL4 fusion proteins by these pathways was sufficient for the transactivation. Overall, this study suggested that different MAPK pathways are involved in the expression of immediate early gene pip92 by growth factors and environmental stresses.

3T3 Cells↗

PubMed2000

Recycling and resensitization of delta opioid receptors.

Exposure to opioids results in the activation of opioid receptors; this is followed by receptor endocytosis. Previously, we showed that delta opioid receptors undergo rapid agonist-mediated internalization and that mutations in the C-tail result in a substantial loss of agonist-mediated internalization. In this study, we investigated the fate of receptors following rapid internalization. We found that the majority of the wild type receptors recycled back to the surface after acute agonist treatment. The kinetics of internalization and recycling of the receptor were virtually identical to the kinetics of internalization and recycling of the radiolabeled agonist. In contrast, the kinetics of internalization and recycling of a C-tail mutant receptor were substantially altered, suggesting an involvement of the C-tail in the recycling process. It is possible that in addition to agonist-mediated internalization, opioid receptors undergo constitutive, agonist-independent internalization. We directly examined this possibility using an antibody-prebinding assay. The wild type delta opioid receptors exhibited agonist-independent internalization via the clathrin-coated pit pathway. We also examined the role of receptor internalization and recycling in the modulation of its function by quantitating the level of opioid-stimulated phosphorylation of MAP kinase (MAPK) under conditions of receptor internalization and recycling. We found that agonist treatment caused a rapid increase in the level of phosphorylated MAPK that was rapidly desensitized. The removal of the agonist, which results in receptor recycling, led to the resensitization of the receptor, as evidenced by the agonist's ability to reinduce MAPK phosphorylation. Mutant receptors that underwent rapid recycling exhibited enhanced resensitization, suggesting a role for receptor recycling in the resensitization process. Taken together, these results indicate that agonist-mediated internalization and recycling modulate opioid receptor function and that the receptor C-tail plays an important role in both processes.

Amino Acid Sequence↗

PubMed2000

Acute ethanol treatment modulates delta opioid receptors in N18TG2 cells.

BACKGROUND: The in vitro adaptive responses of delta opiate receptors (DOR) to chronic ethanol treatment have been well documented. The acute effects of ethanol on these receptors are not well characterized beyond its effect on ligand binding. The aim of this study was to evaluate the acute effects of clinically relevant concentrations of ethanol (50-200 mm) on the saturation binding kinetics, receptor/ligand internalization, and agonist stimulation of G-protein coupling in N18TG2 cells expressing the Flag epitope-tagged mouse DOR. METHODS: Confocal microscopy was used to localize Flag epitope-tagged DOR in N18TG2 cells. Saturation binding assays at 4 degrees C and 37 degrees C were conducted in the absence or presence of ethanol on cells not pretreated or pretreated with ethanol for 30 min at 37 degrees C. Highly specific delta agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), was used in these studies. The effect of ethanol on agonist stimulation of G-protein coupling was examined using [35S]GTPgammaS (guanosine-5'-O-(3-thio)triphosphate) binding to membranes. Agonist-mediated receptor internalization was examined using flow cytometry of cells labeled with the antiserum directed against the Flag epitope, and the ligand internalization was examined using [3H]DPDPE. RESULTS: Ethanol decreased the binding of the agonist [3H]DPDPE, and not the antagonist [3H]diprenorphine, in a dose-dependent manner. These effects were temperature-dependent. Ethanol reversibly inhibited agonist stimulation of [35S]GTPgammaS binding. In non-pretreated cells, ethanol decreased the rate of receptor/ligand internalization, but this effect was not seen in ethanol pretreated cells. Taken together, these results suggest that pretreatment of N18TG2 cells with ethanol induces compensatory mechanisms that allow the receptor to function efficiently in its presence. CONCLUSION: Acute ethanol decreased the binding, agonist-mediated functional coupling and receptor/ligand internalization in N18TG2 cells expressing epitope-tagged DOR. In these cells, 30-min pretreatment with ethanol was sufficient to reverse these effects.

Anesthetics↗

PubMed2000

Cysticercosis in epileptic patients of Mulungu do Morro Northeastern Brazil.

With the aim to study the magnitude of infection by the metacestode of Taenia solium in a population of epileptic patients in the arid region of Bahia, Northeastern Brazil, we examined 200 consecutive cases who attended an ambulatory clinic in the disctrict of Mulungu do Morro. Sixty-six of the patients had a diagnosis of epilepsy. From them 10 (15.2%) presented antibodies against a specific fraction of antigens in Western blot, and 4 (6.0%) had circulating parasite products, as tested by capture ELISA. Only 1 case was positive for antibodies and antigens. We found that the frequency of seropositivity was related to the time without epileptic seizure. We conclude that cysticercosis is endemic in the region of Mulungu do Morro and that it is related to a benign form of epilepsy.

Adolescent↗

PubMed1999

Monoclonal antibody to the delta opioid receptor acts as an agonist in dual regulation of adenylate cyclase in NG108-15 cells.

Monoclonal antibodies generated against multiple antigenic peptides of the N-terminal sequence (3LVPSARAELQSSPLV17) of the cloned delta opioid receptor immunoprecipitated a 58 kDa protein from CHAPS-solubilized NG108-15 membranes. The immunoprecipitates bound [3H]DPDPE--but not [3H]DAMGO--with a Kd of 6.4 nM and a Bmax of 75 pM. Western blot analysis revealed a distinct band of 58 kDa. The antibodies inhibited basal and PGE1-stimulated cAMP levels, and mimicked the effect of agonists manifest in a compensatory increase in cAMP formation. The antibody will be potentially useful in the analysis of functional epitopes on the delta opioid receptor.

Adenylyl Cyclases↗

PubMed1999

Human T lymphotropic virus type I (HTLV-I) infection in neurological patients in Salvador, Bahia, Brazil.

HTLV-I infection represents a major health concern in endemic areas throughout the world, such as Salvador, the main city of Bahia State, with socio-demographic characteristics similar to sub-Saharan African cities, located in the Northeast of Brazil. In order to provide an estimate of the frequency distribution, and range of neurological manifestations potentially related to HTLV-I infection in this city, we conducted a cross-sectional clinical-epidemiological study to determine the prevalence of this infection in patients with neurological diseases. Patients exhibiting vascular diseases, tumoral diseases or trauma were excluded. Over a period of 16 months, we studied 322 consecutive patients with chronic neurological diseases, who attended the neurological clinics of two major hospitals in Salvador. Overall, the prevalence of HTLV-I infection among the patients was 20.9% (67/320). However, the prevalence among the 104 patients with chronic myelopathy was 50.0% (52/104). It was observed that the major prevalence of HTLV-I was between the ages of 40 and 60 years with a female predominance. Our data indicate that, in Salvador city, HTLV-I is associated with chronic myelopathies or myeloneuropathies, which seem to be the only neurological diseases associated with HTLV-I.

Adolescent↗

PubMed1999

Hardy-Weinberg quality control.

An efficient test of deviation from Hardy-Weinberg frequencies with one degree of freedom was applied to 44 marker loci in a genome scan, and 7 loci had a significant excess of apparent homozygotes (chi2 (1) > 6) suggestive of typing error. In this example evidence for linkage did not increase when outliers were censored. Statistical quality control is an essential part of genotyping, and the effect of mistyping and map error should be considered in evaluating any genome scan.

Asthma↗