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Biomedical subjects

S López

Publications and source records attributed to S López.

At least 91 records · Page 5Linked to original sources
PubMed1996

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.

Amino Acid Sequence↗
PubMed1996

Fast-muscle-specific DNA-protein interactions occurring in vivo at the human aldolase A M promoter are necessary for correct promoter activity in transgenic mice.

The human aldolase A tissue-specific M promoter (pM) has served as a model system for identifying pathways that lead to fast-muscle-specialized expression. The current study has delimited the sequences necessary and sufficient for fast-muscle-specific expression in transgenic mice to a short 209-bp fragment extending from bp -164 to +45 relative to the pM transcription start site. Genomic footprinting methods showed that in this proximal region, the same elements that bind muscle nuclear proteins in vitro are involved in DNA-protein interactions in intact muscle nuclei of transgenic mice. Furthermore, these experiments provided the first evidence that different DNA-binding activities exist between slow and fast muscles in vivo. Fast-muscle-specific interactions occur at an element named M1 and at a muscle-specific DNase I-hypersensitive site that was previously detected by in vitro methods. The formation of the muscle-specific DNase I-hypersensitive site reflects binding of proteins to a close element, named M2, which contains a binding site for nuclear factors of the NF1 family. Mutational analysis performed with transgenic mice confirmed the importance of the M1 element for high-level fast-muscle-specific pM activity and suggested that the M2/NF1 element is differently required for correct pM expression in distinct fast muscles. In addition, two other protein binding sites, the MEF3 motif and the USF site, seem to act as stage-specific activators and/or as participants in the establishment of an active chromatin configuration at pM.

Animals↗
PubMed1996

Occupational allergy due to spider mites: Tetranychus urticae (Koch) and Panonychus citri (Koch).

BACKGROUND: Allergy to both house dust and storage mites is well established, but information about other species of mites is scant. OBJECTIVE: One hundred and fifty patients directly exposed to an occupational environment were studied to assess whether spider mites (Tetranychidae) caused their allergic symptoms. We also studied a group of 50 patients from an urban environment, who were not occupationally exposed to spider mites, with a strong sensitization to Dermatophagoides pteronyssinus (RAST class 4). METHODS: Case history (including questions about work-related symptoms), skin tests, RAST and conjunctival provocation tests were performed in both groups using Tetranychus urticae and Panonychus citri extracts as allergens. Cross-reactivity between spider mites and D. Pteronyssinus was determined by RAST inhibition. RESULTS: Fifty-four of 150 rural workers were positive to Tetranychidae and in all cases there was an associated sensitization to D. pteronyssinus. All individuals belonging to the urban group were positive to spider mites. RAST inhibition demonstrated a significant cross-reactivity between Tetranychidae and D. pteronyssinus. Five of fifty-four rural workers sensitized to spider mites developed symptoms only when they handled plants or fruits infested with spider mites and they became asymptomatic when exposure ceased. CONCLUSION: In the rural population studied, 36% of workers were found to be sensitized to spider mites and 10% had symptoms associated with occupational exposure. Since specific IgE antibodies to spider mites could not be detected in the absence of the specific IgE antibodies to D. pteronyssinus, and as all the affected workers were RAST positive to D. pteronyssinus, prior sensitization to house dust mites may be a risk factor for occupational allergy to spider mites.

Adult↗
PubMed1996

Synthesis of pyridopyrimidone derivatives and their activity against P. falciparum in vitro.

Pyridopyrimidone derivatives 4a-d and 5a- were synthesized as potential antimalarial agents on the basis of the pharmacological properties of existing quinolone analogues such as ciprofloxacine IC50 39.8 microM. Meanwhile among these new compounds, only the 3-amino-7-methyl-1(H)pyrazolo[3,4-b]-pyrido [1,2: 1'2']pyrimido-4-ona 4b, produces significant antimalarial activity IC50 0.42 microM against P. falciparum in vitro. The remaining compounds were effective as antimalarial agents leading from IC50 1.0 microM to IC50 4.47 microM.

Animals↗
PubMed1995

Identification of two independent neutralization domains on the VP4 trypsin cleavage products VP5* and VP8* of human rotavirus ST3.

The antigenic structure of the VP4 protein of human rotavirus (HRV) strains Wa and ST3 was studied by using a panel of Wa- and ST3-derived VP4-specific neutralizing monoclonal antibodies (NMAbs) and NMAb-resistant variants. The VP4-coding genes from three Wa and three ST3 variants were sequenced. For Wa VP4, one homotypic and one heterotypic neutralization site, at amino acids 458 and 392, respectively, were identified. For ST3 VP4, three neutralization sites were found at amino acids 72, 217, and 385 that are either homotypic or associated with limited cross-reactivity. Cross-neutralization assays using several pairs of NMAbs and resistant variants showed that Wa VP4 has at least one large neutralization domain on its larger trypsin cleavage product, VP5*, consisting of several operationally related epitopes. VP4 of ST3 has at least two neutralization domains, one located on VP5* that is operationally related to the large neutralization domains on VP5* from HRVs Wa and KU, as well as an independent neutralization domain on VP8*, the smaller trypsin cleavage product of VP4.

Antibodies, Monoclonal↗
PubMed1995

Regulation of inducible nitric oxide synthase expression in rat mesangial cells and isolated glomeruli.

The presence of the inducible isoform of nitric oxide synthase (iNOS) in glomerular mesangial cells facilitates the synthesis of nitric oxide (NO) after stimulation with cytokines or lipopolysaccharide (LPS). As the role of NO within the glomerulus may be important in conditions such as glomerulonephritis, we have studied the effect of dexamethasone (DX) and pirrolidine dithiocarbamate (PDTC), an inhibitor of the nuclear transcription factor, NF-kappa B activation on the induced synthesis of NO in rat mesangial cells (RMC). LPS, tumor necrosis factor-alpha (TNF-alpha) and the combination of both were able to induce NO synthesis in a dose-dependent manner as measured with the determination of NO2- levels. Treatment with LPS (10 micrograms/ml) + TNF-alpha (100 ng/ml) for eight hours was the most potent stimulus for iNOS activity. DX (1 microM) had an inhibitory effect on LPS-, TNF-alpha- and LPS + TNF-alpha-induced NO synthesis (51.2, 42.5 and 68% of inhibition, respectively). The inhibitory effect of DX was confirmed using a reporter cell bioassay, whereas cGMP was measured as a reflection of bioactive NO. DX inhibited induced NO synthesis when RMC were exposed to this agent before (16 hr of pretreatment, 75.7% inhibition) or at the same time (8 hr of cotreatment, 61.2% inhibition) as TNF-alpha + LPS but not four hours after the stimuli. Northern blot analysis showed marked blunting of mRNA expression in RMC treated with DX, in concordance with functional studies. Both actinomycin D and cycloheximide significantly inhibited NO synthesis and iNOS mRNA expression. PDTC (100 microM) was able to inhibit the iNOS activity induced by LPS and TNF-alpha independently (56.8 and 49.9% inhibition, respectively), and in combination (79.1% inhibition). PDTC (1 to 100 microM) inhibited LPS + TNF-alpha-induced NO synthesis and iNOS mRNA expression in a concentration-dependent fashion (69 to 86% inhibition of NO synthesis and 50 to 100% inhibition of mRNA expression). Addition of PDTC four hours after exposure to TNF-alpha + LPS was still able to markedly inhibit NO synthesis. The effects of DX and PDTC were also demonstrated in isolated glomeruli, where two different combinations of inductive stimuli for NO synthesis were employed. Our results establish DX and PDTC as useful tools to study the regulation of NO synthesis in the mesangial cell and glomerulus, and suggest that NF-kappa B is involved in the transcriptional regulation of iNOS in RMC.

Amino Acid Oxidoreductases↗
PubMed1995

Heterogeneity of VP4 neutralization epitopes among serotype P1A human rotavirus strains.

We have used serotype-specific VP4 and VP7 neutralizing monoclonal antibodies (Nt-MAbs), as well as subgroup (SG)-specific MAbs, to characterize by enzyme immunoassay rotavirus strains isolated from diarrheic infants in the city of Monterrey, Mexico, from July 1993 to March 1994. Of a total of 465 children studied, 140 were rotavirus positive, including 3 patients infected with non-group A rotaviruses. The SG and VP7 (G) serotype specificities could be determined for 118 (84%) of the 140 rotavirus-positive stool specimens; 4 rotavirus strains were serotype G1 and SGII; 1 strain was serotype G2 and SGI+II; 112 strains were serotype G3 and SGII; 1 strain was serotype G3 and SGI; and none of the strains was serotype G4. Fifty-eight specimens, representing the 13 different group A rotavirus electropherotypes detected, were chosen for VP4 (P) serotyping. Of these, 48 (83%) strains reacted with the P1A serotype-specific Nt-MAb 1A10. None of the strains reacted with the serotype P2-specific Nt-MAbs tested. Not all viruses that reacted with Nt-MAb 1A10 were recognized by Nt-MAbs 2A3 and 2G1, which also recognize P1A strains, indicating heterogeneity of neutralization epitopes among serotype P1A human rotaviruses. This heterogeneity could be relevant for the specificity of the VP4-mediated neutralizing antibody immune response and indicates the need for antigenic characterization, in addition to genomic typing, of the VP4 proteins of circulating human rotavirus field strains.

Antibodies, Viral↗
PubMed1995

Mapping the hemagglutination domain of rotaviruses.

Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein.

Animals↗
PubMed1994

Mapping the subgroup epitopes of rotavirus protein VP6.

VP6, the most abundant protein of rotaviruses, contains epitopes that allow the classification of these viruses into four subgroups (SG), depending on the presence or absence of two epitopes called I and II. The subgroup-specific epitopes are conformational and appear to be present on trimeric but not monomeric VP6. We have identified on VP6 some of the amino acids that determine the reactivity of the subgroup-specific mAbs 255/60 and 631/9. A single amino acid mutation at positions 172 (Met to Ala) or 305 (Asn to Ala) was sufficient to change the subgroup specificity of the human rotavirus Wa VP6 protein from SGII to SGI/II, since either of these mutations allowed the protein to be recognized by the SGI mAb 255/60, while retaining its capacity to interact with the SGII mAb 631/9. In the case of the SGII epitope, the mutation of two contiguous amino acids (Ala305 Asn306 to Asn305 Ala306) in the porcine rotavirus YM VP6 protein (SGI) enabled the protein to be efficiently recognized by the SGII mAb 631/9, while causing the YM VP6 protein to lose its capacity to interact with mAb 255/60. These results suggest that both subgroup Abs interact with an antigenic domain in VP6 that is composed of at least two regions of the protein that, although distant in the linear sequence, might be in close proximity in the structured VP6 trimer.

Amino Acid Sequence↗
PubMed1994

Osmotic pressure, water kinetics and volatile fatty acid absorption in the rumen of sheep sustained by intragastric infusions.

The effects of changing rumen osmotic pressure (OP) upon water kinetics and volatile fatty acid (VFA) absorption in the rumen of sheep were studied in two 4 x 4 Latin square experiments, each using four lambs with a rumen cannula and an abomasal catheter. In both experiments the lambs were sustained by the intragastric infusion of all nutrients (VFA, Ca, P, Mg and a buffer solution into the rumen, and casein, vitamins and trace elements into the abomasum). On experimental days, which were at least 1 week apart, drinking water and the casein infusion were withdrawn, and the ruminal OP was changed and held constant for 9.5 h, by incorporating NaCl at different concentrations in the buffer solution being infused. In Expt 1 the target OP values were 300, 340, 380 and 420 mosmol/kg, and in Expt 2 were 261 (no saline addition), 350, 420 and 490 mosmol/kg. Using soluble non-absorbable markers (PEG in continuous infusion and Cr-EDTA injected in pulse doses) rumen volume, liquid outflow rates, apparent water absorption through the rumen wall and VFA absorption rates were estimated at six sampling times corresponding to the 1.5 h intervals during the last 7.5 h following the change in rumen OP. Liquid outflow rate (F; ml/h) showed a significant and positive linear relationship with the rumen OP (mosmol/kg), resulting in the equation F = 1.24 OP (SE 0.096)-36.5 (SE 36.6) (r2 0.96). Similarly, water absorption rate (W; ml/h) was significantly affected by rumen OP, and this relationship was given by W = 395 (SE 39.9)-1.16 OP (SE 0.105) (r2 0.95), which means that for an OP of 341 mosmol/kg the net movement of water across the rumen wall would be zero, and either a net efflux or a net influx of water would be observed with lower or higher OP respectively. In Expt 2 there was a significant linear effect of OP on rumen volume (P < 0.01), with higher OP being associated with increases in rumen liquid contents of about 10-20%. As rumen OP was increased there was also a decline in the absorption rate of VFA (from 232 mmol VFA/h for OP 350 to 191 mmol/h for OP 490 mosmol/kg), resulting in the accumulation of VFA (especially acetate) in the rumen and a consequent fall in rumen pH. Rumen OP seems to be important in defining water movement across the rumen wall and, hence, partitioning between absorption and outflow.

Animals↗
PubMed1994

Neutralizing antibody immune response in children with primary and secondary rotavirus infections.

We have characterized the neutralizing antibody immune response to six human rotavirus serotypes (G1 to G4, G8, and G9) in Brazilian children with primary and secondary rotavirus infections and correlated the response with the G serotype of the infecting rotavirus strain. Twenty-five children were studied: 17 had a single rotavirus infection, 4 were reinfected once, and 4 experienced three infections. Two of the reinfections were by non-group A rotaviruses. Among the 25 primary infections, we observed homotypic as well as heterotypic responses; the serotype G1 viruses, which accounted for 13 of these infections, induced mostly a homotypic response, while infections by serotype G2 and G4 viruses induced, in addition to the homotypic, a heterotypic response directed primarily to serotype G1. Two of the primary infections induced heterotypic antibodies to 69M, a serotype G8 virus that by RNA electrophoresis analysis was found not to circulate in the population during the time of the study. The specificity of the neutralizing antibody immune response induced by a virus of a given serotype was the same in primary as well as secondary infections. These results indicate that the heterotypic immune response induced in a primary rotavirus infection is an intrinsic property of the virus strain, and although there seem to be general patterns of serotype-specific seroconversion, these may vary from serotype to serotype and from strain to strain within a serotype.

Antibodies, Viral↗
PubMed1993

Immunological characterization of a rotavirus-neutralizing epitope fused to the cholera toxin B subunit.

A highly conserved neutralizing epitope from the surface protein VP4 (amino acids 296-313) of human rotaviruses was genetically fused to the B subunit of cholera toxin (CTB). Synthetic oligodeoxyribonucleotides encoding the VP4 peptide were inserted between the 3' end of the DNA that codes for the leader peptide, and the 5' end of the gene encoding mature CTB. The hybrid protein synthesized in Escherichia coli was found to maintain the ability of CTB to pentamerize, and to adhere to its cell receptor, the GM1 ganglioside. The chimera was efficiently recognized by a monoclonal antibody (mAb) directed at CTB and by a virus-neutralizing mAb against the VP4 peptide. The hybrid polypeptide was shown to induce high titers of serum antibodies (Ab) against CTB and the synthetic VP4 peptide following subcutaneous immunization; paradoxically, however, the Ab obtained did not recognize the virus by an enzyme-linked immunosorbent assay method, nor had detectable neutralizing activity. Potential implications of these results for future design and evaluation of fusion proteins as immunogens are discussed.

Amino Acid Sequence↗
PubMed1993

Interleukin-1 increases 15-hydroxyeicosatetraenoic acid formation in cultured human endothelial cells.

Interleukin 1 (IL-1) induces prostanoid biosynthesis in endothelial cells by promoting cyclooxygenase expression, but little is known about its activity on the biosynthesis of hydroxyeicosatetraenoic acids (HETEs). We studied the effect of human recombinant IL-1 beta on the conversion of arachidonic acid (AA) to 15-HETE, a powerful inhibitor of the biosynthesis of proinflammatory eicosanoids. Cultured human umbilical vein endothelial cells were incubated with or without IL-1 beta prior to the addition of labeled AA. The eicosanoids produced were analyzed by RP-HPLC. Untreated cells produced little amounts of 15-HETE (6 +/- 3 pmol/10(6) cells), but IL-1 beta treated cells increased 15-HETE formation in a dose-dependent manner (4-5-fold at 10 U/ml IL-1). The production of HETEs by IL-1 beta was dependent on protein synthesis. Aspirin inhibited prostanoids, HHT and 11-HETE dose dependently, whereas it was unable to totally inhibit 15-HETE in IL-1 beta-treated cells (50-60%). Nordihydroguaiaretic acid, a general lipoxygenase inhibitor, preferably inhibited 15-HETE formation but also reduced the synthesis of the other eicosanoids in a dose-dependent manner. Indomethacin and ETYA completely suppressed prostanoids, 11-HETE and 15-HETE formation in resting and IL-1 beta-activated cells. Using specific 15-lipoxygenase oligonucleotides and the reverse transcriptase polymerase chain reaction technique, we were unable to evidence detectable 15-lipoxygenase mRNA both in resting and IL-1-activated endothelial cells. Overall, these results provide evidence that in human endothelial cells IL-1 beta increases 15-HETE production. Data strongly suggest that this effect is mediated by cyclooxygenase rather than 15-lipoxygenase activity or expression.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1993

Influence of fatty acid anilides present in toxic oils on the metabolism of exogenous arachidonic acid in cultured human endothelial cells.

The effect of fatty acid anilides (FAA) on the exogenous arachidonic acid (AA) metabolism and toxicity of isolated human endothelial cells was studied to clarify their possible role in the etiology of toxic oil syndrome. Confluent cells were incubated with and without linoleic acid anilide (LAA), oleic acid anilide (OAA) and two unrelated samples for 2-24 h prior to the addition of [l-14C]AA alone or with calcium ionophore A-23187. The eicosanoids produced were analyzed by RP-HPLC. A dual stimulatory and inhibitory effect on the conversion of exogenous AA as a function of preincubation time with anilides (100 and 1000 microM) was observed. Treated cells significantly increased (1-3-fold) the production of the main cyclooxygenase-derived prostanoids (6-keto-PGF1 alpha and PGF2 alpha) formed by these cells, with a maximum stimulatory effect after 2-3 h, only when AA was used alone. However, afterwards a time- and dose-dependent decrease in prostanoid formation was observed with LAA (P < 0.05 at 24 h), either in the absence or presence of ionophore A-23187 in the incubation mixture. This inhibitory effect on cyclooxygenase was not observed with OAA, which still stimulate after 24 h of treatment. The changes in prostanoid synthesis were not followed with a parallel release in the lactate dehydrogenase activity in the medium (except with unrelated samples). Moreover, anilide treatment increased the appearance of cytosolic lipid droplets or vacuoles after 2 and 5 h of contact with LAA and OAA, respectively. From these results, it was suggested that anilides impair prostanoid synthesis in endothelial cells; their stimulatory effect could be explained by an unspecific effect on cell membrane, not related to cell toxicity and the inhibitory effect by an inhibition of the cyclooxygenase activity. These observations further contribute to our understanding of the possible role of anilides in the etiology of the toxic oil syndrome.

Anilides↗
PubMed1993

Patients' beliefs about their lack of pain control in primary fibromyalgia syndrome.

This study had two aims, first, to determine the expectancies of control over pain experience ('pain locus of control') of patients with primary fibromyalgia syndrome (PFS) and to compare them with other chronic rheumatic diseases. Second, to analyse the relationships between health status and locus of control. We applied the Multidimensional Health Locus of Control-Pain and the Arthritis Impact Measurement Scales (AIMS), by interviewing 137 out patients (32 PFS, 32 RA, 20 SLE, 22 AS and 31 OA). Data were analysed by ANOVA and partial correlation tests. PFS patients believed that their symptoms depended on uncontrollable events and that they could not influence their disease by themselves. PFS patients were the most disabled on the 'Affect' (P < or = 0.001) and 'Symptom' factors (P < or = 0.01). In the PFS group, patients who showed a 'Fate' locus of control orientation reported more disability on 'Affect' and 'Social Interaction' AIMS factors.

Adult↗
PubMed1993

Sequence analysis of rotavirus YM VP6 and NS28 proteins.

We have determined the nucleotide sequence of genes 6 and 10 of porcine rotavirus YM. When the amino acid sequences of VP6 and NS28, the protein products of genes 6 and 10 respectively, were compared with other published sequences it was evident that the proteins of human rotavirus Wa have the highest degree of identity with rotavirus YM. This is in contrast with the observation that when other proteins of these two strains have been compared they have been found to be among the most distantly related pairs of rotavirus strains. This observation is in accordance with the proposed receptor-ligand interaction between NS28 and VP6 during virus morphogenesis, and suggests a specificity in the interaction between these two proteins. In addition, when rotavirus YM VP6, which belongs to subgroup I, was compared with the VP6 proteins of rotavirus strains having different subgroup specificities, it was found to be more closely related to subgroup II rather than subgroup I proteins. This finding allowed us to identify five potential amino acids on VP6 that may contribute to determining the subgroup antigens.

Amino Acid Sequence↗