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Biomedical subjects

M C Ruiz

Publications and source records attributed to M C Ruiz.

At least 37 records · Page 2Linked to original sources
PubMed1997

Productive penetration of rotavirus in cultured cells induces coentry of the translation inhibitor alpha-sarcin.

Internalization of rotavirus in MA104 cells was found to induce coentry of alpha-sarcin, a toxin that inhibits translation in cell-free systems and to which cells are normally impermeable. Entry of the toxin, measured by inhibition of protein synthesis at early times after infection, correlated with virus penetration leading to expression of infectivity, since toxin entry (1) was induced only by trypsin-treated triple-layered virions, to a degree dependent on the toxin and the virus concentration; (2) correlated with the degree of permissivity of different cell lines to rotavirus infection; (3) was inhibited to a similar extent as infectivity by treatment of cells with neuraminidase; and (4) was inhibited by pre- or postadsorption incubation of the virus with neutralizing monoclonal antibodies to VP7 and VP4 (VP8*). Neither the virus infectivity nor the toxin coentry was significantly affected by treatment of cells with bafilomycin A1, an inhibitor of the vacuolar proton ATPase, indicating that both events are independent of the endosomal acid pH. Virus-like particles (VLP), composed of rotavirus proteins 2/6/7/4, but not 2/6/7 or 2/6, were able to induce toxin entry as efficiently as virions. Use of genetically modified VLP in combination with the toxin coentry assay, which measures entry through a productive pathway, should allow identification of the regions of the outer capsid proteins essential for rotavirus penetration.

Animals↗
PubMed1997

Cell lines susceptible to infection are permeabilized by cleaved and solubilized outer layer proteins of rotavirus.

It has previously been shown that trypsinized triple-layered particles of rotavirus induce destabilization of liposomes and membrane vesicles in the absence of Ca2+, a condition which leads to solubilization of the outer capsid proteins of the virus. In this work, we have studied the relationship between outer capsid solubilization and permeabilization of membrane vesicles, monitoring particle and vesicle size simultaneously by changes in light scattering. Permeabilization of intact cells induced by solubilized outer capsid proteins was monitored by following the rate of entry of ethidium bromide into the cells. Solubilized outer capsid proteins separated from double-layered particles induced vesicle permeabilization. Solubilization of the outer capsid preceded and was required for vesicle or cell permeabilization. Membrane damage induced by rotaviral outer proteins was not repaired upon addition of 1 mM Ca2+ to the medium. Rotavirus infection and cell permeabilization were correlated in six different cell lines tested. This phenomenon might be related to the mechanism of virus entry into the cell. We propose a new model for rotavirus internalization based on the permeabilizing ability of outer capsid proteins and the cycling of trapped calcium in the endosomal compartment.

Capsid↗
PubMed1997

Solubilized and cleaved VP7, the outer glycoprotein of rotavirus, induces permeabilization of cell membrane vesicles.

It has been previously shown that rotavirus triple-layered particles induce permeabilization of liposomes and membrane vesicles. These effects were mediated by one or both of the solubilized outer-capsid proteins, VP4 and VP7. Permeabilization was dependent on trypsin treatment of the viral particles, suggesting that VP4 was involved. To analyse the respective roles of the outer-capsid proteins in this permeabilization process, we have used membrane vesicles loaded with carboxyfluorescein and virus-like particles derived from insect cells co-expressing various sets of capsid proteins. Virus-like particles containing VP2, VP6 and VP7 (VLP2/6/7) are as efficient in permeabilizing vesicles as triple-layered particles. As with double-layered particles, virus-like particles made of VP2 and VP6 had no effect on vesicle permeabilization. Permeabilization of membrane vesicles required trypsinization of the VP7 solubilized from VLP2/6/7. These results show that solubilized and trypsinized VP7 is able to induce membrane permeabilization, independently of the presence of VP4.

Antigens, Viral↗
PubMed1996

Calcium, magnesium, sodium, potassium and iron content of infant formulas and estimated daily intakes.

The calcium, magnesium, sodium, potassium and iron content of a total of 22 different infant formulas marketed in Spain were measured by atomic spectrometry, and the mineral intake of infants fed exclusively with these formulas was estimated. The contents (mg/100kJ) are in the following ranges: Ca, 14-24; Mg, 1.1-2.8; Na, 5.6-9.8; K, 19-35; Fe, 0.02-0.50. These values coincide with those recommended by the Codex and European Society for Paediatric Gastroenterology and Nutrition (ESPGAN), and do not exceed the limits established by the European Union (EU). The mean values and ranges of estimated intakes for each formula type and period of infancy (0-1, 1-2, 2-3, 3-4 and 4-5 months) expressed in mg element/kg body weight are tabulated. The mean Ca, Mg, Na, K and Fe daily intakes of infants (0-5 months) fed with infant formulas meet the recommended values (RDA), except for the iron intake when non-iron supplemented formulas were used.

Calcium↗
PubMed1996

The concentration of Ca2+ that solubilizes outer capsid proteins from rotavirus particles is dependent on the strain.

It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.

Animals↗
PubMed1996

[Microbiological study of vulvovaginitis in premenarcheal girls].

BACKGROUND: A prospective microbiological study of the vaginal swab specimens from premenarcheal girls with clinical diagnosis of vulvovaginitis was done from the 1st of September 1991 to the 31st of August 1994. PATIENTS AND METHODS: Vaginal secretions from premenarcheal girls with clinical findings were examined. Most important pathogenic agents were investigated and if there was an inflammatory reaction in the Gram stain and a heavy growth on culture, other potentially pathogenic agents were considered also. RESULTS: In 70 (28.7%) of the 262 patients a potentially pathogenic microorganism was found: eight cases (3.0%) due to Streptococcus pyogenes, four cases (1.5%) due to Candida albicans. Only in one case Neisseria gonorrhoeae was isolated. Among the uncertain etiologic agents, Haemophilus influenzae was the most frequently isolated (7.6%). In 25 (12.6%) of the 198 patients Enterobius vermicularis ova were visualized. CONCLUSIONS: Streptococcus pyogenes was the most frequently related organism with the vulvovaginitis syndrome among the traditionally established pathogens. The role of Haemophilus influenzae should be considered due to the high prevalence of isolation in this group of patients. We consider that differential diagnosis with Enterobius vermicularis infestation should be done in all cases.

Adolescent↗
PubMed1995

Selective depletion of stored calcium by thapsigargin blocks rotavirus maturation but not the cytopathic effect.

Rotavirus matures inside the endoplasmic reticulum (ER), a site of intracellular calcium storage. Total cell Ca2+ depletion has been shown to impair virus maturation, arresting this process at the membrane-enveloped intermediate form following its budding into the ER. On the other hand, rotavirus infection leads to an increase in the internal Ca2+ concentration ([Ca2+]i) and sequestered Ca2+ pools. We have used thapsigargin, an inhibitor of the Ca(2+)-ATPase of the ER, to release stored Ca2+ and to study its role in rotavirus morphogenesis and cytopathic effect. Thapsigargin (0.1 to 1 microM) released stored Ca2+ from MA-104 cells, as measured by chlorotetracycline fluorescence. The concentration of cytoplasmic Ca2+, measured with fura2, increased in infected cells whether treated or not with thapsigargin. Infectivity was decreased dose dependently by thapsigargin (3 log units at 0.25 to 1 microM). In infected cells treated with thapsigargin, glycosylation of VP7 and NS28 was inhibited. Electron microscopy of infected cells treated with thapsigargin showed normal synthesis of viroplasm. However, only membrane-enveloped, not double-shelled, particles could be observed within the ER. The conformation of VP7 in infected cells treated with thapsigargin appeared to be altered, as suggested by decreased immunofluorescence reactivity with monoclonal antibodies to highly conformation-dependent VP7 epitopes. The progression of cell death in infected cells, as measured by penetration of ethidium bromide, was not affected by thapsigargin. These results indicate that rotavirus maturation depends on a high sequestered [Ca2+], specifically in the ER. Cell death is the result of the accumulation of a viral product and is not related to the production of infective particles. This viral product(s) may be responsible for the increase in [Ca2+]i, which in turn leads to cell death.

Animals↗
PubMed1994

[Effect of suppression of apnea on arterial pressure and plasma catecholamines in normotensive patients with sleep apnea].

BACKGROUND: The association of high blood pressure (HBP) and the sleep apnea syndrome (SPS) and the beneficial effect of SAS treatment on HBP are well known. The direct effect of the continuous nocturnal administration of positive air pressure (CPAP) on blood pressure is not, however, well known. The aim of this study was to evaluate the blood pressure (BP), plasma catecholamines (PC) and urinary derivatives of catecholamines (UDC) in 17 normotensive subjects (4 females; age 49 +/- 11 years) with SAS, prior to and after correction of apnea with CPAP. METHODS: Twenty-four hour outpatient registry of blood pressure (OPRBP) and after nocturnal polysomnography were performed both basal and during CPAP administration for two nights. Urine was collected over these 24 hour period for measurement of UDC. At 7 hours a blood sample was collected for measurement of PC. RESULTS: SAS was corrected by CPAP in all the patients with a reduction in mean BP (24 h: 87 +/- 6 vs 84 +/- 6 mmHg, p < 0.05, diurnal, 90 +/- 6 vs 87 +/- 6 mmHg, p < 0.05, nocturnal, 84 +/- 6 vs 82 +/- 7 mmHg, NS) and the percentage of diastolic BP > 90 mmHg (24 h: 10 +/- 7 mmHg vs 6.5 +/- 6 mmHg, p < 0.01, diurnal, 15 +/- 10 vs 10 +/- 10 mmHg, p < 0.05, nocturnal 5.2 vs 5 vs 3 +/- 4 mmHg, p < 0.05). The plasma catecholamines tended to reduce, although not significantly, without changes of urinary metabolites. CONCLUSIONS: There is a significant decrease in blood pressure with the administration of continuous positive air pressure even in normotensive patients. An early correction of sleep apnea syndrome may reduce the high prevalence of hypertension associated with this syndrome.

Adult↗
PubMed1994

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.

Animals↗
PubMed1993

Disruption of the Kluyveromyces lactis GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake.

In the yeast Saccharomyces cerevisiae the GGS1 gene is essential for growth on glucose or other readily fermentable sugars. GGS1 is the same gene as TPS1 which was identified as encoding a subunit of the trehalose-6-phosphate synthase/phosphatase complex and it is allelic to the fdp1, byp1, glc6 and cif1 mutations. Its precise function in the regulation of sugar catabolism is unknown. We have cloned the GGS1 homologue from the distantly related yeast Kluyveromyces lactis. The KlGGS1 gene is 74% and 79% identical at the nucleotide and amino acid sequence level, respectively, to the S. cerevisiae counterpart. We also compared the sequence with the partly homologous products of the S. cerevisiae genes TPS2 and TSL1 which code for the larger subunits of the trehalose synthase complex and with a TSL1 homologue, TPS3, of unknown function. Multiple alignment of these sequences revealed several particularly well conserved elements. Disruption of GGS1 in K. lactis caused the same pleiotropic phenotype as in S. cerevisiae, i.e. inability to grow on glucose or fructose and strongly reduced trehalose content. We have also studied short-term glucose-induced regulatory effects related to cAMP and cAMP-dependent protein kinase, i.e. the cAMP signal, trehalase activation, trehalose mobilization and inactivation of fructose-1,6-bisphosphatase. These effects occur very rapidly in S. cerevisiae and are absent in the Scggs1 mutant. In K. lactis all these effects were much slower and largely unaffected by the Klggs1 mutation. On the other hand, glucose strongly induced pyruvate decarboxylase and activated the potassium transport system in K. lactis and both effects were absent in the Klggs1 mutant. Addition of glucose to galactose-grown cells of the Klggs1 mutant caused, as in S. cerevisiae, intracellular accumulation of free glucose and of sugar phosphates and a rapid drop of the ATP and inorganic phosphate levels. Glucose transport kinetics were the same for the wild type and the Klggs1 mutant in both derepressed cells and in cells incubated with glucose. We have isolated phenotypic revertants of the Klggs1 mutant for growth on fructose. The suppressors that we characterized had, to different extents, diminished glucose uptake in derepressed cells but cells incubated in glucose showed very different characteristics. The suppressor mutations prevented deregulation of glycolysis in the Klggs1 mutant but not the accumulation of free glucose. The mutants with higher residual uptake activity showed partially restored induction of pyruvate decarboxylase and activation of potassium transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Nonparallel secretion of pepsinogen and acid by gastric oxyntopeptic cells of the toad (Bufo marinus).

Pepsinogen and HCl secretion in the amphibian stomach are performed by a single cell type, the oxyntopeptic cell. These functions were studied in gastric mucosae of toads (Bufo marinus) mounted in Ussing-type chambers. HCl and peptic activity of luminal fluid were measured by titration and proteolysis of albumin, respectively. Distribution of pepsinogen in the gastric mucosa was heterogeneous, activity being highest in the proximal part of the stomach. Zymogen granules in the oxyntopeptic cell were more abundant in the deeper cells of the glands and in the fundus. On stimulation, the granules were released into the lumen of the glands by exocytosis. Histamine, forskolin, or carbachol alone each induced an increase in HCl and pepsinogen secretion. Carbachol after maximal histamine or forskolin stimulation produced an extra increase in both secretions that was greater for pepsinogen response. Similarly, joint addition of carbachol and histamine was more potent than histamine alone for both parameters; however, the effect was greater on pepsinogen release. Pretreatment with cimetidine blocked HCl and pepsinogen responses to carbachol but did not affect responses to forskolin. Addition of omeprazole to forskolin-stimulated mucosae uncoupled the two secretions, inducing a total inhibition of HCl secretion with a slight reduction in pepsinogen secretion. Thus pepsinogen release, similar to HCl secretion, is sensitive to cAMP and Ca(2+)-dependent secretagogues. However, the action of Ca2+ would require the previous elevation of cAMP induced by the different secretagogues. In such a case, the increase in intracellular Ca2+ would result in a nonparallel activation of the two secretions.

Animals↗
PubMed1993

[Comparison of acid and pepsinogen secretion control by oxyntopeptic cell of amphibians].

Pepsinogen and HCl secretion in the amphibian stomach is performed by a single cell type, the oxyntopeptic cell. The distribution of pepsinogen in gastric mucosa of Bufo marinus is heterogeneous and higher concentrations are located in the fundus. Both secretions respond to the same secretagogues. Histamine induces the highest response for the two secretions. Carbachol alone, without the endogenous histamine component, has a small effect. Forskolin and 8Br-cAMP have a similar effect to histamine. The Ca2+ ionophore, A23187, and the diacylglycerol analogue, octanoylacylglycerol (OAG) have a small effect on both secretions as carbachol. Joint addition of histamine and carbachol, or forskolin and carbachol, or of OAG and 8Br-cAMP induce a potentiated response for the two secretions. Quantitative analysis of responses and study of the relationship between them during stimulation with histamine plus carbachol revealed a non parallel and uncoupled pattern of stimulation for acid and pepsinogen secretions.

Animals↗
PubMed1992

Home total parenteral nutrition in a "no-bowel" patient in Spain.

OBJECTIVE: To report a case of a patient with no functional bowel who was receiving home total parenteral nutrition in a country that has had a few professional experiences in this area of therapy. CASE SUMMARY: A woman with a history of scattered colonic polyposis developed a mesenteric tumor that caused intestinal obstruction. Tumor withdrawal required the excision of 1.5 m of jejunum-ileum. Postoperative complications required further surgical intervention and subtotal intestinal resection. Duodenocolic anastomosis was not possible and a high output and permanent fistula remained. DISCUSSION: The complications of home parenteral nutrition addressed in the literature are reviewed. The problems encountered in our patient have been resolved. CONCLUSIONS: After three years of postoperative survival, we believe the quality of life of our patient has increased and the parenteral nutrition team members are much better prepared to manage patients with similar problems.

Adult↗
PubMed1991

Evidence for direct coupling of primary agonist-receptor interaction to the exposure of functional IIb-IIIa complexes in human blood platelets. Results from studies with the antiplatelet compound ajoene.

Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide, is a potent antiplatelet compound isolated from alcoholic extracts of garlic. In vitro, ajoene reversibly inhibits platelet aggregation as well as the release reaction induced by all known agonists. In this paper we show that ajoene has a unique locus of action, that is not shared by any other known antiplatelet compound. For example, ajoene inhibits agonist-induced exposure of fibrinogen receptors, as well as intracellular responses such as activation of protein kinase C and the increase in cytoplasmic free calcium induced by receptor-dependent agonists (collagen, ADP, PAF, low-dose thrombin). On the other hand, with agonists that can by-pass (at least partially) the receptor-transductor-effector sequence, such as high-dose thrombin, PMA, NaF, only the exposure of fibrinogen receptors is blocked by ajoene. Binding of fibrinogen to chymotrypsin-treated platelets is only slightly inhibited by ajoene. The results reported here also show that: (a) ajoene does not act as a calcium chelator, does not impair the initial agonist-receptor interaction and does not influence the basal levels of intracellular inhibitors of platelet activation such as cyclic GMP; (b) the locus of action of ajoene is a yet unknown molecular step that links, in the case of physiological agonists, specific agonist-receptor complexes to the sequence of the signal transduction system on the plasma membrane of platelets. In the case of non-physiological, receptor-independent agonists (PMA, NaF), we can only speculate on the hypothesis that they somehow mimic the effect of the agonist-receptor complexes on the signal transduction system; and (c) the exposure of fibrinogen receptors is not a direct consequence of other intracellular processes. These observations clearly show, for the first time, that the exposure of fibrinogen receptors is a membrane event proximally and obligatorily coupled to the occupancy of other membrane receptors by their agonists without any intervention by the cytoplasmic biochemical processes. Additional results support the involvement of G-proteins in these early events of platelet activation. Furthermore, a role of the beta tau subunits of G-proteins in the exposure of fibrinogen receptors is proposed.

Antibodies, Monoclonal↗
PubMed1991

Effect of rotavirus infection on intracellular calcium homeostasis in cultured cells.

The effect of rotavirus infection on intracellular [Ca2+] was studied in a model system (MA-104 cells). In cells infected at high multiplicity with the OSU strain of rotavirus, production of infectious viruses was maximal at 6 hr postinfection. Cell death, as measured by incorporation of ethidium bromide, started at 6 hr and was complete at 15 hr postinfection. At 4 hr postinfection, intracellular [Ca2+], measured by quin2 fluorescence, was not modified, but Ca2+ permeability was increased. With progression of the infection, intracellular [Ca2+] and Ca2+ pools increased due to the failure of regulatory mechanisms to compensate increased Ca2+ entry. These effects were blocked by cycloheximide added up to 5 hr postinfection, but not by actinomycin D. Reduced extracellular [Ca2+] afforded protection of cell death induced by infection, under conditions at which production of infectious viruses was not affected. The cytopathic effect of rotavirus on host cells appears to be mediated by an increase in intracellular [Ca2+] induced by the synthesis of a viral product. The failure of ionic homeostasis of the enterocyte might be involved in the development of diarrhea.

Animals↗