Biomedical subjects

M Villalba

Publications and source records attributed to M Villalba.

At least 91 records · Page 5Linked to original sources

PubMed1993

DR7 and DQ2 are positively associated with immunoglobulin-E response to the main antigen of olive pollen (Ole e I) in allergic patients.

We have studied the relationship between HLA class II haplotypes and alleles, and the IgE antibody response to a highly purified allergen, Ole e I, in allergic patients. Ole e I, is the major antigen from the pollen of olive tree that grows mainly in the Mediterranean. Genomic DNA typing was performed in 40 unrelated patients with seasonal allergic pollenosis who had specific IgE antibodies against Ole e I, detected by double-antibody radioimmunoassay. HLA-DRB and -DQB loci were analyzed by PCR-SSO and RFLP. Phenotypic frequencies were compared with those of 179 healthy unrelated individuals. Significant increases in the phenotypic frequencies of DR7 (pf = 67.5% vs 31.3% in the control population, pc = 0.0023) and DQ2 (pf = 90.0% vs 48.0%, pc = 0.0003) were found, indicating an association between DRB1*0701/2, DQB1*0201 alleles and the IgE antibody response to Ole e I. This is the first time that the HLA-DQ gene has been associated with a positive allergic response.

Allergens↗

PubMed1992

NMDA-induced increase in [Ca2+]i and 45Ca2+ uptake in acutely dissociated brain cells derived from adult rats.

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-D-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca2+]i measured with fluo3 and 45Ca2+ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 microM) and N-methyl-DL-aspartate (200 microM) increased [Ca2+]i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated 45Ca2+ uptake about 16-10% in the same regions. The increases in [Ca2+]i and 45Ca2+ uptake were inhibited by 40% in the presence of 1 mM MgCl2 and by 90-50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca2+]i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca2+]i increase in adult age.

Adenosine Triphosphate↗

PubMed1991

A new distinct group of 2 S albumins from rapeseed. Amino acid sequence of two low molecular weight napins.

Two napins (nIa and nIb), isolated from Brassica napus (rapeseed) seeds, have been sequenced. The two proteins show the common structural pattern of the 2 S albumins, since they are composed of two disulfide-linked chains of different size, yet they exhibit an atypical low molecular weight (12.5 kDa vs. 14.5 kDa of the major napins). High sequence similarity has been found between these 2 proteins, but only 54% similarity can be estimated from their comparison with the 14.5 kDa major napins. Thus, nIa and nIb are considered representatives of a new distinct group of rapeseed napins since all the previously known napins exhibit 95% sequence similarity. Unexpectedly, the similarity increases when compared with the 2 S proteins from other species.

2S Albumins, Plant↗

PubMed1991

Structural analysis of the small chain of the 2S albumin, napin nIII, from rapeseed. Chemical and spectroscopic evidence of an intramolecular bond formation.

Napin nIII is a 2S albumin from rapeseed (Brassica napus L.) homologous to the major mustard allergen. It is composed of two different polypeptide chains linked by two disulphide bonds. The small chain has been isolated by reverse-phase HPLC after reduction of the native protein and its primary structure elucidated. This 37 residue polypeptide contains only two cysteines, at positions 10 and 23, which show a great tendency to form a non-native intramolecular disulphide bridge. The kinetic analysis of this process was performed by measuring the fluorescence emission of the single tryptophan residue of the molecule since its fluorescence intensity is about 30% decreased during disulphide formation. Small changes on the secondary structure of the polypeptide were measured by circular dichroism. The process is delayed in the presence of the reduced large chain of nIII. However, no dimer formation was detected under the conditions used, either between small chains or between the small and the large chains. Thus, the interchain disulphide formation in napin nIII should be considered as an early step during maturation of this multi-subunit seed protein.

2S Albumins, Plant↗

PubMed1991

Prolactin increases cytosolic free calcium concentration in hepatocytes of lactating rats.

PRL at a physiological concentration (10(-8) M) produced a very rapid and transient increase in 45Ca efflux in freshly isolated hepatocytes, which reached the highest value within 5 min and returned to baseline level after 20 min. PRL-induced 45Ca2+ efflux resulted in a loss of 15% of total cell calcium, which was similar to that found in vasopressin-treated cells. However, in contrast with the PRL effect, 45Ca2+ efflux induced by vasopressin was sustained. We demonstrate by using two different approaches, glycogen phosphorylase-a activation and direct cytosolic calcium concentration [( Ca2+]i) measurements, that PRL elicits a [Ca2+]i increase. The treatment of hepatic cells with PRL caused a 4-fold stimulation in glycogen phosphorylase-alpha activity after 2 min of PRL addition. Direct [Ca2+]i determination in fluo-3-loaded hepatocytes showed a 11% increase after 5 min of PRL addition. Similar data were observed in hepatocytes stimulated either with vasopressin (10(-7) M) or calcium ionophore A23187 (200 nM). The increase in [Ca2+]i promoted by PRL was independent of extracellular calcium or voltage-operated calcium channels. The data demonstrate that calcium is involved in the intracellular signaling of PRL in liver cells and that PRL initiates its action by a Ca2+ mobilization from the intracellular stores.

Animals↗

PubMed1990

Isolation of three allergenic fractions of the major allergen from Olea europea pollen and N-terminal amino acid sequence.

A method to isolate the major allergen from olive pollen (Ole e I) in high yield is described. The allergenic fraction has been separated into 3 subfractions by reverse-phase HPLC. All these fractions were reactive to allergic sera from olive-sensitized patients, giving similar responses. No significant differences were observed between the amino acid compositions of these three proteins. The amino acid sequence of the first 27 amino acid residues from the N-terminal end is given. No homologies have been detected between Ole e I and other known allergens obtained from pollen.

Allergens↗

PubMed1990

Mn2(+)-binding properties of a recombinant protein-tyrosine kinase derived from the human insulin receptor.

The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn2+ in the presence or absence of 5 mM Mg2+. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn2+. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn2+ tightly in the absence or presence of MgATP (Kd greater than 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn2+, but not Mg2+, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in alpha-helical content. The addition of ATP to Mn2(+)-bound protein does not further perturb the spectrum. We conclude that Mn2+ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn2+ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.

Circular Dichroism↗

PubMed1990

Hydrolysis of glycosyl-phosphatidylinositol in response to insulin is reduced in cells bearing kinase-deficient insulin receptors.

A glycosyl-phosphatidylinositol (GPI) has been previously identified that serves as a precursor of the polar head group that mimics and may mediate some of the intracellular actions of insulin. Since many of the biological activities of insulin may depend upon the activity of the insulin receptor kinase, we evaluated the requirement for this activity in insulin-dependent GPI hydrolysis. For the analysis we used stably transfected CHO cell lines, expressing either the wild-type human insulin receptor or a mutant receptor that lacks tyrosine kinase activity (Chou et al., 1987) and a stably transfected CHO cell line, expressing the wild-type human insulin-like growth factor I (IGF-1) receptor (Steele-Perkins et al., 1988). A GPI was identified in both types of transfected cells and in both sets of parental cells by metabolic labeling with [3H]glucosamine or [3H]galactose. The isolated glycolipid was sensitive to hydrolysis by phospholipase C and to deamination by nitrous acid. Insulin induced a time- and dose-dependent hydrolysis of the GPI in the parental line and in the transfected cell types. Cells bearing normal human receptors hydrolyzed up to 70% of their radiolabeled GPI within 2 min of the addition of 0.1 nM insulin, whereas parental cells and cells expressing the mutant receptor hydrolyzed only 20-30% in response to 100 nM insulin. IGF-1 (5-50 nM) had little effect on GPI hydrolysis in these cells as well as in CHO cells expressing the human IGF-1 receptor. It is concluded that insulin-dependent GPI hydrolysis is mediated by the normal but not by a kinase-deficient insulin receptor.

Animals↗

PubMed1990

A comparison of cholecysto- and choledochoenterostomy for obstructing pancreatic cancer.

This report compares the efficacy of cholecystoenterostomy and choledochoenterostomy for relief of biliary obstruction due to pancreatic cancer. From 1976 to 1988, 109 biliary enteric bypass procedures were performed on patients with pancreatic carcinoma considered unresectable at exploration. Sixty-four of these patients underwent cholecystoenteric anastomosis (CCEA) and 45 choledochoenteric anastomosis (CDEA). Thirty-day operative mortality was 6.3 per cent for CCEA and 8.8 per cent for CDEA; the mean length of survival after operation was 7.8 months for CCEA and 8.9 months for CDEA. Of the 64 patients who underwent CCEA, all but two experienced short-term (greater than 60 days) relief of jaundice and pruritus. These patients were found to have an obstructed cystic duct and a cholecystectomy and choledochojejunostomy were performed. Five other patients in the CCEA group had recurrence of jaundice for an overall failure rate of 10.9 per cent. Operative morbidity in this group was 14 per cent. One of the patients in the CDEA group had an early recurrence of jaundice and three others experienced late recurrence, for an overall failure rate of 8.8 per cent. Operative morbidity in this group was 16 per cent. We conclude that these procedures have comparable morbidity and mortality. Although a few patients with cholecystoenteric anastomosis will develop recurrent jaundice, the simplicity of the procedure, the shorter operative time, and the equivalent relief of symptoms make it a useful procedure and one we believe preferable in high-risk patients.

Aged↗

PubMed1989

Another version of the human insulin receptor kinase domain: expression, purification, and characterization.

We have overexpressed another insulin receptor kinase molecule, which consists of residues 941-1343 inclusive of the human insulin receptor, by using the baculo-virus expression vector pVL941. Unlike the two previous preparations of insulin receptor kinase in this expression system, this molecule contains the complete unmodified sequence of the cytoplasmic domain of the human insulin receptor beta subunit. Our construct allows high-level expression of the recombinant protein in cultured Sf9 cells and in cabbage looper (Trichoplusia ni) larvae. An improved purification procedure yields greater than or equal to 95% pure protein in 55% yield from the cells. The specific activity of this purified protein is 3.5-fold greater than from the previously described baculovirus insulin receptor kinase (that included residues 946-1343 from the proreceptor). Like the latter molecule, the insulin receptor kinase molecule reported here sediments as a monomer, and its autophosphorylation occurs by an intramolecular process. Preliminary data about the spectroscopic features of the cytosolic domain of the human insulin receptor are presented.

Animals↗

PubMed1989

Ischemic colitis following abdominal aortic reconstruction for ruptured aneurysm. A 10-year experience.

Between January 1, 1978, and December 31, 1987, a total of 103 patients had operations for ruptured abdominal aortic aneurysms. The average age was 73 years (range, 53 to 91 years). Thirty-two patients died during surgery or in the immediate postoperative period. In 19 of the remaining 71 patients ischemic colitis developed, an incidence of 27 per cent. This report reviews the clinical findings and course of these patients. The average age of patients developing ischemic colitis was 72 years (range, 53 to 90 years), not significantly different from the group as a whole. There was no correlation between the type of vascular reconstruction and the development of ischemic colitis. Eleven patients died and eight survived, for a mortality rate of 58 per cent. The most common clinical finding was diarrhea early in the postoperative period, which was noted in 20 patients. Thirteen of these patients had ischemic colitis confirmed by flexible sigmoidoscopy. Eight (62%) of these 13 patients survived; three were managed nonoperatively and five had colectomy. Six patients presented between postoperative days 9 and 20 with signs of increasing sepsis but with no diarrhea or other significant clinical findings; ischemic colitis was confirmed by sigmoidoscopy in all six patients. All of these patients died of septic complications. Seven patients with early postoperative diarrhea had normal sigmoidoscopic findings. None developed septic complications and five survived; two died of cardiac events.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1989

Effect of SMS201-995 (a long-acting somatostatin analog) on bile-induced acute hemorrhagic pancreatitis in the dog.

Recent reports claim a beneficial role for SMS201-995 (SMS) in pancreatitis. To study the effects of SMS in a canine pancreatitis model, four groups of eight dogs each were subjected to laparotomy; and after cannulation of the dorsal pancreatic duct, a mixture of bile and trypsin was infused to induce pancreatitis. Group I constituted the control group and received no SMS. Group II received SMS intravenously at 5 micrograms/hr beginning 1 hour before the induction of pancreatitis. Group III and Group IV received SMS at the same dose starting at 2 hours and 6 hours, respectively, after the induction of pancreatitis. Infusions were maintained until 24 hours after the induction of pancreatitis. Leukocyte counts, serum lipase and amylase levels were obtained preoperatively and at 24 hours. All dogs were killed at 24 hours and autopsies performed. At autopsy, severity of pancreatitis was graded, based on survival, the presence or absence of pancreatic edema, hemorrhage, and necrosis, as well as the presence and severity of bloody ascites. Only on a dog (Group III) died before the 24-hour period. When SMS was used before the induction of pancreatitis, the pancreatitis seen was less severe (Group I vs Group II, P = .022). No effects were found when using SMS after the induction of pancreatitis (Group III or Group IV vs Group I). The serum lipase, amylase, and leukocyte counts changed significantly in all the dogs (P less than .001) with the onset of pancreatitis, but this difference was not significant between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMed1989

Intra-abdominal compartment syndrome as a complication of ruptured abdominal aortic aneurysm repair.

In four patients with ruptured abdominal aortic aneurysms increased intra-abdominal pressure developed after repair. It was manifested by increased ventilatory pressure, increased central venous pressure, and decreased urinary output associated with massive abdominal distension not due to bleeding. This set of findings constitutes an intra-abdominal compartment syndrome caused by massive interstitial and retroperitoneal swelling. The purpose of this report is to establish criteria for this syndrome and suggest a method of treatment. The syndrome developed within 24 hours; in one patient within 5 hours postoperatively. All four patients received more than 25 liters of fluid resuscitation (electrolyte and blood) during and within 16 hours after operation and had massive abdominal distension. Decompressive laparotomies were performed in the Intensive Care Unit with placement of Marlex (Bard Corp., Billerica, MA) mesh. In two additional patients, at the completion of the aneurysmectomy the abdominal incision was left open with interposition Marlex mesh. Opening the abdominal incision was associated with dramatic improvements in central venous pressure, urinary output, ventilatory pressure, arterial carbon dioxide tension, and oxygenation. The authors conclude that some patients with ruptured abdominal aortic aneurysm do not tolerate the closure of the abdominal wall, as manifested by increased ventilatory pressures, decreased oxygenation, and decreased urinary output. Opening the abdominal wound or delayed closure may reverse the oliguria and improve oxygenation. Recognition and treatment of this condition by opening the abdominal wound or delayed closure may affect outcome in some cases.

Abdomen↗

PubMed1988

Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes.

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.

Alkaline Phosphatase↗

PubMed1988

Inhibition of cyclic AMP-dependent protein kinase by the polar head group of an insulin-sensitive glycophospholipid.

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.

Animals↗

PubMed1987

Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed.

Animals↗

PubMed1986

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.

Animals↗

PubMed1986

The effect of imidazole in endotoxin shock in dogs.

The effect of imidazole, a thromboxane A2 inhibitor, in endotoxin shock in dogs was studied. Thromboxane A2 is a vasoconstrictor and enhances platelet and neutrophil aggregation. The purpose of this study was to examine the hemodynamic modifications of endotoxin shock with imidazole. Thirty dogs were studied, all receiving 1 X 10(8) live Escherichia coli/kg over a 30-minute period. The dogs were divided into three groups of ten dogs each. The first group received no treatment. The second group received a concomitant infusion of imidazole 25 mg/kg per hour. In a third group the imidazole infusion was delayed for 1 hour after the E. coli infusion. Mean arterial pressure (MAP), pulmonary wedge pressure (PWP), central venous pressure (CVP), cardiac output (CO), and systemic vascular resistance (SVR) were measured at 30-minute intervals. Hemoglobin (HGB), white blood count (WBC), a-A gradient (a-A grad), A-V 02 difference (AV02 diff), and fluid requirements were measured at 60-minute intervals. The endotoxin shock response in untreated control dogs was characterized by significant changes over time in MAP and SVR (decreased), and CO and a-A grad (increased). The imidazole group was significantly different, as the MAP and SVR did not decrease and the CO did not increase as in the control group. The control group required significantly more fluids than the imidazole group, while the HGB remained stable suggesting more capillary permeability in the control group. The delayed treatment group was statistically similar to the control animals. These data suggest that imidazole significantly modifies the hemodynamic response to endotoxin shock and may reduce the associated capillary permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗