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Biomedical subjects

G Romero

Publications and source records attributed to G Romero.

At least 91 records · Page 5Linked to original sources
PubMed1988

Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes.

The effects of pertussis toxin (PT) treatment on insulin-stimulated myristoyl-diacylglycerol (DAG) generation, hexose transport, and thymidine incorporation were studied in differentiated BC3H-1 myocytes. Insulin treatment caused a biphasic increase in myristoyl-DAG production which was abolished in myocytes treated with PT. There was no effect of PT treatment on basal (nonstimulated) myristoyl-DAG production. Insulin-stimulated hydrolysis of a membrane phosphatidylinositol glycan was blocked by PT treatment. ADP-ribosylation of BC3H-1 plasma membranes with [32P]NAD revealed a 40-kDa protein as the major PT substrate in vivo and in vitro. The time course and dose dependence of the effects of PT on diacylglycerol generation correlated with the in vivo ADP-ribosylation of the 40-kDa substrate. Pertussis toxin treatment resulted in a 71% attenuation of insulin-stimulated hexose uptake without effect on either basal or phorbol ester-stimulated uptake. The stimulatory effects of insulin and fetal calf serum on [3H]thymidine incorporation into quiescent myocytes were attenuated by 61 and 59%, respectively, when PT was added coincidently with the growth factors. Nonstimulated and EGF-stimulated [3H]thymidine incorporation was unaffected by PT treatment. These data suggest that a PT-sensitive G protein is involved in the cellular signaling mechanisms of insulin.

Adenosine Diphosphate Ribose↗
PubMed1988

Phosphatidylinositol-glycan anchors of membrane proteins: potential precursors of insulin mediators.

BC3H1 myocytes release membrane-bound alkaline phosphatase to the incubation medium upon stimulation with insulin, following a time course that is consistent with the generation of dimyristoylglycerol and the appearance of a putative insulin mediator in the extracellular medium. The use of specific blocking agents shows, however, that alkaline phosphatase release and dimyristoylglycerol production are independent processes and that the blockade of either event inhibits the production of insulin mediator. These experiments suggest a new model of insulin action.

Alkaline Phosphatase↗
PubMed1988

The use of nonradioactive DNA probes for the characterization of Leishmania isolates from Peru.

We describe conditions for dot blot DNA hybridization studies using biotinylated kDNA probes from Leishmania. The sensitivity and specificity attained with biotinylated or 32P-labeled probes were equivalent. The lower level of detection obtained was 100 parasites that were blotted on nitrocellulose paper and then treated with Proteinase K. Studies were performed with 112 Leishmania isolates from Andean (uta) and sylvatic mucocutaneous (espundia) patients and all were determined to belong to the Leishmania braziliensis complex.

Animals↗
PubMed1987

The activation of porcine pancreatic phospholipase A2 by dipalmitoylphosphatidylcholine large unilamellar vesicles. Analysis of the state of aggregation of the activated enzyme.

Previous work from this laboratory and others has shown that the hydrolysis of pure dipalmitoylphosphatidylcholine (DPPC) liposomes by porcine pancreatic phospholipase A2 in the vicinity of the gel-to-liquid crystal phase transition is characterized by a slow initial phase followed by an apparent burst of activity. In this article we report a detailed quantitative analysis of the early time course of the hydrolysis of dipalmitoylphosphatidylcholine large unilamellar vesicles at 38 degrees C. Several kinetic models to quantitatively describe the data were considered. The most conservative model consistent with the kinetic data is one in which the enzyme initially binds the bilayer and becomes activated via a process that requires the formation of protein dimers on the surface of the membrane. The relevant kinetic parameters of the model are reported.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1986

Hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by porcine pancreatic phospholipase A2.

The hydrolysis of small unilamellar vesicles made of dipalmitoylphosphatidylcoline by pancreatic phospholipase A2 has been studied under various conditions of temperature and enzyme and substrate concentration using the following three different experimental protocols. When the enzyme was added to the substrate vesicles after being separately adjusted to the temperature of the experiments hydrolysis occurred instantaneously only in the temperature range where the lipid is known to exist in its gel phase, while above the transition range no hydrolysis occurred. Within the transition range, the time course of hydrolysis was characterized by initial very slow rate of hydrolysis (latency phase) followed by an abrupt increase in the rate after a time tau, which is a complex function of temperature and enzyme to substrate ratio. When an enzyme-substrate mixture was first preincubated below Tm and then temperature jumped to a temperature above or within the transition range, the latency phase was markedly shortened. When the temperature jump was to the transition range, this effect is observed even if Ca2+ is absent in the preincubation mixture. However, instantaneous hydrolysis was observed upon temperature jumping the mixture to a temperature high above Tm only if Ca2+ was present in the preincubation medium. In temperature-scanning experiments, hydrolysis was followed while changing the temperature of the enzyme-substrate mixture continuously. Heating an enzyme-substrate mixture from room temperature resulted in an abrupt onset of hydrolysis when the transition range was approached. These results lead us to conclude that two distinctly different steps precede rapid hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by pancreatic phospholipase A2: a Ca2+-independent binding of the enzyme to the substrate vesicles, which for chemically pure bilayers occurs best in the gel phase. This step is followed by a Ca2+-dependent activation of the initially formed enzyme-substrate complex. The latter step only occurs under conditions where the bilayer possesses packing irregularities and probably involves a reorganization of the enzyme-substrate complex. At least one of these two steps appears to involve enzyme-enzyme interaction.

Animals↗
PubMed1986

Hydrolysis of dipalmitoylphosphatidylcholine large unilamellar vesicles by porcine pancreatic phospholipase A2.

The interaction between dipalmitoylphosphatidylcholine large unilamellar vesicles and porcine pancreatic phospholipase A2 has been studied under a variety of conditions. It was found that the presence of large unilamellar vesicles inhibits the hydrolysis of small unilamellar vesicles at room temperature, and reaction calorimetric experiments showed that protein-lipid interactions in the absence of Ca2+ occur in the gel state with a stoichiometry of about 40 phospho-lipid molecules/protein-binding site. However, hydrolysis can be induced in the gel state under conditions of osmotic shock. On the other hand, hydrolysis is usually observed within the lipid transition temperature range, but then it occurs only after a latency phase during which the hydrolysis is very slow. The duration of this latency phase reaches a minimum near the phase transition temperature. However, if the enzyme-substrate mixture is heated from low temperatures (continuously or by a temperature jump) to a temperature within the phase transition region, hydrolysis occurs instantaneously. These results are in accordance with the conclusions of the preceding paper (Menashe, M., Romero, G., Biltonen, R. L., and Lichtenberg, D. (1986) J. Biol. Chem. 261, 5328-5333) that effective binding of the enzyme to lipid vesicles occurs relatively rapidly in the gel state and that activation of the enzyme-substrate complex requires the existence of structural irregularities in the lipid bilayer. Although hydrolysis products may have a pronounced effect on the time course of the reaction in the transition range, instantaneous hydrolysis can be induced in the phase transition region in the absence of reaction products by appropriate manipulation of the experimental conditions during which no reaction products are produced. Thus reaction products are not essential for activation of porcine pancreatic phospholipase A2. Furthermore, it is shown that the fraction of lipid hydrolyzed during the latency period is a function of the initial substrate concentration in a manner inconsistent with the proposition that the accumulation of a constant critical fraction of reaction products is the basis for activation. Comparison of the results of this study with those of the preceding paper strongly support the previously proposed reaction scheme.

Animals↗
PubMed1985

Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA.

The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.

Guanine Nucleotides↗
PubMed1982

Comparison of oxygen kinetics in young and old subjects.

Five older men (aged 60-69 yr) and five young men (aged 21-29 yr) with approximately equal levels of age-corrected VO2 max were compared with respect to oxygen kinetics at equal absolute workloads (100 watts) and at equal relative workloads (45% VO2 max) on a cycle ergometer. At 45% VO2 max, half times for VO2 response to instantaneous transition from unloaded pedalling were 30.0 s and 27.4 s for old and young respectively (t = 0.260, p less than 0.80). No significant differences were found in the VE response and by inference none existed in O2 extraction. Mean half times for heart rate responses at a workload of 100 W were 24.2 s and 20.6 s for old and young groups respectively (t = 0.722, p less than 0.49). Mechanical efficiency estimated from steady state data at 100 W was 19.8% and 20.5% for old and young groups respectively (t = 0.574). The close similarity in responses to submaximal work in old and young subjects of equivalent fitness suggests caution in the interpretation of agewise decrements observed in physiological variables which may be sensitive to physical fitness status.

Age Factors↗