Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation.

Insulin-induced differentiation of 3T3 L1 cells to adipocytes can be mimicked by the expression of transfected ras oncogenes but not of the tyrosine-kinase oncogenes src and trk. Expression of two different transfected, dominant inhibitory ras mutants resulted in significant inhibition of insulin-induced differentiation, suggesting that endogenous Ras proteins are mediators of insulin signaling in these cells. Exposure of untransfected 3T3 L1 cells to insulin resulted in significant formation of the active Ras.GTP complex, at levels comparable with those resulting from exposure to platelet-derived growth factor. However, whereas exposure of the same cells to platelet-derived growth factor resulted in significant tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP), insulin-treated cells did not show any detectable levels of de novo GAP tyrosine phosphorylation. Interestingly, insulin caused tyrosine phosphorylation of the p62 polypeptide coprecipitated with GAP by anti-GAP antibodies. Insulin-induced activation of cytosolic MAP kinase activity in untransfected 3T3 L1 cells was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with an inducible ras construct. These results confirm that Ras proteins participate in insulin signaling pathways in these mammalian cells and indicate that activation of cytosolic MAP kinases is an early event occurring downstream from Ras activation. However, tyrosine phosphorylation of GAP appears not to be a significant upstream regulatory event in the activation of Ras by insulin.     

Semicontinuous ethanol fermentation of sugar cane blackstrap molasses by pressed yeast

Summary A mathematical model is proposed to explain the influence of the volume fraction of inoculum on the fermentation time and ethanol productivity in semicontinuous ethanol fermentation of sugar cane blackstrap molasses by pressed yeast.Nomenclature a, b, c, d constants, see equation (5) - E_o initial ethanol concentration - E_f final ethanol concentration - K₁, K₂, K₃ constants, see equation (1) - P ethanol productivity - P_c calculated values of P - P_e experimental values of P - r correlation coefficient - S_o initial TRS concentration - S_m TRS concentration of the feeding mash - T fermentation time (average of the experimental values) - T_c calculated value of T - T_e experimental value of T - TRS total reducing sugars calculated as glucose - U_o initial urea concentration - U_m urea concentration of the feeding mash - V reactor working volume - V_i volume of the inoculum - volume fraction of inoculum=V_i/V     

Blood genetic systems in four Amazonian tribes.

Data on 31 genetic systems were obtained for 421 individuals belonging to the Arara, Araweté, Mundurucu, and Jamamadi tribes of northern Brazil. The Jamamadi depart farthest, and the Mundurucu least, from South American Indian averages. These data are analyzed together with those of 24 other Amazonian groups. Genetic distances and corresponding dendrograms indicate a cluster of 14 related tribes living north of the Amazon river. These genetic results show only a modest correlation with linguistic and geographic relationships among these groups.     

Vagal function impairment after exercise training.

The present investigation was undertaken to evaluate the vagal function of trained (T) and sedentary (S) rats by use of different approaches in the same animal. After 13 wk of exercise training (treadmill for 1 h 5 times/wk at 26.8 m/min and 15% grade), T rats had a resting heart rate (HR) slightly but significantly lower than S rats (299 +/- 3 vs. 308 +/- 3 beats/min). T rats had marked reduction of the intrinsic HR (329 +/- 4 vs. 369 +/- 5 beats/min) after blockade by methylatropine and propranolol. They also exhibited depressed vagal and sympathetic tonus. Baroreflex bradycardia (phenylephrine injections) was reduced, bradycardic responses produced by electrical stimulation of the vagus were depressed, and responses to methacholine injection were decreased in T rats. Therefore several evidences of vagal function impairment were observed in T rats. The resting bradycardia after exercise training is more likely to be dependent on alterations of the pacemaker cells, inasmuch as the intrinsic HR was markedly reduced.     

Permeabilization of yeast for in situ determination of α-glucosidase

A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass.     

The plasma membrane of Saccharomyces cerevisiae. Isolation and some properties.

The isolation of Saccharomyces cerevisiae plasma membrane was carried out after hypotonic lysis of yeast protoplasts treated with concanavalin A by two independent methods: a, at low speed centrifugation and b, at high speed centrifugation in a density gradient. Several techniques (electron microscopic, enzymic, tagging, etc.) were used to ascertain the degree of purification of the plasma membranes obtained. The low speed centrifugation technique as compared with the other method gave a higher yield of plasma membranes with a similar degree of purification. Analysis of the yeast plasma membrane of normally growing cells by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed at least 25 polypeptide bands. Twelve glycoprotein bands were also found, and their apparent molecular weights were determined. Treatment of the protoplasts with cycloheximide resulted in a significant decrease in the carbohydrate and protein content of the plasma membrane. The electrophoretic pattern of the plasma membrane of cycloheximide-treated cells showed a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff-positive bands. The isoelectric point of the most abundant proteins was low (pI 4) or lower than expected from previous data. A large part of the mannosyl transferase activity found in the cell (80%) was associated with the internal membranes, the remaining activity (20%) was located in the plasma membrane preparation. Part of the mannosyl transferase activity of the cells is located at the plasma membrane surface. Invertase (an external mannoprotein) is found in both the plasma and internal membranes, and as the specific activity dropped significantly following cycloheximide treatment of the cells, it is suggested that these membranes systems are the structures for the glycosylation of a precursor invertase and its subsequent release into the periplasmic space. Other transferase found in the plasma membrane preparation transfers glucose residues from UDPglucose to a poly(alpha(1 leads to 4) polymer identified as glycogen.     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.