Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.

Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.

The cholesterol-lowering effect of guar gum in rats is not accompanied by an interruption of bile acid cycling.

A viscous hydrocolloid (guar gum, GG; 2.5% of the diet) or a steroid sequestrant (cholestyramine; 0.5% of the diet) was included in semipurified diets containing 0.2% cholesterol to compare the cholesterol-lowering effects of each agent in rats. In the present model, GG significantly lowered plasma cholesterol (-25%), especially in the density < 1.040 kg/L fraction, whereas cholestyramine was less potent. Bile acid fecal excretion significantly increased only in rats fed cholestyramine, similar to the cecal bile acid pool; the biliary bile acid secretion was accelerated by GG, but not their fecal excretion, whereas GG effectively enhanced neutral sterol excretion. As a result, the total steroid balance (+13 micromol/d in the control) was shifted toward negative values in rats fed the GG or cholestyramine diets (-27 or -50 micromol/d, respectively). Both agents induced liver 3-hydroxy-3-methylglutaryl-CoA reductase, but cholestyramine was more potent than GG in this respect. The present data suggest that, at a relative low dose in the diet, GG may be more effective than cholestyramine in lowering plasma cholesterol by impairing cholesterol absorption and by accelerating the small intestine/liver cycling of bile acids, which is interestingly, accompanied by reduction of bile acid concentration in the large intestine.

The cholesterol-lowering effect of guar gum is not the result of a simple diversion of bile acids toward fecal excretion.

The effects of partially hydrolyzed, nonviscous, guar gum (PHGG) on cholesterol metabolism and digestive balance have been compared with those of native guar gum (GUAR) in rats adapted to 0.4% cholesterol diets. Both types of guar gum elicited acidic fermentations in the large intestine, but only GUAR effectively lowered plasma cholesterol (P < 0.001), chiefly in the triglyceride-rich lipoprotein fraction. The biliary bile acid excretion was significantly enhanced in rats fed GUAR (P < 0.05), as well as the intestinal and cecal bile acid pool (P < 0.001). In rats fed GUAR and to a lesser extent in those fed PHGG, the fecal excretion of bile acids and neutral sterol was higher than in controls (P < 0.01). The digestive balance (cholesterol intake-steroid excretion) was positive in control rats (+47 mumol/d), whereas it was negative in rats fed GUAR (-20 mumol/d), which could involve a higher rate of endogenous cholesterol synthesis. In rats fed PHGG, the steroid balance remained slightly positive. Liver 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was very low (22 pmol/min/mg protein), owing to cholesterol supplementation, in control rats or in rats fed PHGG, whereas it was markedly higher (+463%) in rats fed GUAR. In conclusion, even if PHGG does alter some parameters of the enterohepatic cycle of cholesterol and bile acids, its effects are not sufficient to elicit a significant cholesterol-lowering effect. The intestinal (ileal or cecal) reabsorption of bile acids was not reduced, but rather increased, by GUAR; nevertheless the intestinal capacities of reabsorption were overwhelmed by the enlargement of the digestive pool of bile acids. In the present model, induction of HMG-CoA reductase probably takes place in the presence of elevated portal bile acid concentrations.

Studies on the cell-free biosynthesis of beta-lactam antibiotics.

Cell walls of Cephalosporium acremonium mycelia were lysed by enzyme preparations from either Helix pomatia (snail) digestive juice or Cytophaga. The yield of protoplasts depended on the lytic-enzyme preparation and the age of the culture, and it increased after the mycelia were pretreated with dithiothreitol. A cell-free preparation, obtained by osmotic lysis of protoplasts, synthesized labelled penicillin N from L-[14C]valine. Approx. 0.03-0.06% of the amino acid was incorporated into penicillin N. Under conditions of penicillin N synthesis, the broken-protoplast preparation failed to produce significant amounts of cephalosporin C or its precursors, deacetylcephalosporin C and deacetoxycephalosporin C.

RP 55778, a PAF receptor antagonist, prevents and reverses LPS-induced hemoconcentration and TNF release.

Platelet-activating factor (PAF) and tumor necrosis factor (TNF) are present in the plasma of animals injected with endotoxin (LPS). Furthermore, when exogenously administered to animals, PAF and TNF induce similar pathological effects. Thus, in order to explore a possible link between these two factors, the effects of a PAF receptor antagonist, RP 55778, and a glucocorticoid, dexamethasone, were studied on LPS-induced hemoconcentration in rats and on the release of TNF induced by exposing isolated murine macrophages to LPS. RP55778 administered either before or after LPS inhibited these endotoxin effects whereas dexamethasone was effective only when given prior to the LPS challenge. Additionally, in murine macrophages the strong TNF mRNA signal induced by LPS was abolished by RP 55778 and dexamethasone treatment. These results indicate that PAF and TNF can mediate the functional manifestations associated with endotoxemia and only RP 55778 appears to show potential for activity against an already established LPS response.