Major outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages.

Brucella spp. can establish themselves and cause disease in humans and animals. The mechanisms by which Brucella spp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-alpha) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) of Brucella suis 1330 (wild-type [WT] B. suis) on TNF-alpha production. For this purpose, omp25 and omp31 null mutants of B. suis (Deltaomp25 B. suis and Deltaomp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-alpha. We showed that, in contrast to WT B. suis or Deltaomp31 B. suis, Deltaomp25 B. suis induced TNF-alpha production when phagocytosed by human macrophages. The complementation of Deltaomp25 B. suis with WT omp25 (Deltaomp25-omp25 B. suis mutant) significantly reversed this effect: Deltaomp25-omp25 B. suis-infected macrophages secreted significantly less TNF-alpha than did macrophages infected with the Deltaomp25 B. suis mutant. Furthermore, pretreatment of WT B. suis with an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-alpha production during macrophage infection. These observations demonstrated that Omp25 of B. suis is involved in the negative regulation of TNF-alpha production upon infection of human macrophages.

Low molecular weight serine protease inhibitors from insects are proteins with highly conserved sequences.

A low molecular weight protease inhibitor peptide found in ovaries of the desert locust Schistocerca gregaria (SGPI-2), was purified from plasma of the same locust and sequenced. It was named SGCI. It was found active towards chymotrypsin and human leukocyte elastase. SGCI was synthesized using a solid-phase procedure and the sequence of its reactive site for chymotrypsin was determined. Compared with an inhibitor purified earlier from another locust species, the total sequence of SGCI showed 88% identity. In particular, the sequence of the reactive site of these inhibitors was identical. Our search for a closely related peptide in an insect species far removed from locusts, the lepidopteran Spodoptera littoralis, was unfruitful but a different chymotrypsin inhibitor, belonging to the Kazal family, was found whose mass is greater than that of SGCI (20 vs 3.6 kDa). Its N-terminal sequence shares 80% identity with that of a chymotrypsin inhibitor purified earlier from the haemolymph of another lepidopteran. Conservation of the amino acid sequence in the reactive site seems to be an exception among protease inhibitors.

Insect immunity: two proteinase inhibitors from hemolymph of Locusta migratoria.

Two protease inhibitors were isolated from the plasma of Locusta migratoria and sequenced. They were 35 and 36 amino acids long and revealed very little similitude for the protease inhibitors isolated from other arthropods. They inhibit the proPhenoloxidase Phenoloxidase proteolytic activation cascade in hemocyte extracts of the same insect. This inhibiting activity resulted in a lower production of PO, a key enzyme for the defence mechanism in arthropods. Both peptides however showed a strong in vitro inhibiting activity toward alpha-chymotrypsin and elastase, LMCI I inhibits the human leukocyte enzyme while LMCI II mostly the pancreatic one, a difference explainable on the basis of the active site sequence changes.

Purification of a protease inhibitor which controls prophenoloxidase activation in hemolymph of Locusta migratoria (insecta).

A protein which inhibits the prophenoloxidase----phenoloxidase (EC 1.14.18.1) proteolytic activation in hemocyte extracts of Locusta migratoria was isolated from the plasma of the same insect and partially characterized. It shows a molecular weight of 14,000, an inhibiting activity toward the cascade system in the insect hemocytes, which resulted in a lower production of phenoloxidase, a key enzyme for the defence mechanism in arthropods. To identify the specificity of the Locusta inhibitor and consequently the specificity of its target enzyme, inhibitory tests were performed against a number of known serine-proteases. A strong in vitro inhibiting activity toward chymotrypsin and, to a lesser extent, toward human leukocyte elastase was present, while trypsin, Carlsberg subtilisin, human thrombin and pancreatic elastase failed to react. The lack of trypsin inhibition by the isolated inhibitor suggested that the trypsin-catalysed activation of the system in the hemocyte extract takes place under different controls or at an earlier stage of the cascade. The N-terminal sequence of the inhibitor reveals that this molecule is different from the protease inhibitors isolated from other arthropods.

Molecular recognition between serine proteases and new bioactive microproteins with a knotted structure.

Microproteins with proteinase inhibitory activity, 28 to 30 amino acids long, with 3 disulfide bridges have been isolated from Ecballium elaterium seeds. A peptide (EETI II) was isolated and behaved as a powerful trypsin inhibitor (Kd = 10(-11) to 10(-12) M). It was sequenced, chemically synthesized and the 3-D structure determined by 2-D 1H NMR. The information gained in the process enabled us to synthesize modified derivatives with inhibitory activity towards pancreatic elastase, chymotrypsin and human leucocyte elastase (Kd = 10(-7) to 10(-9) respectively). The most striking characteristic that appeared during the synthetic approach was the unfailing ability of the 28 amino acid peptides to refold and correctly close the 3 disulfide bridges, giving in each case an active compound. These disulfide bridges are assembled in a particular knotted structure, shared by few other bioactive peptides and called the 'knottin' structure. Molecular modeling of the peptide and a comparison with the other active molecules with similar topology allowed the synthesis of a chimaeric peptide, bearing 1 active site against a seryl-protease (trypsin), and 1 against a metallo-protease (carboxypeptidase A). The bis-headed peptide was able to inhibit both enzymes separately and concomitantly.

N-terminal domain of pepsin as a model for retroviral dimeric aspartyl protease.

Autolysis of porcine pepsin at pH 4 affords a derivative possessing intrinsic proteolytic activity. This derivative was isolated by alumina pseudo-affinity chromatography and gel-filtration and was found to result from the tight association of two identical molecules, 135 amino acids long, emerging from the N-terminal domain of pepsin. This finding emphasizes a similarity with the only aspartyl-proteases known to act as dimers, the retroviral proteases.

Na+-coupled D-glucose uptake and membrane order of enterocyte brush border membrane vesicles, under the effect of a series of N-phenylcarbamates.

The importance of the hydrophobic effect of exogenous substances and of modifications of membrane order on D-glucose uptake are still poorly defined. Our results show that the concentrative Na+ -coupled D-glucose uptake of rat enterocyte brush border membrane vesicles is inhibited by N-phenylcarbamates increase the membrane order. However, since the concentrations required for membrane order increase are much greater than those active on D-glucose uptake, the effects on lipid order cannot be responsible for the inhibition of D-glucose uptake. Measurements of D-glucose uptake under conditions of Na+ equilibrium show that these carbamates do not act directly on the carrier but indirectly by favouring the dissipation of the Na+ gradient.

[Nutritional status of the rat in refeeding following massive mobilization of DDT by fasting]. / Etat nutritionnel du rat au cours d'une réalimentation faisant suite à une mobilisation massive du DDT par le jeûne.

Rats were subjected to fasting-induced mobilization of a quantity of DDT equivalent to 45% of the LD50. They were then fed for 1 week for a nutritional and toxicological study. Mobilization of DDT by starvation had no apparent effect on the activity of the various enzymes or the weight of the organs measured at the end of the refeeding period. The animals which had received DDT pretreatment showed higher protein efficiency than the control animals. Only the level of body lipids was lower in the animals pretreated with DDT, although the quantities of lipids stored during the study period were identical in both groups of animals. Overall, the results show that the nutritional state of the two groups of animals was very similar at the end of the refeeding period.

Thermodynamic interpretation of effects of alcohols on membrane lipid fluidity.

The effect of a series of amphiphilic compounds, the first eight n-aliphatic alcohols, on the fluidity of rat enterocyte brush border was determined by ESR using 5-doxyl stearic acid as a lipid spin probe. Packing order variations are compared to the relative hydrophobic effect of the alcohols. The concentrations, [Ci]5 of each alcohol that decrease the membrane 2T' value by 5%, vary by a factor of 1500 from methanol to octanol. From [Ci]5, the membrane concentrations Cm and the variation of free energy delta F degree due to the incorporation of the alcohols in the lipids, were calculated. These calculations were performed taking into account the respective volumes of the aqueous phase and the membrane lipids. Cm is of the order of 0.18 mol/kg for the odd chain length alcohols and of 0.27 mol/kg for the even alcohols. The value of delta F degree in cal/mol -CH2- is -687 cal on average for the eight alcohols. This work shows that for all the alcohols, the concentrations at equilibrium in the membrane and in the aqueous phase are respectively in agreement with Meyer and Overton's theory and with the gradient of free energy which constitutes the most general index of interaction of lipophilic substances with membranes.

Sensitivity of Na+-coupled D-glucose uptake, Mg2+-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols.

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg2+-ATPase or sucrase activity and Na+-coupled D-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the D-glucose uptake were therefore measured under the action of n-aliphatic alcohols and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the D-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations (C) of the eight alcohols inhibiting the D-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1/C and log P, P being octanol/water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg2+-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg2+-ATPase and sucrase, but they modulate the Na+-coupled D-glucose uptake.

[Deperoxidation of hydroperoxylinoleic acid by the intestinal mucosa]. / Déperoxydation de l'acide hydroperoxylinoléique par la muqueuse intestinale.

In the presence of glutathione, a homogenate of rat intestinal mucosa transforms linoleic acid hydroperoxide (LOOH) into the corresponding alcohol (LOH). The Km of the enzyme involved (a glutathione peroxidase) is 5.7 x 10(-6) M. The specific activity, measured as generated LOH, was found to be 0.058 mumol/mg protein/min, six times lower than that of the liver. A mitochondrial supernatant of the mucosal homogenate had 1.5 times the activity of the initial homogenate. The reduction of 1 mol LOOH requires 2 mol glutathione. Besides this enzymatic deperoxidation, 5% of the LOOH was decomposed in both the intestinal mucosa and liver by a non-enzymatic pathway, probably involving the Fe3+ of the haemoproteins.

[Effect of preincubation of hepatic microsomes in the presence of chloramphenicol on microsomal lipid peroxidation]. / Effects d'une préincubation anaérobie de microsomes hépatiques on présence de chloramphénicol sur la péroxydation des lipides microsomaux.

The influence of anaerobic preincubation of hepatic microsomes in the presence of chloramphenicol, in the view of reduction of nitro-group has been studied on later peroxidative degradation and activity of several enzymatic systems on lipids of pretreated microsomes. An inhibition of malonaldehyde production and conjugated dienes occurs in these conditions. Activity of methylaniline N-demethylase and Neotetrazolium reductase were not affected. Possible relationships of chloramphenicol and its reduced metabolites on lipoperoxidation are discussed.

Evaluation of the toxic risk of DDT in the rat: during accumulation.

An investigation was undertaken on the accumulation of DDT and its metabolites in the rat. Rats received 14.5 mg DDT/kg b.w. every day for 52 days. Growth, food intake, body composition, and the activities of various enzymes were little affected. However, the level of total lipids fell 30% and the weight of the liver rose 20% due to cellular hypertrophy induced by the DDT. The quantity of DDT and its metabolites found in the carcass was 24 mg/rat i.e. three times that found in rats dead after a single dose of 200 mg/kg. Liver and brain contained 130 micrograms/rat and 10 micrograms/rat, respectively i.e. five times lower than those found in the rats which died from an acute dose of DDT. In the carcass, p,p' DDT accumulates more than p,p' DDE or p,p' DDD; the latter is preponderant in the liver.