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Proteins inhibiting pancreatic lipase in vitro have been isolated from wheat germ and wheat bran, with relative molecular mass ranging from 24,400 to 27,500. Inhibition of pancreatic lipase by the wheat germ proteins is related to their ability to interact with the emulsified substrate and to hinder the adsorption of the enzyme on the interface. The extent of inhibition depends on the amount of substrate and is independent of the enzyme concentration. Bile salts forming micelles in the concentration range used are able to partially reverse the inhibition of pancreatic lipase by the wheat germ proteins. The nutritional significance of the data obtained is discussed. [ABSTRACT FROM AUTHOR]

The natural killer group 2 member D (NKG2D) receptor is a C-type lectin-like activating receptor mainly expressed by cytotoxic immune cells including NK, CD8 + T, γδ T and NKT cells and in some pathological conditions by a subset of CD4 + T cells. It binds a variety of ligands (NKG2DL) whose expressions is finely regulated by stress-related conditions. The NKG2DL/NKG2D axis plays a central and complex role in the regulation of immune responses against diverse cellular threats such as oncogene-mediated transformations or infections. We generated a panel of seven highly specific anti-human NKG2D single-domain antibodies targeting various epitopes. These single-domain antibodies were integrated into bivalent and bispecific antibodies using a versatile plug-and-play Fab-like format. Depending on the context, these Fab-like antibodies exhibited activating or inhibitory effects on the immune response mediated by the NKG2DL/NKG2D axis. In solution, the bivalent anti-NKG2D antibodies that compete with NKG2DL potently blocked the activation of NK cells seeded on immobilized MICA, thus constituting antagonizing candidates. Bispecific anti-NKG2DxHER2 antibodies that concomitantly engage HER2 on tumor cells and NKG2D on NK cells elicited cytotoxicity of unstimulated NK in a tumor-specific manner, regardless of their apparent affinities and epitopes. Importantly, the bispecific antibodies that do not compete with ligands binding retained their full cytotoxic activity in the presence of ligands, a valuable property to circumvent immunosuppressive effects induced by soluble ligands in the microenvironment., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The genome of Pseudomonas aeruginosa PAO1 contains three type VI secretion systems (T6SSs) called H1-, H2-, and H3-T6SS. The H1-T6SS secretes three identified toxins that target other bacteria, providing a fitness advantage for P. aeruginosa, and likely contributes to bacterial pathogenesis in chronic infections. However, no specific substrates or defined roles have been described for the two other systems. Here, we demonstrate that the expression of H2-T6SS genes of strain PAO1 is up-regulated during the transition from exponential to stationary phase growth and regulated by the Las and Rhl quorum sensing systems. In addition, we identify two putative Fur boxes in the promoter region and find that H2-T6SS transcription is negatively regulated by iron. We also show that the H2-T6SS system enhances bacterial uptake into HeLa cells (75% decrease in internalization with a H2-T6SS mutant) and into lung epithelial cells through a phosphatidylinositol 3-kinase-dependent pathway that induces Akt activation in the host cell (50% decrease in Akt phosphorylation). Finally, we show that H2-T6SS plays a role in P. aeruginosa virulence in the worm model. Thus, in contrast to H1-T6SS, H2-T6SS modulates interaction with eukaryotic host cells. Together, T6SS can carry out different functions that may be important in establishing chronic P. aeruginosa infections in the human host.

Bacterial pathogenesis often depends on regulatory networks, two-component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine-tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti-anti-sigma factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS-dependent but HptB-independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine-tuned regulation of gene expression.

Bacterial attachment to the substratum involves several cell surface organelles, including various types of pili. The Pseudomonas aeruginosa Tad machine assembles type IVb pili, which are required for adhesion to abiotic surfaces and to eukaryotic cells. Type IVb pili consist of a major subunit, the Flp pilin, processed by the FppA prepilin peptidase. In this study, we investigated the regulatory mechanism of the tad locus. We showed that the flp gene is expressed late in the stationary growth phase in aerobic conditions. We also showed that the tad locus was composed of five independent transcriptional units. We used transcriptional fusions to show that tad gene expression was positively controlled by the PprB response regulator. We subsequently showed that PprB bound to the promoter regions, directly controlling the expression of these genes. We then evaluated the contribution of two genes, tadF and rcpC, to type IVb pilus assembly. The deletion of these two genes had no effect on Flp production, pilus assembly, or Flp-mediated adhesion to abiotic surfaces in our conditions. However, our results suggest that the putative RcpC protein modifies the Flp pilin, thereby promoting Flp-dependent adhesion to eukaryotic cells.

Pseudomonas aeruginosa is an opportunistic pathogen involved in several diseases, including cystic fibrosis and nosocomial infections. Although the behavior of this bacterium at 37 degrees C has been intensively studied, little is known about its capacity to adapt and survive at suboptimal temperatures, such as those encountered in hospitals. In this work, transcriptomic and proteomic analyses were used to identify factors that allow P. aeruginosa to become established at room temperature (close to 25 degrees C) and thus facilitate host infections. Since the virulence of this pathogen is multifactorial and dependent on the extracellular release of toxins and degradative enzymes targeted to the host by several secretory systems, the study focused on genes activated at 25 degrees C, namely, those encoding either components of the secretory machinery or secreted proteins. These observations were enhanced by 2D-PAGE analyses, which showed that the production of effectors from type I and type II secretion systems (respectively, proteases AprA and PrpL) and of a hemolysin co-regulated protein (Hcp) related to the type VI secretion system was specifically stimulated when the growth temperature was lowered from 37 to 25 degrees C. The results provide a fundamental basis for investigating the processes that allow P. aeruginosa to adapt to suboptimal growth temperatures and which thereby promote nosocomial infection.

Although preB cell-receptor (pre-BCR) formation and cell-surface expression is essential for B cell development, pre-BCR generation of signal transduction remains elusive. Here, we report that recombinant pre-BCRs and the surrogate light chain bind specifically to the bone marrow stromal cell galectin-1 (GAL1), an S-type lectin. The surrogate light chain/GAL1 association is a direct protein-protein interaction (K(a) = 2 x 10(6) M(-1)), and the NH(2) extra loop of lambda-like is the major binding element. Pre-BCR binding to stromal cells depends upon GAL1 anchoring to glycosylated counter-receptors, and these complexes completely relocalize to form a synapse at the contact zone between preB and stromal cells. This immune developmental synapse is accompanied by the initiation of intracellular tyrosine kinase activity and signal transduction from the pre-BCR.

The effects of increased dietary n-3 polyunsaturated fatty acids on gastric mucosal lipid metabolism were studied in rats fed for 8 weeks with different combinations of fish and corn oils. Lipid composition, ex vivo prostaglandin E2 (PGE2) production and enzymatic activities involved in phospholipid metabolism and peroxisomal oxidative catabolism of fatty acids and PGE2 were examined. With dietary n-6/n-3 compositional ratios ranging between 75 and 3.3 it was observed that: (i) the arachidonic acid-to-eicosapentaenoic acid ratio (AA/EPA) fell from infinity to 3.1 and 5.1 in phosphatidylcholines (PC) and phosphatidylethanolamines (PE), respectively; (ii) ex vivo production of PGE2 was lowered by a factor of about 2; and (iii) gastric phospholipase A2 activity was enhanced by 32%. With dietary n-6/n-3 ratio lower than 3.3, stimulation of PGE2-CoA oxidase activity was observed whilst the PGE2 level remained constant. These data suggest that the fish oil-induced decrease in ex vivo PGE2 production is more closely related to a decrease in the membrane AA level than to an enhanced oxidative catabolism of PGE2.

In the present study, we have performed experiments to gain some insight into the subcellular localization and biochemical properties of gastric mucosal phospholipase A2. After classical subcellular fractionation of whole glandular stomach mucosa, we found that gastric phospholipase A2 was essentially enriched in the 105,000 x g pellet that contains microsomes and plasma membranes. Except for the cytosol, all the subcellular fractions exhibited similar phospholipase A2 activity (i.e., optimum of pH, calcium dependence, apparent Km and positional specificity). The high-speed pellet was further characterized by ultracentrifugation on a sucrose gradient. Data showed that the sedimentation profile of phospholipase A2 was quite similar to those of plasma membrane markers and more specifically to an apical membrane marker. These results, taken together, showed that a gastric phospholipase A2 is distributed among the various subcellular fractions (as a result of cross-contamination) together with the membrane fraction on which it is associated. It is proposed that this fraction is the apical plasma membrane which would be the main site of phospholipase A2 action for arachidonic acid release. Lysophospholipase showed the same sedimentation profile as phospholipase A2, whereas acyl CoA-lysophosphatidylcholine: acyltransferase mainly sedimented with heavy microsomes. The substrate specificity of the enzyme was assessed by endogenous hydrolysis of gastric mucosal phospholipids. We were able to show that the enzyme acts at nearly the same rate on two major gastric membrane phospholipids, namely phosphatidylcholine and phosphatidylethanolamine.

Fatty acid composition of membrane phospholipids of cultured cardiomyocytes can be modified by the type of polyunsaturated fatty acids (n-3 or n-6 PUFA) constituting the culture medium. In this study, we investigated the effect of fatty acid modification on the activities of the key enzymes involved in the deacylation-reacylation cycle of membrane phospholipids. Results showed that cardiomyocytes grown in the presence of n-6 PUFA exhibited a higher specific alkaline phospholipase A (mainly A2) activity (+34%) and a moderately lower lysophospholipase activity (-17%) than when incubated with n-3 PUFA. AcylCoA:lysophosphatidylcholine acyltransferase, acid lysosomal phospholipase A1 and acylCoA synthetase activities were not significantly altered by changes in cellular PUFA composition. It was demonstrated that the differences between phospholipase A activities of the two types of cultured cells were linked neither to a differential leakage of enzyme nor to oxidative injury to the enzyme through blockage of essential sulfhydryl groups. One likely explanation is that the PUFA-induced changes in membrane composition alter membrane physical properties which, in turn, affect membrane-bound phospholipase A activity. Possible beneficial effects of the n-3 PUFA-induced changes on membrane stability are discussed.

Two methods were used to estimate the intracellular phospholipase activity in rat heart: one using exogenous radioactive substrate dispersed as unilamellar vesicles; the other using endogenous membrane hydrolysis and subsequent phospholipid and lysophospholipid separation by high-performance liquid chromatography and quantification by phosphorus determination. We found that the endogenous method provided a higher hydrolysis rate than the exogenous method and that phosphatidyl ethanolamine was a better substrate than phosphatidyl choline.

A simple and rapid method for assaying acyl-CoA:lysophosphatidylcholine acyltransferase is described. This method is based on silicic acid microcolumn chromatography using labelled lysophosphatidylcholine (lysoPC) as substrate. The reaction was stopped by conventional Folch extraction. The chloroform extract (2 ml) was deposited on the silica gel and pushed through with air, and then elution was performed with methanol/water (50:50, v/v). Under these conditions, only the labelled phosphatidylcholine (PC) synthesized was retained on the gel, and this was then removed from the column and counted immediately. This method gave enzyme activities comparable to those obtained with the TLC method, and has proved to be reproducible. The new method, however, is both faster and safer than the classical TLC method.

A method combining enzymatic and electrochemical detection of cholesterol in biological fluids was compared with conventional detection methods: two chromatographic methods--gas chromatography or high performance liquid chromatography--and two enzymatic methods with colorimetric detection either by kinetic measurement or at the end of the reaction. For serum, enzymatic determination is accurate with both colorimetric detection methods; but for bile, colorimetric detection is difficult to perform due to interference from bile pigments. Enzymatic cholesterol determination, combined with electrochemical detection, is simpler and gives results in good agreement with those of chromatographic methods.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) assays are usually performed by incubation of the enzyme with a labeled substrate followed by thin-layer chromatography separation and subsequent quantification of cholesteryl esters (CE) formed. Herein, a method is described for rapid separation of CE from other lipids, by elution from a silica gel column with a solvent mixture of petroleum ether/diethyl ether (98:2, v/v). Silica gel column chromatography is reliable and more rapid and safer than TLC. The best results were obtained when the reaction was stopped by Dole extraction followed by CE separation on a silica gel column. Assays for ACAT from rat intestinal microsomes showed that the specific activity values obtained using this method were reproducible and in good agreement with those obtained by conventional TLC method.

In this study, we investigated the effect of various types of fats on heart lipid peroxidation status and on blood lipid parameters. Rats were fed either a low-fat diet (2.2% lard plus 2.2% corn oil), a corn oil diet (17%), a salmon oil diet (12.5%) supplemented with 4.5% corn oil, or a lard diet (15%) supplemented with 2% corn oil. All diets were supplemented with 1% cholesterol. Rats were fed for eight weeks. When compared with the low-fat diet, the salmon oil-diet intake resulted in a lower blood cholesterol, triglyceride and phospholipid concentrations (-50, -56 and -30%, respectively). Corn oil only tended to lower blood lipids; this decrease was significant for triglycerides only (-40%). The hypocholesterolemic effect of salmon oil diet is even more pronounced, if blood cholesterol values are compared with those of rats fed the lard diet. Heart lipid composition was not affected by dietary manipulations. Fatty acid composition of cardiac phosphatidylcholines and phosphatidylethanolamines, however, were altered by high-fat diets. In phosphatidylcholine, salmon oil induced a twelvefold decrease in the n-6/n-3 ratio and a 26% increase in the unsaturation index. For phosphatidylethanolamine, the n-6/n-3 ratio decreased 7.7-fold and the unsaturation index increased by 13%. A 50% decrease of the n-6/n-3 ratio was observed in animals fed the lard diet. Ultramicroscopic examination of ventricles revealed that those of the salmon oil group significantly accumulated lipofuscin-like or ceroid material, whereas this accumulation was barely detectable in hearts of the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyunsaturated fatty acids are known to lower plasma cholesterol concentrations. We studied their effect on intestinal acyl-CoA:cholesterol acyltransferase (ACAT) activity in rats fed either salmon oil or corn oil (17% fat) with or without 1% cholesterol. After an 8-week feeding period we confirmed the hypolipidemic effect of salmon oil and we established its ability to stimulate ACAT activity in rats fed low-cholesterol diets. The most striking effect of 1% dietary cholesterol on ACAT activity was obtained in the control group (34% enhancement), whereas cholesterol supplementation had no effect on ACAT activity in the salmon oil group. The results enable us to suggest that n-3 fatty acids have an effect per se on ACAT activity; the regulation of enzyme activity by dietary cholesterol probably involves independent processes.

The influence of mineral NPK fertilizers and organic fertilizers such as manure compost, woodchip compost and blood meal on the yields and the nutritive value of leeks and turnips were compared. Fertilizers were applied on the two crops grown successively in 200-1 containers. Mineral fertilizers, manure compost and blood meal provided equivalent vegetable yields. Contents in dry matter, ascorbic acid and minerals of leeks and turnips were not strongly affected by the different fertilization regimes. The nitrate content of both crops was significantly lower under the application of manure compost and woodchip compost, while mineral fertilizers and blood meal induced similar, higher nitrate levels. The present work thus provides new data showing the usefulness of composts in growing vegetables with low nitrate contents.

Rats were fed lard-enriched (17%) or corn oil-enriched (17%) diets and were compared with rats fed a low fat (4.5%) diet. Cardiac protein, DNA, phospholipid (PL) and fatty acid (FA) compositions were analyzed. Neutral phospholipase A, lysophospholipase and creatine kinase activities in the membrane and cytosolic compartments were also investigated. No significant modification of cardiac protein, DNA nor PL was observed among the three groups. Some alterations appeared in the FA composition. A lard-enriched diet induced a significant increase of 22:5n-3 and 22:6n-3 in heart phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas a linoleic acid-rich diet induced a specific increase of 22:4n-6 and 22:5n-6 in these two major PL. Compared to rats fed the low fat diet, membrane-associated phospholipase A activity, measured by endogenous hydrolysis of membrane PC and PE, showed a significant increase (+45%) for both PL in rats fed corn oil. However, the activity of membrane-associated phospholipases, measured with exogenous [1-14C]dioleoyl PC, was not different among the three groups of rats. Cytoplasmic activity was decreased in rats fed corn oil, and lysophospholipase and creatine phosphate kinase activities were not significantly affected by diet. FA modification of the long chain n-6 FA induced by corn oil may be responsible for the observed increase in phospholipase activity. Physiological implications are suggested in terms of membrane degradation and prostaglandin production.

For 2 mo rats were fed a salmon oil diet (12.5%, wt/wt) supplemented with 4.5% (wt/wt) corn oil, a corn oil diet (17%, wt/wt) or a low fat diet (4.4%, wt/wt). Cardiac lipids were analyzed and fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was determined. Ventricular biopsies were taken for ultramicroscopic examination. Serum cholesterol, triglyceride, phospholipid and vitamin E concentrations were significantly lower in rats fed salmon oil than in those fed the other two diets, whereas serum transaminases and vitamin A were not significantly affected. Cardiac protein, phospholipid, triglyceride and cholesterol concentrations were unaffected by diet. Cardiac phospholipid composition remained unchanged and no significant changes in lyso-PC or lyso-PE levels were observed. However, the salmon oil diet produced a markedly lower n-6/n-3 ratio in both PE and PC than in the other two diets. This was the result of replacement of n-6 polyunsaturated fatty acids (PUFA), primarily 20:4n-6 with n-3 PUFA, primarily 22:6n-3. The unsaturation index of PC and PE was higher with the salmon oil diet than with the other two diets. Ventricular biopsies of rats fed salmon oil showed mild lipid accumulation associated with some lipofuscin-like material. It is suggested that, in rat heart, fish oil led to a moderate accumulation of lipids, the composition of which may include long-chain monounsaturated fatty acids and a degradative form of peroxidized lipids.