Conformation of beta-lactoglobulin at an oil/water interface as determined from proteolysis and spectroscopic methods

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Limited proteolysis of beta-lactoglobulin using thermolysin. Effects of calcium on the outcome of proteolysis

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Synergistic Inhibition of Influenza a Virus Replication by a Bacterial Protease Inhibitor and a Plant Preparation

International audienceWe screened the NIH MLSCN 100,000 compound library and discovered a novel scaffold that shows sub-micromolar activity against H5N1and H1N1 influenza viruses in vitro. Cheminformatics and medicinal chemistry analyses were performed of the hit compounds and SAR led to the synthesis of several second-generation compounds with potent nanomolar activity and increased polarity for hit-to-lead optimization. Of the second-generation compounds, several met our activity criteria for identification of lead compounds: an efficacy EC50 value of 10 in secondary assays. We screened several lead compounds against 15 influenza A and B viruses in cell culture. They were active against H1N1 and H5N1 viruses, but not against H3N2 and B viruses. Interestingly, neuraminidase assays reveal that this scaffold did not inhibit viral neuraminidase. Real-time q-RT-PCR results revealed that these chemotypes significantly reduced RNA levels as compared to the no drug influenza infected MDCK cells. This suggests that these compounds target an early event in the viral life cycle in agreement with time-of-addition experiments whereby a significant reduction in virus-specific protein synthesis occurred 6 h post-infection in the presence of compound. Indirect immunofluorescence studies suggest these compounds affect the nuclear export of the N proteins from H1N1 virus. To supplement our in vitro studies, we have developed an HPLC procedure to measure the concentration of lead agents in mouse plasma after IP injection. Preliminary pharmacokinetic results indicate that significant plasma levels (μM) were achieved with five of the eight compounds that have been tested. These compounds were retained in the plasma for over 1 h. Because of the nM potency of these compounds, these results suggest that these five compounds are candidates for evaluation of efficacy in animal models. We are in the process of determining the maximally tolerated dose of these five agents. One or more of these will be selected for initial in vivo evaluation and the data will be presented

Characterization of the Maillard reaction products of beta-lactoglobulin glucosylated in mild conditions

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Hexanal and t-2-hexenal form covalent bonds with whey proteins and sodium caseinate in aqueous solution

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Kinetics of beta-casein hydrolysis by wild-type and engineered trypsin

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Emulsifying properties and adsorption behavior of egg yolk lipoproteins (LDL and HDL) in oil-in-water emulsions

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Characterization of mare caseins. Identification of alpha S1- and alpha S2 caseins

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Antiviral potential of a proteolytic inhibitor from Streptomyces chromofuscus 34-1

International audienceThe novel proteinaceous protease inhibitor, isolated from the culture supernatants of Streptomyces chromopfuscus 34-1 (SS 34-1) demonstrated a specific and selective anti-influenza virus effect. By the use of complementary virological assays it was demonstrated that the expression of the viral haemagglutinin (HA) on the surface of infected cells, the virus-induced cytopathic effect (CPE) and the infectious virus yields (IVY), used as measures of A/Germany/34, strain Rostock (H7N1) (A/Rostock) virus growth, were all reduced at non-toxic concentrations of SS 34-1 in a dose-dependent way. In a single cycle of viral growth, the penetration of viral particles and the late stages of viral replication were the most susceptible to inhibition. The preparation protected mice from mortality in the experimental A/Aichi/2/68 (H3N2) influenza virus infection. The SS 34-1-treatment (0.16 mg/mouse/day) of virus-infected mice led to a considerable reduction of mortality rate (index of protection = 71.0%) and marked prolongation of mean survival time (+ 5.5 days). The lesions of the lungs due to infection as well as lung infectious virus titres were significantly reduced - delta log(10)TCID(50)/ml up to 4.5. The application of SS 34-1 induced a continuous rise of the anti-protease activity but did not cause substantial changes in the lung protease activity of healthy mice. The infection triggered a slight reduction of protease activity in the lungs at 5 and 48 h p.i. and a marked increase at 24 hand 6 day p.i. Protease inhibition in the lungs was reduced at 24 and 48 h p.i. and an increase was observed at 5 h, 6 and 9 day p.i. SS 34-1-treatment brought both activities to normal levels. It was assumed that SS 34-1 exerted its inhibitory effect indirectly, through an increase of the protease-inhibitory activity. Histopathological examination of the lungs showed that under SS 34-1-treatment pulmonary lesions were less severe than those of untreated controls. The isolated novel protease inhibitor was purified by anion-exchange chromatography and reversed phase-HPLC. Analysis by differential scanning calorimetry (DSC) revealed that at pH 7.5 the denaturation temperature was 75 degrees C and the denaturation itself was irreversible. Analysis by circular dichroism (CD) in the UV region 190-250 nm showed that at pH 7.5 the spectrum presented 2 minima at 208 and 222 nm, typical for an a-helical structure. At pH 2.5 and after heating the spectrum corresponded to an unordered structure

Factors influencing pepsinolysis of methyl-, ethyl- and propyl-ester derivatives of beta-lactoglobulin

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