Impairment of cardiac contractility and sarcoplasmic reticulum Ca2+ ATPase activity by hypochlorous acid: reversal by dithiothreitol.

Isolated rat hearts perfused with 100 microM hypochlorous acid (HOCl), a powerful oxidant produced by activated neutrophils, exhibited progressive impairment of contractile performance suggestive of a cytosolic Ca2+ overload (increased left ventricular end-diastolic pressure, increased aortic root perfusion pressure, and depressed pulse pressure). Sarcoplasmic reticulum (SR) enriched microsomal preparations isolated from HOCl-perfused hearts showed a significant decline, when compared with control hearts, in both Ca2+ ATPase activity (123 +/- 40 vs. 473 +/- 46 nmol Pi.mg-1 protein.min-1) and Ca2+ uptake (12 +/- 5 vs. 46 +/- 4 nmol Ca2+.mg-1 protein.min-1). The sulfhydryl content in Ca2+ ATPase and other proteins, as determined by [14C]iodoacetamide binding, was also progressively depleted in HOCl-perfused hearts. Perfusion of the HOCl-treated hearts with dithiothreitol (DTT), a disulfide reducing agent, resulted in a time-dependent attenuation, and eventual partial reversal, of the dysfunction in both contractility and SR Ca2+ ATPase activity. Protein thiol levels were concomitantly restored to near control values. The data indicate that HOCl-induced contractile dysfunction in heart is related to the inactivation of the SR Ca2+ ATPase as a result of thiol oxidation and suggest that DTT is capable of reversing this dysfunction in situ by reducing the oxidized sulfhydryls in the Ca2+ ATPase.     

2,3-butanedione as a photosensitizing agent: application to alpha-amino acids and alpha-chymotrypsin

2,3-Butanedione sensitized the rapid photodestruction of free alpha-amino acids, and the photoinactivation of alpha-chymotrypsin, in the presence of ultraviolet light and oxygen. These reactions showed "pseudo-first-order" kinetics at 2,3-butanedione concentrations approximating those employed for the chemical modification of arginine residues in proteins. The photoreactions were inhibited in anoxic media or in the presence of azide; findings were consistent with a singlet oxygen mechanism for these reactions. No enhancement in the rate of reaction was observed in D2O. The rate of 2,3-butanedione-sensitized photodestruction of free amino acids increased with increasing pH. However, the rate constants for the photosensitized inactivation of alpha-chymotrypsin, as well as those for the photodestruction of the tryptophan residues of this enzyme, decreased linearly with increasing pH.     

Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a steroid hormone receptor

DnaJ-like proteins function in association with Hsp70 molecular chaperones to facilitate protein folding. We previously demonstrated that a yeast DnaJ-like protein, Ydj1p, was important for activation of heterologously expressed steroid hormone receptors (Caplan, A. J., Langley, E., Wilson, E. M., and Vidal, J. (1995) J. Biol. Chem. 270, 5251-5257). In the present study, we analyzed Ydj1p function by assaying hormone binding to the human androgen receptor (AR) heterologously expressed in yeast. We analyzed hormone binding in strains that were wild type or deleted for the YDJ1 gene. In the deletion mutant, the AR did not bind hormone to the same extent as the wild type. Introduction of mutant forms of Ydj1p to the deletion strain revealed that the J-domain is necessary but not sufficient for Ydj1p action, and that other domains of the protein are also functionally important. Of three human DnaJ-like proteins introduced into the deletion mutant, only Hdj2, which displays full domain conservation with Ydj1p, suppressed the hormone binding defect of the deletion mutant. By comparison of the domains shared by these three human proteins, and with mutants of Ydj1p that were functional, it was deduced that the cysteine-rich zinc binding domain is important for Hdj2/Ydj1p action in hormone receptor function. A model for the mechanism of DnaJ-like protein action is discussed.     

Hepatitis A virus attachment to agri-food surfaces using immunological, virological and thermodynamic assays

AIMS: This study was designed to investigate the ability of hepatitis A virus (HAV) to attach to various food contact surfaces. METHODS AND RESULTS: HAV attachment was demonstrated after elution of attached viruses from solid surfaces by an immunofluorescent method using anti-HAV-specific antibodies and confocal microscopy. Attachment and survival of HAV on stainless steel, copper, polythene and polyvinyl chloride (PVC) at 20 and 4 degrees C after 2 and 4 h were quantified by plaque assay. HAV was shown to attach almost instantaneously to all four surfaces tested. Attachment of HAV depended on initial viral concentration and was slightly greater at 4 degrees C. The total surface energy (gammaTOT), nonpolar Lifshitz-Van der Waals (gammaLW) and polar short range (gammaSR) hydrogen-bonding components for HAV and each surface as well as total free energy of the system were determined by contact angle measurements using an extended Young equation [Young (1805) Philosophical Transactions of The Royal Society (London) 95, 65-87). The calculation of these parameters predicted the favourable conditions for attachment of HAV to all four surfaces tested. CONCLUSION: HAV particles attach to stainless steel, copper, polythene and PVC at 20 and 4 degrees C and the total free energy of the interaction is optimal for this attachment. SIGNIFICANCE AND IMPACT OF THE STUDY: Comprehension of viral attachment to the solid surfaces will permit to successfully disinfect these surfaces and to establish a better surveillance programme for control of viral food-borne illnesses.     

The Cdc37 protein kinase-binding domain is sufficient for protein kinase activity and cell viability

Cdc37 is a molecular chaperone required for folding of protein kinases. It functions in association with Hsp90, although little is known of its mechanism of action or where it fits into a folding pathway involving other Hsp90 cochaperones. Using a genetic approach with Saccharomyces cerevisiae, we show that CDC37 overexpression suppressed a defect in v-Src folding in yeast deleted for STI1, which recruits Hsp90 to misfolded clients. Expression of CDC37 truncation mutants that were deleted for the Hsp90-binding site stabilized v-Src and led to some folding in both sti1Delta and hsc82Delta strains. The protein kinase-binding domain of Cdc37 was sufficient for yeast cell viability and permitted efficient signaling through the yeast MAP kinase-signaling pathway. We propose a model in which Cdc37 can function independently of Hsp90, although its ability to do so is restricted by its normally low expression levels. This may be a form of regulation by which cells restrict access to Cdc37 until it has passed through a triage involving other chaperones such as Hsp70 and Hsp90.     

Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes

AIMS: This study aimed to characterize new isolates of human bifidobacteria, evaluate some of their probiotic potential and to screen these isolates for their effectiveness at inhibiting Listeria monocytogenes in vitro. METHODS AND RESULTS: Thirty-four Bifidobacterium isolates from infant faeces were identified by fructose-6-phosphate phosphoketolase and PCR. Six isolates, coded RBL67, RBL68, RBL69, RBL70, RBL85 and RBL86, showed higher antagonistic activity against L. monocytogenes. Neutralized culture supernatants of these strains did not inhibit L. monocytogenes when tested by agar diffusion method. However, the concentration of supernatant by speed-vac resulted in the formation of an inhibitory effect with supernatants from strains RBL67, RBL68 and RBL70. This effect was shown to be related to heat-stable proteinaceous compound(s) which were resistant to heating at 100 degrees C for 5 min but not to pronase-E, proteinase-K or trypsin. The extraction of the inhibitory compounds by methanol-acetone extraction procedure indicated that four strains (RBL67, RBL68, RBL69 and RBL70) were mostly soluble in acetone. However, strain RBL85 produced inhibitory substances that were soluble in methanol. CONCLUSION: Infant bifidobacterial isolates produce heat-stable proteinaceous compounds active against L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of antibacterial substances by bifidobacteria would improve intestinal bacterial ecology and inhibit intestinal pathogens.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells

Aim:  To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form.
Methods and Results:  Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD₅₀ showed that the fibroblasts were able to tolerate up to 80  μ g ml⁻¹ for 24 h, dropping thereafter to 62  μ g ml⁻¹ after 72 h of contact, compared to 160  μ g ml⁻¹ after 24 h, and 80  μ g ml⁻¹ after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25  μ g ml⁻¹) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition.
Conclusion:  These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans .
Significance and Impact of the Study:  This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.     

BACTIBASE second release: a database and tool platform for bacteriocin characterization

Background  

BACTIBASE is an integrated open-access database designed for the characterization of bacterial antimicrobial peptides, commonly known as bacteriocins.     

Action of divergicin M35, a class IIa bacteriocin, on liposomes and Listeria

AIMS: The mode of action of divergicin M35, a class IIa bacteriocin, was studied against Listeria monocytogenes with sensitive (DivS) and resistant (DivM) phenotypes, as well as on synthetic phospholipid liposomes. METHODS AND RESULTS: Divergicin-induced release of 1,6-diphenyl-1,3,5-hexatriene (DPH) from zwitterionic (DMPC) and anionic (DMPC/DMPG, 4:1) liposomes, divergicin binding to liposomes, intracellular ATP concentration, cation efflux, cell affinity for hydrocarbons and cell lysis were measured and cell damage was visualized by fluorescence imaging and transmission electron microscopy. Divergicin M35 at 5 microg ml(-1) induced DPH efflux from anionic and zwitterionic liposomes at rates of about 2.58% and 1.61% per minute, respectively. DPH efflux rate from anionic liposomes was reduced by about 1.83% and 2.1% per minute in the presence of Li+ and Ca2+, respectively. Binding affinity of divergicin M35 to anionic and zwitterionic liposomes was about 86% and 63%, respectively. Intracellular ATP decreased in the sensitive and the resistant strains by 96.7% and 72.8%, respectively after 20 min of exposure to 5 microg ml(-1) divergicin M35. Lysis of the sensitive strain reached 57% in 18 h at a concentration of 5 microg ml(-1) when compared with the lysis of the divergicin-resistant strain (38.8%). The K+ and Na+ efflux from the divergicin-sensitive strain reached 87% and 80% of the total ion content within 5 min of exposure. This strain also showed higher affinity for hydrocarbons. CONCLUSIONS: The cell death of listerial strains upon addition of divergicin M35 could result from ATP depletion, K+ and Na+ efflux, and bacteriolysis. This triple biological effect was attenuated in the DivM strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to the understanding of the mode of action of divergicin M35, a pediocin-like bacteriocin.