Isolation and characterization of an extracellular lipase from the conidia of Neurospora cvassa

A triacylglycerol lipase (EC 3.1.1.3) from the conidia of Neurospora crassa was purified and characterized. The enzyme was purified by Sephadex G-100 column chromatography. Homogeneity was checked by PAGE, and isoelectric focusing gave a single band corresponding to a pI of 6.4. The enzyme had an apparent Mr 54000≥1000 as determined by gel filtration. SDS-PAGE gave a single band of Mr 27000, suggesting the presence of two identical subunits. This lipase preferred triglycerides with C16- and C18-fatty acyl chains. It cleaved only the primary groups of triglycerides. The lipase also exhibited a marked preference for substrates containing endogenously occurring fatty acids and so may prove useful in detailed studies on the physiological relevance of fatty acyl specificity of lipases. The enzyme was not affected by detergents, or thiol-binding agents. Modification of free amino groups caused 90% inhibition, suggesting a role of these groups in the maintenance of lipase activity

Mycotin: a lectin involved in the adherence of Mycobacteria to macrophages

Pathogenic Mycobacteria colonize host macrophages. Attachement of these organisms to macrophages is the preliminary step prior to invasion of the macrophages by the bacteria. Western blot confirmed that walls of Mycobacterium avium and Mycobacterium tuberculosis contain molecules which are immunologically related to mycotin, a lectin found in Mycobacterium smegmatis. We have demonstrated that the adherence of Mycobacteria to macrophages is significantly inhibited by anti-mycotin antibody or the mycotin-specific sugar, mannan. These observations suggest that prevention of the interaction of mycotin-related molecules on the surfaces of Mycobacteria with mannose-specific receptors on macrophages, offers an important approach for blocking attachment of pathogenic Mycobacteria to macrophages, thereby preventing infection

Human erythrocyte membrane protein 4.2 is palmitoylated

Protein 4.2 is a major protein of the human erythrocyte membrane. It has previously been shown to be N-myristoylated. After labeling of intact human erythrocytes with [3H]palmitic acid, radioactivity was found to be associated with protein 4.2 by immunoprecipitation of peripheral membrane proteins extracted at pH 11 from ghosts with anti-(4.2) sera, followed by SDS/PAGE and fluorography. The fatty acid linked to protein 4.2 was identified as palmitic acid after hydrolysis of protein and thin-layer chromatography of the fatty acid extracted in the organic phase. Protein 4.2 could be depalmitoylated with hydroxylamine, suggesting a thioester linkage. Depalmitoylated protein 4.2 showed significantly decreased binding to protein-4.2-depleted membranes, compared to native protein 4.2

Overexpression and functional characterization of an ABC transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis

The genes encoding ABC transporters occupy 2.5% of the genome of Mycobacterium tuberculosis . However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential in order to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2',7'-bis-(2-carboxyethyl)-5(-and 6)-carboxyfluorescein (BCECF), a neutral compound also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters

An atypical cyclin-dependent kinase controls Plasmodium falciparum proliferation rate

Malaria parasites multiply in human erythrocytes through schizogony, a process characterised by nuclear divisions in the absence of cytokinesis, leading to the formation of a multinucleated schizont from which individual daughter cells are subsequently generated. Here, we provide evidence that parasites lines lacking Pfcrk-5, an atypical cyclin-dependent kinase, display a reduced parasitemia growth rate linked to a decrease in the number of daughter nuclei produced by each schizont. We show that in vitro activity of recombinant Pfcrk-5 is indeed cyclin-dependent and that the enzyme localises to the nuclear periphery. Thus, Pfcrk-5 is part of a regulatory pathway that mediates the proliferation rate of Plasmodium falciparum through the control of nuclear divisions during schizogony

An Oligopeptide Transporter of Mycobacterium tuberculosis Regulates Cytokine Release and Apoptosis of Infected Macrophages

Background: The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA(MTB). Methodology/Principal Findings: OppA(MTB) was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA(MTB) confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1 beta, IL-6 and TNF-alpha was also compromised after inactivation of oppD. Conclusions: Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis

Altered lipid peroxidation and antioxidant potential in human uterine tumors

439-443A comparative study of lipid peroxidation and antioxidant potential has been made in human uterus and uterine tumor. Two types of uterine tumor used are : tumor (I), a fibroid which is the commonest benign solid tumor in uterus and tumor (II), an adenomyoma. Tumor microsomes are less susceptible to lipid peroxidation induced by both enzymic (NADPH-ADP- Fe3+ and xanthine-xanthine-oxidase) and non-enzymic (ascorbate-Fe2+) systems except in the ease of tumor (II) microsomes when Induced with xanthine-xanthine oxidase. Resistance of tumor microsomes to lipid peroxidation is associated with the low content of substrates in the form of polyunsaturated fatty acids (PUFAs), higher level of α-tocopherol , reduced glutathione and protein thiols and altered enzymic antioxidant potential (catalase and superoxide dismutase)

Effect of fatty acid supplementation on thermotropic behavior of membrane lipids and leucine transport in Saccharomyces cerevisiae

An unsaturated fatty acid-requiring mutant (KD 115) of Saccharomyces cerevisiae shows altered phospholipid composition, transport behavior, and physical properties of membrane lipids when grown in the presence of different cis- and trans-unsaturated fatty acids. There is an increase in phosphatidyl ethanolamine content and a concomitant decrease in phosphatidyl choline content in the cells supplemented with trans-unsaturated fatty acids. The affinity for uptake of L-leucine is higher in the cis-unsaturated fatty acid-supplemented cells compared with the trans-unsaturated fatty acid-supplemented cells. The temperature-dependence of L-leucine uptake bears a reasonably good correlation with the thermotropic behavior of the membrane lipids as studied by the steady-state fluorescence polarization technique. The present findings are discussed in light of the importance of the lipid environment in modulating membrane-associated functions

Purification of a phosphatidylinositol/phosphatidylcholine transfer protein from Neurospora crassa

This paper reports, for the first time, the purification of a phospholipid transfer protein (PLTP) from a fungus, Neurospora crassa. The protein was purified from the post-microsomal supernatant of N. crassa by successive chromatography on DEAE-cellulose, Sephadex-G75 and PBE 94 (pH 4-7). The purified protein (Mr38 000) was found to transfer phosphatidylinositol preferentially over phosphatidylcholine, like the PLTP from the yeast, Saccharomyces cerevisiae. PC transfer was completely inhibited by inactivation of free amino groups or tryptophan residues. Surprisingly, the protein did not cross-react with antibodies against the bovine brain P1TP. The cellular content of the protein was maximal during the logarithmic phase of growth. However, no direct correlation between the content of the protein and PC transfer activity could be demonstrated

Release of a lectin from a fatty acid auxotroph of Saccharomyces cerevisiae grown in presence of oleic acid

The unsaturated fatty acid-requiring mutant KD 115 of Saccharomyces cerevisiae secretes a lectin when grown in presence of oleic acid. This lectin is homogeneous on PAGE at pH 8.3, has an approximate molecular weight of 320,000, pI of 4.2 and contains about 60% sugar. It agglutinates chicken and different mammalian erythrocytes, but lyses rabbit red cells only. It is D-galactose-specific. To our knowledge, this is the first report of a hemagglutinin from yeast