Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.     

Plasmodium falciparum origin recognition complex subunit 5: functional characterization and role in DNA replication foci formation

The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum . Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiae o rigin r ecognition c omplex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.     

Mechanisms of ligand discrimination by heme proteins

Several classes of heme proteins have been identified whose primary role is to sense and transport one of the three gaseous ligands: O(2), NO, or CO. A common feature shared by all of these proteins is the need to not only recognize its target ligand, but also discriminate against other heme ligands of similar size and shape. This review describes the mechanisms that each class of heme protein sensor and transporter utilizes to promote this discrimination. The common factors utilized in the recognition of each ligand are discussed.     

Immunological unresponsiveness in leprosy and its relevance to immunoregulation in man

The varied forms of leprosy form a clinical and immunological spectrum which offers extraordinary possibilities for insight into immunoregulatory mechanisms in man. At one pole, tuberculoid leprosy, patients develop high levels of cell-mediated immunity which ultimately results in killing of bacilli in the tissues, albeit often with damage to nerves. At the lepromatous pole, patients exhibit selective immunological unresponsiveness to antigens ofMycobacterium leprae. Even though all currently known protein species ofMycobacterium leprae and BCG are cross-reactive, lepromatous patients unreactive toMycobacterium leprae antigens frequently respond strongly to tuberculin.In vitro experiments suggest the existence of lepromin-induced suppressor activity, mediated by both monocytes andT cells. TheT suppressor cells have the T₈ phenotype of which 50% express the activation markers,Ia and FcR. The one unique species of antigen of the leprosy bacillus is a phenolic glycolipid, and it appears that theT _s cells largely recognize the terminal trisaccharide of this unique antigen. Depletion ofT _s cells restoresin vitro reactivity of lymphocytes to lepromin in a portion of lepromatous patients, and addition of IL-2 containing supernatants partially restores responsiveness toMycobacterium leprae antigens. Vaccination of lepromatous patients with a mixture ofMycobacterium leprae and live BCG restores cell-mediated immunity in the majority of lepromatous patients, and concomitantly reduces thein vitro suppressor activity and number of activated T₈ cells. These experiments suggest the existence of stage-of-disease related suppressor cells in leprosy which appear to block the responsiveness ofT _H capable of responding to either specific or cross-reactive mycobacterial antigens. The mode of action of theseT _s appears to be the inhibition of production of IL-2 and other lymphokines. Successful immunotherapeutic vaccination appears to overcome this block in the majority of patients.     

The major native proteins of the leprosy bacillus

This study addresses a major obstacle to vaccine development for leprosy, the isolation and characterization of the native protein antigens of the leprosy bacillus. Mycobacterium leprae harvested from armadillos was subjected to a simple fractionation protocol to arrive at the three major subcellular fractions, cell walls, cytoplasmic membrane, and soluble cytoplasm. The application of extensive detergent phase separations to membrane fractions allowed removal of lipoarabinomannan and the mannosyl phosphatidylinositols, and the recognition and purification of two major membrane proteins (MMP) of molecular mass 35 kDa (MMP-I) and 22 kDa (MMP-II); recovery of these proteins was about 0.5 mg each per g of M. leprae. MMP-I is N-blocked and is perhaps a lipoprotein. End group analysis on MMP-II indicates a new protein. Three major cytoplasmic proteins (MCP) of molecular mass 14 kDa (MCP-I), 17 kDa (MCP-II), and 28 kDa (MCP-III) were also recognized. MCP-I, the most abundant protein in M. leprae, represents 1% of the bacterial mass. End group analysis of the first 30 residues and immunoblotting studies demonstrate sizeable structural homology to a protein from Mycobacterium tuberculosis but immunological distinctiveness. MCP-I, which also occurs in highly immunogenic peptidoglycan-bound form, is a primary candidate for future vaccine development. The cell walls of M. leprae are also characterized by one major extractable protein, also of molecular mass 17 kDa. Thus the major antigens of the leprosy bacillus, protein and carbohydrate alike, are now nearer to complete definition.     

Studies on the metabolism of rat liver copper-metallothionein.

The degradation of purified 35S-labelled rat liver isometallothioneins (MT) by lysosomal extracts was studied. Zn-MT-I was more readily hydrolysed than Zn-MT-II, but no significant degradation of the Cu-containing metallothioneins could be detected, even after 24 h incubation. The susceptibility of MT to degradation in vitro may be related to the strength of the metal-thiolate bonds. However, the turnover rates of cytosolic MT in vivo, as established by pulse-labelling techniques, are apparently subject to different controls. The half-lives of MT-I and -II in the liver cytosol of Cu2+-injected rats were only 15.4 +/- 1.5 and 18.2 +/- 1.1 h respectively. Approx. 25% of the total liver MT was present in particulate fractions (probably in lysosomes) of the liver and had a half-life of 25.1 +/- 4.1 h.     

Fingernails as biological indices of metal exposure

Metal determination in human tissues is the most common application of biological monitoring for screening, diagnosis and assessment of metal exposures and their risks. Various biopsy-materials may be used. This paper deals with the quantitative determination of Cd, Pb, Cr, Mn, Fe, Ni, Cu, and Zn concentrations in nails of male subjects exposed to these metals alongwith their respective controls, while working in locomotive, carriage and roadways workshops, and lead battery factories. The levels of Cd, Pb, Cr, Mn, Fe, Ni, Cu and Zn in fingernails, assayed by atomic absorption spectrophotometry, were compared with their respective controls by student ‘t’ test. All the obtained values were correlated to the personal and medical history of the subjects under study. Significantly high levels of Cd, Pb, Cr, Fe, Ni, Cu and Zn were present in smokers, compared to nonsmokers. The concentrations of Cd, Pb, Cr, Mn and Fe were not significantly high in vegetarian subjects. It was also observed that there is no contribution of liquor towards nail-metal concentration. Significant correlations were observed between skin disease and Cr, Mn, Fe, Cu; hypertension and Cd, Mn, Cu; mental stress and Cd, Pb, Mn, Ni, Cu, Zn; diabetes and Cr, Mn, Ni; chest pain and Pb; respiratory trouble and Cr, Mn, Fe, Ni, Zn; tuberculosis and Zn; acidity and Cd; and ophthalmic problems and Mn, Fe, Ni, and Zn     

Antigenic properties of peptide mimotopes of HIV-1-associated carbohydrate antigens

The glycan shield of the human immunodeficiency virus (HIV) envelope protein presents many potential epitopes for vaccine development. To augment immune responses to HIV, type 1 (HIV-1), envelope-associated carbohydrate antigens, we are defining peptide mimics of HIV-associated carbohydrate antigens that function as antigen mimotopes that upon immunization will induce antibodies cross-reactive with carbohydrate antigens. We have previously defined peptides with a putative sequence tract RYRY that mimic concanavalin A-binding glycans. To imitate the multivalent binding of carbohydrates, we compared the avidity of a linear (911) and cyclic peptide (D002) reactive with concanavalin A presented in a multiple antigen peptide (MAP) format. The affinity of the MAP-D002 peptide was higher than that of the peptide MAP-911, whereas the avidity of D002 peptide was lower than that of 911. Serum from mice immunized with MAP-911 had lower titer for oligomannose-9 than those elicited by MAP-D002 under the same conditions, but both immunogens elicited antibodies that can block the binding of GP120 to dendritic cells. Antibodies that bind to the studied MAPs were found in a preparation of normal human immunoglobulin for intravenous use. Those that were purified on 911 bound back to 911 and D002, whereas anti-D002 antibodies were specific only for D002. Human antibodies reactive with both mimotopes and with a mannosyl preparation were observed to bind to envelope protein. These results suggested the potential to fine-tune the antibody response to carbohydrate antigens by modifying structural features of peptide mimotope-based immunogens.     

The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFbeta family signaling pathways

Wishful thinking (Wit) is a Drosophila transforming growth factor-beta (TGFbeta) superfamily type II receptor most related to the mammalian bone morphogenetic protein (BMP) type II receptor, BMPRII. To better understand its function, we undertook a biochemical approach to establish the ligand binding repertoire and downstream signaling pathway. We observed that BMP4 and BMP7, bound to receptor complexes comprised of Wit and the type I receptor thickveins and saxophone to activate a BMP-like signaling pathway. Further we demonstrated that both myoglianin and its most closely related mammalian ligand, myostatin, interacted with a Wit and Baboon (Babo) type II-type I receptor complex to activate TGFbeta/activin-like signaling pathways. These results thereby demonstrate that Wit binds multiple ligands to activate both BMP and TGFbeta-like signaling pathways. Given that myoglianin is expressed in muscle and glial-derived cells, these results also suggest that Wit may mediate myoglianin-dependent signals in the nervous system.     

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.