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Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis.

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.

Pharmacological modulation of the nonselective voltage-dependent cation (NSVDC) channel from human erythrocytes was studied. Using the inorganic cations ruthenium red and La3+, as well as the organic thiol group reagents iodoacetamide (IAA) and N-ethylmaleimide (NEM), it was possible to demonstrate a concentration-dependent decrease in the voltage-activated conductance, reflecting an inhibition or inactivation of the channel. Initial voltage activation was achieved by injecting human red cells into sucrose-substituted Ringers with a low chloride concentration, which causes a strongly positive membrane potential to develop, initially determined by the equilibrium potential for Cl- ( approximately +100 mV). Due to the voltage- and time-dependent activation of the cation channel, net effluxes, minimized by addition of a chloride conductance blocker, occurred and Vm gradually decreased and stabilized at a value less positive than E(Cl), reflecting the increased cation conductance, g+, reaching 1.5-2.0 microS/cm2. In the presence of inhibitors of the NSVDC channel, both the membrane potential repolarization and the cation efflux were diminished.

We describe the purification of a cysteine proteinase from latex of Carica candamarcensis, hereby designated CC23. The enzyme has been purified by ion-exchange chromatography and behaves electrophoretically as a monomer of M(r) 23,000 and optimal pH of 8.0. It displays a basic isoelectric point, has one cysteine residue in the active site by titration with E-64, confirmed by DNA sequencing, and responds to proteinase inhibitors as a classic cysteine proteinase. The K(m) and k(cat)/K(m) for CC23 using BAPNA were respectively 14.7 +/- 1.8 x 10(-4) M and 1.3 x 10(3) M(-1) s(-1). Therefore, the catalytic efficiency of CC23 is sixfold higher than that of CC-I, another proteinase from the same plant. DNA primers were designed to amplify by PCR a genomic sequence related to this enzyme. An 895-bp DNA fragment was cloned and sequenced. It shows strong homology with chymopapain isoform IV from C. papaya. The translated sequence is similar to that of chymopapain isoform II (73%) and CC-III (77%) from C. candamarcensis., (Copyright 2001 Academic Press.)

Recent studies indicate that polymicrobial sepsis can markedly increase inducible macrophage Ao (nonnecrotic cellular suicide) and that this is associated with decreased M phi function. In vitro studies suggest that M phi Ao can be induced by IL-1 beta via interleukin-1 beta-converting enzyme (ICE, a cysteine protease), prostanoids, or reactive oxygen/nitrogen. However, the mechanism(s) underlying this process in septic M phi remains unknown. To determine this, male C3H/HeN mice were subjected to sepsis (cecal ligation and puncture, CLP) or sham-operation. Twenty-four hours thereafter, M phi were isolated from the peritoneum (PM phi) and liver (LM phi). Macrophage monolayers were treated with LPS (10 micrograms/ml) alone (Cont) or in the presence of iodoacetamide (Iodo, 5 mM), N-methylmalamide (meth, 5 mM), ibuprophen (Ibu, 40 micrograms/ml), N-methyl-L-arginine (LNMA, 0.4 mM), or superoxide dismutase (SOD, 60,000 U/ml) for 24 hr. The extent of Ao was determined using an enzyme-linked immunosorbent cell-death assay, which detects the presence of cytoplasmic oligonucleosomes measured as optical density. The results indicate that both PM phi and LM phi from septic animals exhibit increased Ao over cells from sham animals. However, only the nonspecific cysteine protease inhibitors (Iodo and meth) and the NO inhibitor LNMA blocked septic mouse M phi Ao. Furthermore, only PM phi from CLP mice treated with Iodo, but not LNMA or IBU, showed an improved capacity to release IL-6. We conclude that increased M phi Ao seen during sepsis appears to be mediated by both ICE-like cysteine protease activation and NO release but the level/mechanism of action of these inhibitors differs.

Previously we have shown that Lelystad virus (LV) contains five structural proteins, a nucleocapsid protein N, an integral membrane protein M, and three glycoproteins GP3, GP4, and GP5. In this study we identified a sixth structural protein of Lelystad virus. The protein has an apparent molecular weight of 29 to 30 kDa, was recognized by an ORF2-specific antipeptide serum in Western immunoblotting using sucrose gradient purified LV virions, and was shown to be N-glycosylated. It was therefore designated GP2. The GP2 protein was also immunoprecipitated by ORF2-specific antipeptide serum from lysates and extracellular virus of CL2621 cells infected with LV. A fraction of the GP2 protein present in these lysates contained an intrachain disulfide bond. Endoglycosidase H treatment of immunoprecipitates indicated that the endoglycosidase H-sensitive N-glycans of the GP2 protein become endoglycosidase H-resistant during passage through the Golgi compartment in cells infected with LV. In contrast, the N-glycans of the GP2 protein expressed individually in a recombinant Semliki Forest virus remained endoglycosidase H-sensitive, indicating that the GP2 protein was retained in the endoplasmic reticulum. LV is the first arterivirus which has been demonstrated to contain six virion-associated proteins, one nucleocapsid protein, one integral membrane protein, and four glycoproteins.

The gene encoding phenylethanolamine N-methyltransferase (PNMT) has been amplified from a human adrenal medulla cDNA library. Following ligation of the gene into a pET3a-derived expression vector and transformation into Escherichia coli BL21(DE3)pLysS, PNMT was expressed, yielding about 10% of the soluble protein. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by ion-exchange chromatography and gel filtration. The Km for phenylethanolamine and S-adenosyl-L-methionine were determined to be 130 and 16 microM, respectively. The enzyme could be inhibited by reagents expected to modify cysteine, arginine, tyrosine, and histidine residues, but not by methyl acetimidate, a reagent expected to modify lysine residues.

The mechanisms by which sulfhydryl (SH-) binding reagents modulate the light-sensitive conductance of retinal rods were investigated by current recording from single rods, by patch clamp recording from the plasma membrane of the rod outer segment (ROS), and by biochemical study of their effects on the light-induced hydrolysis of cyclic GMP. The electrophysiology, as well as measurements of the reagents' ability to traverse the ROS plasma membrane, was done on amphibian (Rana and Ambystoma) rods, and the biochemistry on bovine rods. The main SH-reagents used were N-ethyl-maleimide (NEM) and iodoacetamide (IAA). Both transiently increased rod current, but part of the large current could not be turned off by light. After a few minutes' exposure, NEM, but not IAA, caused a continuous decay of the rod's light sensitivity. In patch-clamp recordings from the ROS plasma membrane, the reagents increased conductivity both in the presence and absence of cGMP, consistent with the observation that the drug-induced current increase in intact rods involved both light-sensitive and light-insensitive components. In vitro, NEM was found to be a powerful inhibitor of cGMP hydrolysis, which can explain the gradual loss of light sensitivity in the rod and could initially contribute to the increased dark current via elevated cGMP levels. Thus, SH-reagents act both by modifying the light-sensitive channel and by inhibiting phototransduction inside the rod.

When rat liver nuclei are isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, digested with DNase I and RNase A, and extracted with 1.6 M NaCl, nuclear envelope (NE) spheres depleted of intranuclear material, as analysed by thin-section electron microscopy, are obtained. Two-dimensional isoelectric focusing (IEF)/SDS-PAGE and non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE reveal that the predominant polypeptides are lamins A, B and C. Nuclei isolated in the absence of sulfhydryl blocking reagents yield salt- and nuclease-resistant structures which contain sparse but demonstrable intranuclear material. A number of non-histone polypeptides are seen in addition to the lamins. Nuclei treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) yield, after exposure to nucleases and 1.6 M NaCl, nuclear matrix-like structures containing an extensive intranuclear network and components of the nucleolus in addition to the NE. Increased amounts of the non-lamin, non-histone polypeptides are recovered with these structures. Subsequent treatment of these NaTT-cross-linked structures with reducing agents in 1.0 M NaCl selectively solubilizes the intranuclear components but leaves the nuclear envelope apparently intact. The lamins remain sedimentable and are virtually absent from the soluble (intranuclear) material. Instead, the major solubilized polypeptides are (a) 68 and 63 kD polypeptides which migrate in the vicinity of lamins B and C, respectively, but are distinguishable from the lamins by immunoblotting and by uni-dimensional peptide mapping; (b) a series of basic 60-70 kD polypeptides (pI greater than 8.0) which are not recognized by anti-lamin antisera; (c) an acidic (pI 5.3) 38 kD polypeptide; and (d) a number of high molecular mass (greater than 100 kD) polypeptides. These observations not only suggest a convenient method for fractionating matrix structures from rat liver nuclei into biochemically and morphologically discrete components, but also identify a subset of major non-lamin, non-histone nuclear polypeptides (comprising approx. 20% of the total nuclear protein) whose intermolecular interactions can be reversibly stabilized apparently by intermolecular disulfide bond formation by NaTT.

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 degrees C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 degrees C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9-18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60-70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (greater than 95%) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 degrees C or with NaTT transferred most (greater than 95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamide-treated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.

The effect of thyrotropin-releasing hormone (TRH), TRH metabolites, and a TRH analog on the secretion of 35S-labeled sulfated polysaccharides from in vitro incubated foot integument of the pond snail Lymnaea stagnalis palustris was assessed. Whereas TRH significantly inhibited the secretion of 35S-labeled polysaccharides, its metabolite deamido TRH significantly stimulated polysaccharide secretion. Histidyl-proline-diketopiperazine did not alter the secretion of 35S-labeled sulfated polysaccharides. Foot integument exhibited considerable TRH-peptidase activity in vitro. The principal proline-containing TRH metabolites were deamido-TRH and proline. The deamido-TRH content of the foot, mantle, and hemolymph was determined by radioimmunoassay. All tissues contained deamido-TRH. These findings suggest that the secretory activity of skin mucus glands of L. stagnalis palustris is the result of the stimulatory action of deamido-TRH and the inhibitory action of TRH. 3Methyl-2histidyl-TRH, TRH, vasotocin, and bombesin did not alter the polysaccharide secretion by in vitro incubated foot integument, indicating that the changes in mucus secretion after TRH and deamido-TRH administration are specific and that the gastropod "TRH receptors" may differ from the mammalian brain and anterior pituitary gland TRH receptors.

The receptor for the minor group of human rhinoviruses was solubilized from HeLa cell membranes with various detergents. Virus binding activity was determined in a filter binding assay using 35S-labeled human rhinovirus 2 (HRV2) as a probe. The receptor protein was enriched on Lens culinaris lectin columns and the active fractions were further purified by gel permeation and anion exchange chromatography. The receptor has an apparent molecular weight of 450 kDa in the presence of detergent. The binding activity is sensitive to trypsin, sulfhydryl modifying agents, but insensitive to neuraminidase. Divalent cations are essential for virus binding.

Extracts of poliovirus-infected HeLa cells promoted the in vitro assembly of 14 S subunits into empty capsids antigenically indistinguishable from procapsids. When infected cells were treated with iodoacetamide, the extract lacked the assembly promoting activity. This activity was restored by the addition of heat-disrupted virions, but the empty capsids formed in this system were antigenically different from procapsids. This and other observations introduce a distinction between the "assembly promoting" and "antigenicity conferring" activities of infected-cell extracts.