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PubMed1980

DNA wrapping in nucleosomes. The linking number problem re-examined.

Chromatin was assembled in vitro from relaxed closed circular DNA (SV40) and core histones at histone to DNA ratios of 0.2 to 0.3 (g/g) and incubated with topoisomerase I to relax supercoils in DNA regions not constrained by protein. Addition of histones H1 + H5 to the chromatin at an ionic strength of 0.1 M, in the presence of the solubilizing agent, polyglutamic acid, and topoisomerase I, increased the magnitude of the DNA linking number change, relative to protein-free DNA. No change in the linking number distribution occurred for relaxed protein-free DNA under these conditions. Control experiments indicated that the increase in the absolute value of the DNA linking number change in the chromatin could not be attributed to an increase in the number of nucleosomes per DNA molecule. These data suggest a solution to the linking number problem associated with models of chromatin structure.

Animals↗
PubMed2002

Circle ligation of in vitro assembled chromatin indicates a highly flexible structure.

Evidence is provided that some condensed linker histone-containing chromatin structures are highly flexible in solutions containing 2 mM Mg2+. Chromatin assembled in vitro +/- histone H5 on a 6.3 kb linear DNA fragment in 90 mM NaCl using the polyglutamic acid method sedimented fairly homogeneously. The H5-containing sample had s(20, w) values that were 58-69% greater than the sample lacking H5. Chromatin assembled on linear pUC19 plasmid DNA was treated with T4 DNA ligase in solutions containing 2 mM Mg2+ over a range of DNA concentrations. It was found that the intramolecular DNA ends of the chromatin could be joined together more efficiently than the intramolecular ends of the naked DNA at the higher DNA concentrations. This result could not be attributed to the effective reduction in DNA length by nucleosome formation. The chromatin structures formed did not have naked DNA tails extending from the ends as assessed by exonuclease III digestion. Chromatin assembled on DNA shortened by up to 420 bp gave very similar results, suggesting that the structure was a flexible one, rather than a rigid one having DNA ends that were fortuitously juxtaposed.

Animals↗
PubMed1980

Failure to increase the in vivo immunosuppressive activity of antilymphocyte globulin by conjugation with melphalan.

An attempt was made to increase the in vivo immunosuppressive powers of antilymphocyte globulin (ALG) by conjugating it with malphalan (MEL), an alkylating agent, via an inert intermediate carrier (polyglutamic acid). Careful controls to distinguish between increased activity attributable to the conjugate per se, as opposed to synergy between the components of the complex, were included. Conjugation did not destroy the alkylating properties of the drug nor the cytotoxic activity of the antibody. The effect of MEL-ALG complexes on skin allograft survival in both inbred and outbred strains were appraised. In neither system did the immunosuppressive powers of the conjugate exceed those of ALG alone, regardless of the dose used. We conclude that alkylating drugs are not suitable for this particular purpose.

Animals↗
PubMed1986

Structure and properties of casein kinase-2 from Saccharomyces cerevisiae. A comparison with the liver enzyme.

A type-2 casein kinase (YCK-2), lacking the 25-kDa autophosphorylatable beta subunit characteristic of animal casein kinases-2, has been obtained in a nearly pure form from Saccharomyces cerevisiae and was compared with liver casein kinase-2 (LCK-2). A 22-kDa phosphorylatable protein, copurifying with YCK-2, can be removed by ultracentrifugation at low ionic strength and is shown by several criteria to be unrelated to the beta subunit of LCK-2. The native Mr of YCK-2, deprived of the 22-kDa phosphoprotein, is about 150 000. Limited proteolysis experiments show that YCK-2 included 37-kDa catalytic subunits, which can be converted into still active 35-kDa proteolytic derivatives. These data are consistent with a homotetrameric quaternary structure as opposed to the heterotetrameric subunit composition alpha 2 beta 2 of LCK-2 and other animal casein kinases-2. Although many properties of YCK-2 and LCK-2, including substrate specificity, inhibition by heparin, polyglutamic acid and quercetin and stimulation by polyamines, are similar; their stability under denaturing and dissociating conditions and their response to polybasic peptides are quite different. In particular YCK-2 is more readily denatured than LCK-2 by heating and exposure to urea, sodium dodecylsulphate and deoxycholate while its activity is inhibited by 100-150 mM NaCl, which conversely stimulates LCK-2 activity 2-3-fold. The Km value of the synthetic peptide substrate Ser-(Glu)5 for YCK-2 is not significantly changed by the addition of polylysine. On the contrary the Km value of the same peptide substrate for LCK-2 decreases approximately tenfold upon addition of polylysine, which also prevents the fast autophosphorylation of the kinase at its beta subunit. These data suggest that the beta subunit of animal CK-2 may play a role in determining both the stability of the enzyme and its regulation and that, consequently, the different properties of YCK-2 may be at least in part accounted for by its lack of beta subunits.

Animals↗
PubMed2006

Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin.

Proteolytic but chitinase-deficient microbial cultures were isolated from shrimp shell waste and characterized. The most efficient isolate was found to be a mixed culture consisting of two Bacillus licheniformis strains, which were first determined microscopically and physiologically. Molecular characterization was carried out by sequencing the 16S rRNA gene of both strains. According to the residual protein and ash content, the chitin obtained by fermentation of such a mixed culture was found to be comparable to a commercially available, chemically processed product. However, the strikingly high viscosity (80 versus 10 mPa of the commercially available sample) indicates its superior quality. The two strains differed in colony morphology and in their secretion capabilities for degradative extracellular enzymes. Sequencing of the loci encoding amylase, cellulase, chitinases, and proteases, as well as the degS/degU operon, which is instrumental in the regulation of degradative enzymes, and the pga operon, which is responsible for polyglutamic acid production, revealed no differences. However, a frameshift mutation in chiA, encoding a chitinase, was validated for both strains, providing an explanation for the ascertained absence of chitinolytic activities and the concomitant possibility of producing highly viscous chitin in a fermentational deproteinization process.

Amino Acid Sequence↗
PubMed1982

Monocyte-mediated damage to Rhizopus oryzae hyphae in vitro.

Clinicopathological correlations from human cases and experimental animal studies suggest that neutrophils are critical components of the host response to mucormycosis but that other cellular defense mechanisms appear to be important as well. Since our previous studies demonstrated that Rhizopus oryzae hyphae which are too large to be ingested completely can be damaged and probably killed by human neutrophils, we studied the antihyphal activity of human monocytes. As with neutrophils, light and electron microscopic studies indicated that monocytes attached to hyphae and appeared to destroy them in the absence of serum. As judged by our previously described assay for the leukocyte-induced inhibition of [14C]uracil uptake by hyphae, quantitative damage to hyphae by monocytes was 40.8 +/- 2.2% in 54 experiments. Neither attachment to nor damage of hyphae by monocytes was augmented by the presence of 10% human serum. As with neutrophils, monocyte-mediated damage of R. oryzae was significantly decreased by some inhibitors of oxidative metabolism and scavengers of the potentially microbicidal oxidative leukocyte products, which included 10(-4)M sodium azide, 10 (-3) M sodium cyanide, catalase, 10(-3) M histidine, 10(-3) M tryptophan, and 10(-4) M 1,4-diazobicyclo[2.2.2]octane but not superoxide dismutase, 1.4 X 10(-2) M dimethyl sulfoxide, and 4.0 X 10(-1) M mannitol. Moreover, monocytes from three patients with chronic granulomatous disease failed to damage hyphae at all. In contrast to our previous data for neutrophils, polyanions (10(-5) M polyaspartic or polyglutamic acid) did not inhibit monocyte-mediated hyphal damage. Thus, monocytes can damage and probably kill R. oryzae hyphae by oxidative mechanisms and so may be involved in host defense mechanisms against mucormycosis.

Azides↗
PubMed1989

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases.

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.

ADP Ribose Transferases↗
PubMed1981

Transcription of vesicular stomatitis virus activated by pardaxin, a fish toxin that permeabilizes the virion membrane.

The toxic protein, Pardaxin, of the Red Sea flatfish Pardachirus marmoratus readily induced transcription of vesicular stomatitis virus by making the virion membrane permeable to nucleoside triphosphates in the absence of nonionic detergents. Virion transcription was activated over a wide range of Pardaxin concentrations, but at optimal concentrations, the rate of transcription exceeded that induced by Triton X-100. The inhibitory effect of M protein was manifested for both Pardaxin-induced and Triton-induced transcription at high concentrations of vesicular stomatitis virions; however, unlike the Triton-induced reaction, the inhibitory effect of M protein was not reversed by polyglutamic acid added to the Pardaxin-induced transcription reaction. We propose that activation of virion transcription by Pardaxin resembles more closely intracellular transcription initiated by virion penetration than does detergent-activated transcription of vesicular stomatitis virus.

DNA-Directed RNA Polymerases↗
PubMed1990

Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract.

We report here a mammalian cell-free system that can support chromatin assembly. Effective nucleosome assembly in HeLa cell extracts occurred at 125 to 200 mM KCl or potassium glutamate. At this physiological K+ ion concentration, two types of chromatin assembly were observed. The first was interfered with by Mg2+. Other cations such as Mn2+, Ca2+, Fe3+, and spermidine also inhibited this type of nucleosome assembly. The second type of assembly occurred in the presence of Mg2+ and at least equimolar ATP. However, even in the presence of ATP, excess Mg2+ inhibited assembly and promoted catenation of DNA; these effects could be circumvented by excess ATP, GTP, EDTA, or polyglutamic acid. The critical DNA concentration for optimum assembly in both pathways suggested a stoichiometric association of histones with DNA. The spacing of nucleosomes formed by both types of assembly on linear and circular DNA was reasonably regular, but chromatin assembled in the presence of ATP and Mg2+ was more stable.

Adenosine Triphosphate↗
PubMed1994

Investigations on the role of flagella in adhesion of Pseudomonas aeruginosa to mouse and human corneal epithelial proteins.

Antiflagellar monoclonal antibodies (MAbs) were used in an overlay assay to determine whether flagella bind to blots of mouse and human corneal epithelial proteins (CEPs). The role of carbohydrates and surface charge was also explored by preincubation of blots with periodate or neuraminidase, or flagella with monosaccharides or charged compounds, respectively. Periodate slightly decreased binding of flagella, while sialic acid inhibited binding, and the effect was dose dependent. Neuraminidase treatment of blots or incubation of flagella with a negatively, but not a positively charged compound, also blocked binding. The data suggest that flagella interact with mouse and human CEPs by electrostatic mechanisms.

Animals↗
PubMed1992

Identification and developmental expression of a novel low molecular weight neuronal intermediate filament protein expressed in Xenopus laevis.

Xenopus laevis is a valuable model system for the study of vertebrate neuroembryogenesis. However, very few well-characterized nervous system-specific molecular markers are available for studies in this organism. We screened a X. laevis adult brain cDNA library using a cDNA probe for mouse low molecular weight neurofilament protein (NF-L) in order to identify neuron-specific intermediate filament proteins. Clones for two distinct neuron-specific intermediate filament proteins were isolated and sequenced. One of these encoded for a Xenopus NF-L (XNF-L) and the other for a novel neuron-specific Xenopus intermediate filament protein (XNIF) that was present earlier and more abundantly than XNF-L during development. XNIF contained a central rod domain with multiple sequence features characteristic of IF proteins. The XNF-L was very similar to mouse NF-L, with a 77% sequence identity in the rod domain and the presence of a polyglutamic acid region in the tail domain, characteristic of type IV neurofilament proteins. In contrast, XNIF showed only 60% identity to mouse NF-L in the rod domain and lacked the glutamic acid-rich sequence in the tail domain. XNIF also had a very low (approximately 38%) sequence identity in the head and tail domains as compared to NF-L and other neurofilament proteins (45% identity to the head domain of alpha-internexin). In the adult frog, XNIF mRNA is detected by Northern blots only within the nervous system and by in situ hybridization histochemistry exclusively in neurons, particularly in the medullary reticular system and spinal cord. Antisera raised against the unique tail region of XNIF detected a single distinct 60 kDa band in Western blots of nervous system cytoskeletal preparations, and this XNIF immunoreactivity was concentrated in axons in the PNS and in small perikarya in the dorsal root ganglion. In contrast, NF-L immunoreactivity was principally in the large perikarya in the dorsal root ganglion. In development, XNIF mRNA appears more abundant than XNF-L mRNA in all premetamorphic stages examined. XNIF mRNA is first detectable at stage 24 (26 hr), whereas stable expression of XNF-L is at stage 35/36 (50 hr). XNIF immunoreactivity is detectable within the cement gland, within many neuronal cell bodies and axon tracts within the developing nervous system, and within all cellular layers of the developing retina. The availability of these two distinct neuron-specific intermediate filament proteins, with different temporal and spatial expression patterns, should provide new markers as well as targets for functional perturbation in the developing X. laevis nervous system.

Amino Acid Sequence↗
PubMed1991

Nature of the inhibitory effect of complex saccharide moieties on the tight binding of human spermatozoa to the human zona pellucida.

Fucoidin and heparin sulfate inhibit binding of human sperm to the human zona pellucida under hemizona assay (HZA) conditions. Here we used the HZA to further assess tight sperm binding with/without preincubation of the sperm with other sulfated and nonsulfated glycoconjugates and charged polymers. Fucoidin significantly inhibited binding compared with controls (greater than 75% inhibition), even if sperm were washed after preincubation with the saccharide. Dextran sulfate also produced significant inhibition, although to a lesser extent (54% inhibition). Chondroitin sulfates A and B, heparin, and dextran did not affect binding. Sodium sulfate and polyglutamic acid did not affect HZA results; polyphosphates produced only moderate inhibition. The potent inhibitory effect of the sulfated carbohydrates fucoidin and dextran is probably competitive (receptor-ligand type) in nature. However, the lack of significant effects of simple charged molecules (nonspecific effects) suggests that the degree of sulfation (charge) may not be crucial to its inhibitory action.

Adult↗
PubMedDate not supplied

Macrophage interaction with mycobacteria including M. leprae.

Resistance properties of pathogenic mycobacteria to macrophage bactericidal activity seems to be due mostly to the composition and constitution of their cell walls. In the case of Mycobacterium tuberculosis, sulfatides and polyglutamic acid could be implicated in the phenomenon of fusion inhibition between phagosomes and lysosomes. M. leprae and M. lepraemurium, which do not seem to inhibit fusions are protected by a thick electron transparent zone (ETZ) that seems to be composed of mycosides. This layer would inhibit lysosomal enzyme diffusion inside phagosomes. As ETZ does not exist in mycobacteria before their phagocytosis, we have tried to see when and how it is formed inside macrophages. We have compared ETZ formation in M. leprae and M. avium which both contain mycosides. These two species were allowed to be phagocytized by mouse bone-marrow derived macrophage and samples were taken for electron microscopy during the first hours of phagocytosis and also during several weeks of incubation. In M. avium ETZ appeared within 1 to 2 hours after phagocytosis. It seems to be formed by a sort of swelling of the thin electron transparent layer of the bacterial cell wall. This swelling occurs only in regions where the external polysaccharide layer of M. avium starts to disappear. After 1 to 3 hours, this layer was completely absent and all bacteria were enveloped in a thick ETZ. In M. leprae, the ETZ is also formed within one hour after ingestion. However, the presence in some bacteria of a very thin dense layer located at the original place of the outer dense layer of the cell wall does not fit well with the idea of ETL swelling. In addition, the appearance of a thick dense layer located between the ETZ and the phagosome membrane is not yet understood. The ETZ formed also rapidly in macrophages infected with heat killed cells of M. avium or M. leprae. This shows that its formation does not require the active participation of the bacterium. As already proposed ETZ seems to lessen considerably the diffusion of lysosomal enzymes towards the bacterium in both species. In M. leprae it seems especially efficient because despite acid phosphatase activity found in many phagosomes, neither the number of bacteria per macrophage nor their state of degradation changed during 3 and a half months of macrophage culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Immunologically induced peliosis hepatis in rats.

Peliosis hepatis has been induced immunologically with anti-rat glomerular basal membrane rabbit serum in rats pre-sensitized with a rare earth metal complex, neodymium pyrocatechin disulphonate (NPD). This is the first experimental evidence that peliosis hepatis may develop as a result of an immunological process. It is noteworthy that in this experimental form of peliosis hepatis and in that observed earlier in rats treated with basic polyglutamic acid derivatives, severe defibrination was detected and, as in most human cases, not only the liver but other organs were also involved in the peliotic lesions. Since the rare earth metal compounds, among them the pyrocatechin disulphonate complex of neodymium, depress the reticulo-endothelial activity, a role of the reticulo-endothelial system in the pathogenesis of this experimental form of peliosis hepatis is suggested.

Animals↗
PubMed1994

Quantitative analyses of electrostatic interactions between NADPH-cytochrome P450 reductase and cytochrome P450 enzymes.

A decline in the ionic interactions in the medium with increasing ionic strength (decrease in the ionic activity coefficients) was accompanied by an increase in the fast phase rate constants of CYP2B4 and CYP1A2 reduction. The stimulations were observed both in reconstituted P450 systems and in microsomes. An increase in the ionic strength from 10 to 100 mM sodium phosphate resulted in a 7-fold decrease in the Km of CYP1A2 for NADPH-cytochrome P450 reductase, while the Vmax was unchanged. When ionic interactions were neutralized without changing the ionic strength by addition of charged oligopeptides (polylysine and polyglutamic acid), stimulations of CYP1A2 and CYP2B4 reduction were observed. Increase in the ionic strength also enhanced the rate of cytochrome P450 reduction in control and phenobarbital-induced rat liver microsomes and in reconstituted systems containing purified rat liver CYP2C6, CYP2C12, CYP2C13, and CYP2E1, and rat reductase. A method was devised for the quantification of the number of charges involved in protein-protein interactions based on the estimation of the ionic activity coefficients. Different numbers of charged residues are involved in the repulsion between different P450 forms and the reductase. The product of the number of charges involved in the interaction between rabbit reductase and CYP2B4 is 10.84 compared with the value of 6.64 for the reductase-CYP1A2 interaction.

Animals↗
PubMed1994

Molecular cloning and expression of alternatively spliced PITSLRE protein kinase isoforms.

Minimal ectopic expression of the p58GTA protein kinase results in a provocative phenotype involving cell cycle delay, mitotic catastrophe, and decreased cell viability. In addition, this kinase is well conserved evolutionarily, ubiquitously expressed, and its genes map to a position on human chromosome 1 frequently deleted in the late stages of tumorigenesis. Here we report that the p58GTA protein kinase is a member of a larger subfamily of proteins. The mRNAs encoding these proteins are generated by alternative splicing from multiple duplicated genes. These isoforms range in size from 50 to 110 kDa. Divergence between the alternatively spliced isoforms is localized to the amino-terminal region of the molecule. The entire p58GTA open reading frame is conserved in most of these p58GTA isoforms. The predicted sequences of the larger isoforms encode bipartite nuclear localization signal sequences and extensive polyglutamic acid domains. Antibodies to the p58GTA isoform were used to confirm the presence of the alternatively spliced isoforms in different cell types as well as identify two additional isoforms that appear to arise from a separate gene(s). Cellular fractionation studies indicate that one of the isoforms is found only in the nucleus, and the remainder are found in both the cytoplasm and the nucleus. Expression and localization of some p58GTA isoforms suggest that they may have specialized cellular functions. Because of the large number of isoforms generated from multiple genes we propose naming these kinases PITSLRE alpha 1, alpha 2-1, alpha 2-2, alpha 2-3 alpha 2-4, beta 1, beta 2-1, and beta 2-2 based on the conserved sequence of the PSTAIRE box unique to p34cdc2 kinases and the gene from which they are transcribed.

Alternative Splicing↗
PubMed1993

The effect of heparin and related proteoglycans on allergen and PAF-induced eosinophil infiltration.

Exposure of normal guinea pigs to an aerosol of PAF induced a selective increase in the percentage of eosinophils in bronchoalveolar lavage (BAL) fluid 24 h after challenge. Challenge of actively sensitised guinea pigs with an aerosol of ovalbumin also induced a selective increase in the percentage of eosinophils recovered in BAL fluid 24 h post challenge. Pretreatment of actively sensitised guinea pigs or normal guinea pigs with unfractionated heparin significantly reduced such eosinophil infiltration induced by allergen or PAF challenge respectively, although higher amounts of heparin were required to inhibit antigen induced eosinophil infiltration. Similar effects were also observed following treatment with the low molecular weight heparin-like material ORG 10172 but not the anionic molecule polyglutamic acid or high molecular weight dextrans. These results suggest that proteoglycans may possess anti-allergic activity that is not necessarily related to either such molecules being anionic in nature nor to anti-coagulant activity.

Aerosols↗
PubMed1993

Characterization of the interactions between procoagulant albumin and human endothelial cells.

Normal human plasma contains procoagulant albumin (PC-Al), an anionic form of albumin that induces tissue factor (TF) activity in human umbilical vein endothelial cells (HUVEC) and monocytes. In this study, we investigated both the interactions between HUVEC and PC-Al and the mechanism by which PC-Al induces TF activity. Binding of PC-Al to HUVEC was specific and reversible. Further studies indicated that membrane-bound PC-Al was not internalized by HUVEC. A potential receptor on HUVEC was suggested by studies in which the capacity of a variety of reagents to inhibit the activity of PC-Al was quantitated. Induction of TF activity by PC-Al was antagonized by dextran sulfate, heparin, fucoidan, and concanavalin A but not by ovalbumin, polyglutamic acid, or polyvinyl sulfate. This competition profile bears similarities to those reported for scavenger receptors that have been identified on both HUVEC and monocytes. Involvement of protein kinase C (PKC) in the PC-Al-induced enhancement of TF activity was suggested by experiments in which staurosporine, an inhibitor of PKC, suppressed the activity of PC-Al. The induction of TF activity by PC-Al was further characterized by using a quantitative polymerase chain reaction assay. Increased TF mRNA was first seen after 1 hour of incubation with PC-Al. Maximal observed expression occurred at 2 hours, but at 5 hours, expression had significantly decreased. Monocytes could also be induced to express TF mRNA after a 2-hour incubation with PC-Al. These results suggest that the functionally relevant binding of PC-Al to HUVEC may be mediated through interactions with a membrane constituent that has some of the properties of a scavenger receptor and that this interaction augments TF activity by enhancing transcription of TF mRNA, at least in part, by a mechanism that is dependent on activation of PKC.

Base Sequence↗