[Pharmacological studies on decanohydroxamic acid. 3. General pharmacological action. (2)].
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Animals↗
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Siderophores: diverse roles in microbial and human physiology.
Siderophores, defined as high affinity iron(III) ion transport agents, and their cognate membrane-bound receptor complexes, occur in the enteric bacteria Escherichia coli and Salmonella typhimurium. The total system is tightly regulated by iron repression. The transport properties of the specific siderophores enterobactin and ferrichrome (which is not made by these particular enteric bacteria) have been examined in detail. In E. coli the outer membrane receptor for ferrichrome is programmed by the tonA gene; the receptor also serves as the binding site for T1, T5, phi80, albomycin and colicin M. Similarly, in S. typhimurium phage ES18, ferrichrome and albomycin compete for the genetic equivalent of the tonA locus. The ability of ascorbic acid to protect against atherosclerosis as well as rhinovirus infection in humans may be related to the role of the vitamin in iron metabolism. Deferrisiderophores are clinically useful in the treatment of acute and chronic iron poisoning but, on the other hand, they could constitute a natural hazard by transporting actinides, such as 239Pu, through the food chain.
Arteriosclerosis↗
Structures of the "open" and "closed" state of trypanosomal triosephosphate isomerase, as observed in a new crystal form: implications for the reaction mechanism.
The structure of trypanosomal triosephosphate isomerase (TIM) has been solved at a resolution of 2.1A in a new crystal form grown at pH 8.8 from PEG6000. In this new crystal form (space group C2, cell dimensions 94.8 A, 48.3 A, 131.0 A, 90.0 degrees, 100.3 degrees, 90.0 degrees), TIM is present in a ligand-free state. The asymmetric unit consists of two TIM subunits. Each of these subunits is part of a dimer which is sitting on a crystallographic twofold axis, such that the crystal packing is formed from two TIM dimers in two distinct environments. The two constituent monomers of a given dimer are, therefore, crystallographically equivalent. In the ligand-free state of TIM in this crystal form, the two types of dimer are very similar in structure, with the flexible loops in the "open" conformation. For one dimer (termed molecule-1), the flexible loop (loop-6) is involved in crystal contacts. Crystals of this type have been used in soaking experiments with 0.4 M ammonium sulphate (studied at 2.4 A resolution), and with 40 microM phosphoglycolohydroxamate (studied at 2.5 A resolution). It is found that transfer to 0.4 M ammonium sulphate (equal to 80 times the Ki of sulphate for TIM), gives rise to significant sulphate binding at the active site of one dimer (termed molecule-2), and less significant binding at the active site of the other. In neither dimer does sulphate induce a "closed" conformation. In a mother liquor containing 40 microM phosphoglycolohydroxamate (equal to 10 times the Ki of phosphoglycolohydroxamate for TIM), an inhibitor molecule binds at the active site of only that dimer of which the flexible loop is free from crystal contacts (molecule-2). In this dimer, it induces a closed conformation. These three structures are compared and discussed with respect to the mode of binding of ligand in the active site as well as with respect to the conformational changes resulting from ligand binding.
Amino Acid Sequence↗
Biochemical and pharmacological properties of a new topical anti-inflammatory compound, 9-phenylnonanohydroxamic acid (BMY 30094).
Drugs which block the biosynthesis of leukotrienes and prostaglandins may have potential in the treatment of psoriasis and other skin diseases. The biochemical and anti-inflammatory activity of 9-phenylnonanohydroxamic acid (BMY 30094) is described. BMY 30094 inhibited human neutrophil 5-lipoxygenase with an IC50 of 5.7 microM. BMY 30094 also blocked human platelet cyclooxygenase and lipoxygenase with IC50 values of 15.2 and 15.0 microM, respectively. Topical application of this compound blocked arachidonic acid and 12-O-tetradecanoylphorbol ester-induced mouse skin inflammation with activity comparable to that observed for lonapalene. The topical ED50 for BMY 30094 in the arachidonic acid-induced inflammation model is 2.2 mumoles/ear. In the sub-cutaneous carrageenan sponge assay in rats, BMY 30094 blocked LTB4 and PGE2 production and inhibited neutrophil migration. This compound would be a useful tool to determine the role of arachidonic acid metabolites in the etiology of inflammatory dermatoses.
Administration, Cutaneous↗
In-situ enrichment of ARGs and their carriers in soil by hydroxamate siderophore: A promising biocontrol approach for source reduction.
Pathogenic microorganisms with antibiotic resistance genes (ARGs) pose a serious threat to public health and soil ecology. Although new drugs and available antibacterial materials can kill ARG carriers but accidentally kill beneficial microorganisms. Therefore, the rapid enrichment and separation of ARGs and their carriers from soil is becoming an important strategy for controlling the diffusion of ARGs. Hydroxamate siderophore (HDS) has gained widespread attentions for its involvement in trace element transfer among microorganisms in the soil environment, we thus explored an in-situ trapping-enrichment method for ARGs and their carriers via a small molecular HDS secreted by Pseudomonas fluorescens HMP01. In this study, we demonstrate that HDS significantly in-situ traps and enriches certain ARGs, including chloramphenicol, MLS, rifamycin, and tetracycline resistance genes in the soil environment. The enrichment efficiencies were 1473-fold, 38-fold, 17-fold, and 5-fold, respectively, higher than those in the control group. Specifically, the primary enriched ARGs were rpoB, mphL, catB2, and tetA(60), and Bacillus, Rhizobium, Rossellomorea, and Agrobacterium were hosts for these ARGs. This enrichment was caused by the upregulation of chemotaxis genes (e.g., cheW, cheC, and cheD) and rapid biofilm formation within the enriched bacterial population. Notably, representative ARGs such as cat, macB, and rpoB were significantly reduced by 36%, 85.7%, and 72%, respectively, in the paddy soil after HDS enrichment. Our research sheds light on the potential application of siderophore as a rapping agent for the eco-friendly reduction of ARGs and their carriers in soil environments.
Soil Microbiology↗
Crystal structure of the catalytic domain of human ADAM33.
Adam33 is a putative asthma susceptibility gene encoding for a membrane-anchored metalloprotease belonging to the ADAM family. The ADAMs (a disintegrin and metalloprotease) are a family of glycoproteins implicated in cell-cell interactions, cell fusion, and cell signaling. We have determined the crystal structure of the Adam33 catalytic domain in complex with the inhibitor marimastat and the inhibitor-free form. The structures reveal the polypeptide fold and active site environment resembling that of other metalloproteases. The substrate-binding site contains unique features that allow the structure-based design of specific inhibitors of this enzyme.
ADAM Proteins↗
Enhanced expression of uridine diphosphate-N-acetylglucosaminyl transferase (OGT) in a stable, tetracycline-inducible HeLa cell line using histone deacetylase inhibitors: kinetics of cytosolic OGT accumulation and nuclear translocation.
We have created a stable, tetracycline-inducible HeLa cell line that overexpresses murine uridine diphosphate-N-acetylglucosaminyl transferase (OGT). Tetracycline increased cytosolic OGT activity about 4-fold in a dose-dependent manner (ED(50)=0.03 microg/ml) with enhanced activity observable at 8h and maximal activity observable by 40h. Enhanced OGT activity was due to overexpression of OGT protein as determined by Western analysis. Trichostatin A (TSA), a potent and specific histone deacetylase inhibitor (HDI), markedly enhanced tetracycline-induced OGT gene expression, resulting in a >10-fold increase in OGT activity (>50-fold compared to that of uninduced cells). Other HDIs such as butyrate (ED(50)=1.6mM) and propionate (ED(50)=8mM) were similarly effective, but less potent than TSA (ED(50)=120 nM). We next examined the appearance of recombinant OGT in cytosol and nucleosol at various times (10 min to 6h) after inducing OGT gene. Within 2h, recombinant OGT was detected by Western analysis in both cytosol and nucleosol. This indicates rapid biosynthesis and accumulation of recombinant OGT in the cytosol and subsequent nuclear translocation. Entry of OGT into the nucleus was closely correlated with enhanced O-linked glycosylation of nuclear proteins, indicating that recombinant OGT was enzymatically active. The ability to rapidly induce OGT expression in a stable cell line provides an excellent model system to study the mechanism(s) underlying OGT nuclear translocation and a useful system to elucidate the cascade of signaling events related to O-linked glycosylation.
Animals↗
Gene expression within a chromatin domain: the role of core histone hyperacetylation.
Scaffold-attached regions (SAR elements) increase transcriptional rates for integrated but not episomal templates, and this effect can be potentiated by using an epigenetically active reagent, butyrate. The action of butyrate is a direct one, not involving de novo protein synthesis, and can be mimicked by using a novel and highly specific inhibitor of histone deacetylases, (R)-trichostatin A. This leads to a model in which SAR elements serve to stabilize the chromosomal topology arising as a consequence of hyperacetylation of histone cores. The synergistic effects of histone hyperacetylation and SARs are mediated by promoter upstream elements since, for a simple TATA box, the response to both parameters is an additive one.
Acetylation↗
Tripeptide (glutathione) synthetase. Purification, properties, and mechanism of action.
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Adenine Nucleotides↗
Structures of Cys319 variants and acetohydroxamate-inhibited Klebsiella aerogenes urease.
Cys319 is located on a mobile flap covering the active site of Klebsiella aerogenes urease but does not play an essential role in catalysis. Four urease variants altered at position C319 range from having high activity (C319A) to no measurable activity (C319Y), indicating Cys is not required at this position, but its presence is highly influential [Martin, P. R., & Hausinger, R. P. (1992) J. Biol. Chem. 267, 20024-20027]. Here, we present 2.0 A resolution crystal structures of C319A, C319S, C319D, and C319Y proteins and the C319A variant inhibited by acetohydroxamic acid. These structures show changes in the hydration of the active site nickel ions and in the position and flexibility of the active site flap. The C319Y protein exhibits an alternate conformation of the flap, explaining its lack of activity. The changes in hydration and conformation suggest that there are suboptimal protein-solvent and protein-protein interactions in the empty urease active site which contribute to urease catalysis. Specifically, we hypothesize that the suboptimal interactions may provide a significant source of substrate binding energy, and such hidden energy may be a common phenomenon for enzymes that contain mobile active site loops and undergo an induced fit. The acetohydroxamic acid-bound structure reveals a chelate interaction similar to those seen in other metalloenzymes and in a small molecule nickel complex. The inhibitor binding mode supports the proposed mode of urea binding. We complement these structural studies with extended functional studies of C319A urease to show that it has enhanced stability and resistance to inhibition by buffers containing nickel ions. The near wild-type activity and enhanced stability of the C319A variant make it useful for further studies of urease structure-function relationships.
Amino Acid Sequence↗
Specificity in enzyme inhibition. 2. -Aminohydroxamic acids as inhibitors of histidine decarboxylase and 3,4-dihydroxyphenylalanine decarboxylase.
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Alanine↗
Levels and molecular forms of MMP-7 (matrilysin-1) and MMP-8 (collagenase-2) in diseased human peri-implant sulcular fluid.
OBJECTIVES: Matrix metalloproteinases (MMPs) play crucial role in various tissue destructive inflammatory processes by degrading almost all peri-cellular and basement membrane components. MMP-8 (collagenase-2) is the major MMP in periodontitis. MMP-7 (matrilysin-1), in addition to its ability to degrade matrix and basement membrane components, activates other latent pro-MMPs and defensins, host cell-derived antimicrobial cryptidins. The aim of the present study was to characterize the relationship, levels and molecular forms of MMP-8 and MMP-7 in diseased peri-implant sulcular fluid (PISF). MATERIALS AND METHODS: Seventy-two human dental implant fluid samples were collected with filter paper strips from peri-implant sulci from healthy and untreated diseased implant sites. Gingival index (GI) and/or bone resorption (BR) were also recorded. Western immunoblot method with polyclonal anti-human-MMP-8 and monoclonal anti-human-MMP-7 antibodies was used, and immunoreactivities were quantified with computer scanning program. The effects of MMP inhibitors (doxycycline, chemically modified tetracycline-3, clodronate, CTT-peptide and marimastat) were studied on the activity of recombinant human matrilysin-1 (MMP-7) using beta-casein degradation assay. RESULTS: The levels of active forms of MMP-8 and MMP-7 were significantly elevated in diseased PISF in relation to healthy PISF. Furthermore, MMP-8 and MMP-7 levels correlated significantly to each other and GI. MMP-8 was present not only as bands corresponding to 75-kDa polymorphonuclear leukocyte (PMN) -type pro- and 65-kDa active forms, but also as 55-kDa non-PMN-type pro- and 45-kDa active forms. Immunoreactivities > 80 kDa most likely represented dimeric and/or inhibitor-bound MMP-8 complexes and the low molecular weight (< 30 kDa) species were apparently degraded fragments. In diseased PISF, 19-21-kDa active MMP-7 and 28-30-kDa pro-MMP-7 species were detected, and the active 19-21-kDa forms of MMP-7 predominated in diseased PISF. Doxycycline (50 micro m and 250 micro m), chemically modified non-antimicrobial tetracycline (CMT-3) (50 micro m and 100 micro m), clodronate (a bisphosphonate, 20 micro m and 500 micro m) and the cyclic CTT (CTTHWGFTLC)-peptide (125 micro m and 250 micro m), all known broad-spectrum or selective MMP-inhibitors, did not inhibit the activity of human recombinant MMP-7; only marimastat (1 micro m and 5 micro m) inhibited MMP-7. DISCUSSION: Increased immunoreactivities of the active MMP-8 and MMP-7 species in PISF from diseased peri-implantitis lesions eventually reflect the stage and course of peri-implantitis; MMP-7 may potentially act as MMP-8 and defensin activator in diseased PISF. CONCLUSION: The elevated levels of MMP-8 and matrilysin-1/MMP-7 were identified in active forms in diseased PISF, but MMP-7 was less prominent. MMP inhibitors, potential future tissue protective drugs, seemingly do not interfere with the defensive antibacterial action of MMP-7.
Adult↗
Studies on mammalian intestinal peroxidase.
A peroxidase, purified from rat small intestine to apparent homogeneity as judged by polyacrylamide-gel electrophoresis, exhibited an absorbance ratio (A412/A280) of 0.783. Its Mr (44000 +/- 1000) and spectral properties were similar to those of the pig intestinal enzyme. The velocity constant for the reaction between rat intestinal peroxidase and hydrogen peroxide was found to be 1.8 x 10(7) M-1 . s-1. Benzhydroxamic acid inhibited the peroxidative oxidation of guaiacol by intestinal peroxidase from both species but the concentration required to cause half-inhibition of the enzyme from the rat was higher by one order of magnitude than for the pig enzyme. The amino acid composition of highly-purified pig intestinal peroxidase showed a relative abundance of basic amino acids (lysine and arginine) and was similar to that of lactoperoxidase, but not that of myeloperoxidase. The initial ten amino acid residues of this enzyme (the first reported partial sequence for a mammalian peroxidase) were also determined.
Amino Acid Sequence↗
Histone acetylation is required to maintain the unfolded nucleosome structure associated with transcribing DNA.
Nucleosomes associated with transcribing chromatin of mammalian cells have an unfolded structure in which the normally buried cysteinyl-thiol group of histone H3 is exposed. In this study we analyzed transcriptionally active/competent DNA-enriched chromatin fractions from chicken mature and immature erythrocytes for the presence of thiol-reactive nucleosomes using organomercury-agarose column chromatography and hydroxylapatite dissociation chromatography of chromatin fractions labeled with [3H]iodoacetate. In mature and immature erythrocytes, the active DNA-enriched chromatin fractions are associated with histones that are rapidly highly acetylated and rapidly deacetylated. When histone deacetylation was prevented by incubating cells with histone deacetylase inhibitors, sodium butyrate or trichostatin A, thiol-reactive H3 of unfolded nucleosomes was detected in the soluble chromatin and nuclear skeleton-associated chromatin of immature, but not mature, erythrocytes. We did not find thiol-reactive nucleosomes in active DNA-enriched chromatin fractions of untreated immature erythrocytes that had low levels of highly acetylated histones H3 and H4 or in chromatin of immature cells incubated with inhibitors of transcription elongation. This study shows that transcription elongation is required to form, and histone acetylation is needed to maintain, the unfolded structure of transcribing nucleosomes.
Acetylation↗
Helicobacter pylori containing only cytoplasmic urease is susceptible to acid.
Helicobacter pylori, an important etiologic agent in a variety of gastroduodenal diseases, produces large amounts of urease as an essential colonization factor. We have demonstrated previously that urease is located within the cytoplasm and on the surface of H. pylori both in vivo and in stationary-phase culture. The purpose of the present study was to assess the relative contributions of cytoplasmic and surface-localized urease to the ability of H. pylori to survive exposure to acid in the presence of urea. Toward this end, we compared the acid resistance in vitro of H. pylori cells which possessed only cytoplasmic urease to that of bacteria which possessed both cytoplasmic and surface-localized or extracellular urease. Bacteria with only cytoplasmic urease activity were generated by using freshly subcultured bacteria or by treating repeatedly subcultured H. pylori with flurofamide (1 microM), a potent, but poorly diffusible urease inhibitor. H. pylori with cytoplasmic and surface-located urease activity survived in an acid environment when 5 mM urea was present. In contrast, H. pylori with only cytoplasmic urease shows significantly reduced survival when exposed to acid in the presence of 5 mM urea. Similarly, Escherichia coli SE5000 expressing H. pylori urease and the Ni2+ transport protein NixA, which expresses cytoplasmic urease activity at levels similar to those in wild-type H. pylori, survived minimally when exposed to acid in the presence of 5 to 50 mM urea. We conclude that cytoplasmic urease activity alone is not sufficient (although cytoplasmic urease activity is likely to be necessary) to allow survival of H. pylori in acid; the activity of surface-localized urease is essential for resistance of H. pylori to acid under the assay conditions used. Therefore, the mechanism whereby urease becomes associated with the surface of H. pylori, which involves release of the enzyme from bacteria due to autolysis followed by adsorption of the enzyme to the surface of intact bacteria ("altruistic autolysis"), is essential for survival of H. pylori in an acid environment. The ability of H. pylori to survive exposure to low pH is likely to depend on a combination of both cytoplasmic and surface-associated urease activities.
Benzamides↗
Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster.
The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Bordetella interspecies allelic variation in AlcR inducer requirements: identification of a critical determinant of AlcR inducer responsiveness and construction of an alcR(Con) mutant allele.
Previous studies established the critical roles of AlcR and alcaligin inducer in positive regulation of alcaligin siderophore biosynthesis and transport genes in Bordetella pertussis and Bordetella bronchiseptica. Transcriptional analyses using plasmid-borne alcR genes of B. pertussis UT25 and B. bronchiseptica B013N to complement the alcR defect of B. bronchiseptica strain BRM13 (Delta alcR1 alcA::mini-Tn5 lacZ1) revealed interspecies differences in AlcR inducer requirements for activation of alcABCDER operon transcription. Whereas the B. pertussis UT25 AlcR protein retained strong inducer dependence when produced from multicopy plasmids, B. bronchiseptica B013N alcR partially suppressed the alcaligin requirement for transcriptional activation. Functional analysis of AlcR chimeras produced by interspecies domain swapping and interspecies reciprocal site-specific mutagenesis determined that the phenotypic difference in AlcR inducer dependence was due to a single amino acid difference within the proposed inducer-binding and multimerization domain of AlcR. Structural predictions guided the design of a mutant AlcR protein with a single amino acid substitution at this critical position, AlcR(S103T), that was fully constitutive not only when produced from multicopy plasmids but also at a single-copy gene dosage. These results indicate that AlcR residue 103 affects a critical determinant of alcaligin inducer dependence of AlcR-mediated transcriptional activation. The alcR(S103T) mutant allele is the first alcR(Con) mutant allele identified.
Alleles↗
Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii.
A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes. It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov. The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.
Amino Acid Sequence↗