| 17464077 |
Uberolysin: a novel cyclic bacteriocin produced by Streptococcus uberis |
10.1099/mic.0.2006/005967-0. |
Microbiology (Reading) |
Uberolysin: a novel cyclic bacteriocin produced by Streptococcus uberis
Abstract
- Streptococcus uberis is commonly found in the environment and in association with various bovine body sites and is a major cause of bovine mastitis. Moreover, S. uberis is known to produce a variety of bacteriocin-like inhibitory substances, antimicrobial agents that generally inhibit closely related bacterial species. In this respect, S. uberis strain 42 has previously been shown to produce a novel nisin variant named nisin U. This paper reports that, in addition to nisin U, S. uberis strain 42 produces a second bacteriocin that induces the lysis of metabolically active, susceptible target bacteria and which has therefore been named uberolysin. Isolation of the native active antimicrobial agent revealed that uberolysin is a 7048 Da peptide that is refractory to sequence analysis by Edman degradation. Transposon mutagenesis was used to generate a uberolysin-negative mutant of S. uberis 42 and sequencing of DNA flanking the insertion site revealed, in addition to the structural gene (ublA), several open reading frames likely to be involved in post-translational modification, transport and producer self-protection (immunity), and possibly in regulation of the biosynthetic gene cluster. In addition, a pair of direct repeats that may be involved in bacteriocin acquisition were identified; indeed, ublA could be identified in 18 % of tested S. uberis strains. Enzymic hydrolysis of uberolysin was used to confirm that ublA does indeed encode the precursor of uberolysin, that an unusually short leader sequence of only six amino acids is cleaved during processing of the mature peptide and that uberolysin is post-translationally covalently modified to form a head-to-tail monocycle. Thus, uberolysin is a unique cyclic bacteriocin, belonging to the same family of bacteriocins as enterocin AS-48 and circularin A.
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| 17464953 |
Type 1 Gaucher disease: null and hypomorphic novel chitotriosidase mutations-implications for diagnosis and therapeutic monitoring. |
10.1002/humu.20524 |
Hum. Mutat. |
Type 1 Gaucher disease: null and hypomorphic novel chitotriosidase mutations-implications for diagnosis and therapeutic monitoring.
Abstract
- Human plasma chitotriosidase (Chito) is a useful diagnostic and therapeutic biomarker for Type 1 Gaucher disease (GD). However, approximately 40% of Caucasians are heterozygous or homozygous for a common null mutation, c.1049_1072dup24 (dup24) in the chitotriosidase gene (chitinase 1, CHIT1), that complicates interpretation for heterozygotes and precludes use for null homozygotes. 320 Type 1 GD patients were screened for CHIT1 genotype and plasma Chito enzyme levels; 37% were heterozygous and 4% were homozygous for the CHIT1 dup24 allele. Four patients who had no or very low plasma Chito activities had wild-type (wt)/dup24 or wt/wt CHIT1 genotypes, suggesting the presence of other mutations. Sequencing their CHIT1 genes revealed three novel mutations: p.E74K (E74K), p.G102S (G102S), and a complex exon 10 lesion (c.[1060G>A; 1155G>A; 1156+5_1156+8delGTAA], p.[G354R; L385L; missplicing], designated "complex E/I-10"). The G102S mutation was common in Type 1 GD patients and controls ( approximately 30% of alleles). In contrast, the E74K mutation was rare, present only in three Type 1 GD patients ( approximately 1% of alleles), all of Ashkenazi Jewish (AJ) descent, but it was not found in normal controls. The complex E/I-10 mutation occurred in two Caribbean Hispanic/African Type 1 GD patients and was present in 0 to 6% of alleles among normal controls from different populations. In vitro expression demonstrated that the E74K and G102S alleles had approximately 51% and approximately 23% of wild-type Chito catalytic efficiency, respectively. Expression of the G354R allele alone or with the L385L silent substitution did not produce detectable Chito activity or protein. RNA studies indicated that the complex E/I-10 allele also caused missplicing. Recognition of these mutations, particularly G102S, will facilitate the use and interpretation of plasma Chito activities for disease diagnosis, estimating disease severity, and monitoring therapeutic efficacy in GD.
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| 17480000 |
Single-walled tubulin ring polymers |
10.1002/bip.20752. |
Biopolymers |
Single-walled tubulin ring polymers
Abstract
- An unusual class of nanoscopic, ring-shaped, single-walled biopolymers arises when alphabeta-tubulin is mixed with certain small peptides obtained from various marine organisms and cyanobacteria. The single-ring structures, whose mean molecular weight depends on the specific peptide added to the reaction mixture, usually have sharp mass distributions corresponding, e.g., to rings containing eight tubulin dimers (when the added peptide is cryptophycin) and 14 dimers (e.g., with dolastatin). Although the ring-forming peptides have been shown to possess antimitotic properties when tested with cultured eukaryotic cells (and thus have generated considerable interest as possible agents to be used in the treatment of cancer), it is not our intention to extensively discuss the potential pharmacological properties of the peptides. Rather, we will review the polymeric structures that form and illustrate how certain physical techniques can be used to characterize their properties and interactions. The nanoscopic size and particular geometry of the individual rings make them appropriate targets for scattering and hydrodynamic techniques that provide details about their structure in solution, but it is necessary to relate measured data to postulated structures by nontrivial, albeit straight-forward, mathematical, and computational means. We will discuss how this is done when one uses such methods as small angle neutron scattering, dynamic light scattering, fluorescence correlation spectroscopy, and sedimentation velocity measurements. Moreover, we show that, by using several techniques, one can eliminate degeneracy to provide better discrimination between model structures.
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| 17482713 |
CsV03-3 is a member of a novel gene family from citrus that encodes a protein with DNA binding activity and whose expression is responsive to defense signals and abiotic stress |
10.1016/j.jplph.2007.01.009. |
J Plant Physiol |
CsV03-3 is a member of a novel gene family from citrus that encodes a protein with DNA binding activity and whose expression is responsive to defense signals and abiotic stress
Abstract
- We have identified a novel gene designated CsV03-3 from Citrus sinensis (L.) Osbeck that encodes a 50 amino acid polypeptide with a predicted molecular mass of 5.4kDa and a pI of 3.7. CsV03-3 expression is up-regulated by application of methyl jasmonate, salicylic acid, and abscisic acid as well as by abiotic stress and insect herbivory. CsV03-3 belongs to a small gene family consisting of at least three other closely related members (CsV03-1, CsV03-2, and CsV03-4) whose expression is also responsive to phytohormone application and abiotic stress. Sequence similarity searches of the public databases were unsuccessful in finding sequence homologs to CsV03-3 or any CsV03 family member; however, structural prediction models coupled with model comparison to Protein Data Bank folds indicated that the predicted polypeptide encoded by CsV03-3 has structural similarity to proteins with nucleic acid binding activity. Gel mobility shift assays performed on recombinant CsV03-3 protein demonstrated active binding with dsDNA and, to a lesser extent, ssDNA. Based on the phytohormone-inducible expression patterns, the ability to bind nucleic acids, and the lack of significant sequence homology to public databases, we propose that CsV03-3 and its related homologs defines a new class of nucleic acid binding proteins that are responsive to defense and stress signaling in woody perennials.
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| 17487921 |
Toward a global characterization of the phosphoproteome in prostate cancer cells: identification of phosphoproteins in the LNCaP cell line |
10.1002/elps.200600782. |
Electrophoresis |
Toward a global characterization of the phosphoproteome in prostate cancer cells: identification of phosphoproteins in the LNCaP cell line
Abstract
- Protein phosphorylation plays a major role in most cell-signaling pathways in all eukaryotic cells. Disruptions in phosphorylation-mediated cell-signaling events are associated with various diseases, including cancer. Here, we applied a fully non-gel-based methodology to obtain an initial panel of phosphoproteins from the LNCaP human prostate cancer cell line. The analytical strategy involved enrichment of phosphopeptides by immobilized metal ion affinity chromatography, the use of POROS Oligo R3 to capture phosphopeptides that were not retained with a C18 packing, and gas-phase fractionation in the m/z dimension to extend the dynamic range of the LC-MS/MS analysis. In this pilot investigation, 137 phosphorylation sites in 81 phosphoproteins were identified. The characterized phosphoproteins include kinases, co-regulators of steroid receptors, and a number of cancer-related proteins.
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| 17495006 |
Pharmacological and physiological characterization of d[Leu4, Lys8]vasopressin, the first V1b-selective agonist for rat vasopressin/oxytocin receptors |
10.1210/en.2006-1633. |
Endocrinology |
Pharmacological and physiological characterization of d[Leu4, Lys8]vasopressin, the first V1b-selective agonist for rat vasopressin/oxytocin receptors
Abstract
- Recently, we synthesized and characterized the first selective V(1b) vasopressin (VP)/oxytocin receptor agonist, d[Cha(4)]arginine vasopressin. However, this agonist was only selective for the human receptors. We thus decided to design a selective V(1b) agonist for the rodent species. We started from previous observations showing that modifying [deamino(1),Arg(8)]VP in positions 4 and 8 altered the rat VP/oxytocin receptor selectivity. We synthesized a series of 13 [deamino(1),Arg(8)]VP analogs modified in positions 4 and 8. Among them, one seemed very promising, d[Leu(4), Lys(8)]VP. In this paper, we describe its pharmacological and physiological properties. This analog exhibited a nanomolar affinity for the rat, human, and mouse V(1b) VP receptors and a strong V(1b) selectivity for the rat species. On AtT20 cells stably transfected with the rat V(1b) receptor, d[Leu(4), Lys(8)]VP behaved as a full agonist on both phospholipase C and MAPK assays. Additional experiments revealed its ability to induce the internalization of enhanced green fluorescent protein-tagged human and mouse V(1b) receptors as expected for a full agonist. Additional physiological experiments were performed to further confirm the selectivity of this peptide. Its antidiuretic, vasopressor, and in vitro oxytocic activities were weak compared with those of VP. In contrast, used at low doses, its efficiency to stimulate adrenocorticotropin or insulin release from mouse pituitary or perfused rat pancreas, respectively, was similar to that obtained with VP. In conclusion, d[Leu(4), Lys(8)]VP is the first selective agonist available for the rat V(1b) VP receptor. It will allow a better understanding of V(1b) receptor-mediated effects in rodents.
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| 17496129 |
Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin |
10.1128/AEM.00485-07. |
Appl Environ Microbiol |
Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin
Abstract
- Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.
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| 17497764 |
Conformational templates for rational drug design: flexibility of cyclo(D-Pro1-Ala2-Ala3-Ala4-Ala5) in DMSO solution |
10.1021/jm070084n. |
J Med Chem |
Conformational templates for rational drug design: flexibility of cyclo(D-Pro1-Ala2-Ala3-Ala4-Ala5) in DMSO solution
Abstract
- Long MD simulations (100 ns) for the important model cyclopentapeptide cyclo(D-Pro1-Ala2-Ala3-Ala4-Ala5) were performed in explicit DMSO solution using both OPLS-AA and AMBER03 force fields. Simulations revealed conformational transitions between two main conformers, a predominant one (population 93-99%) and a minor conformer (population 0.4-6.7%). These results are in excellent agreement with 20 experimental proton-proton distances estimated for this cyclopentapeptide. The previously discussed gamma-turn-like conformation for Ala4 was present only in a minor conformer.
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| 17504145 |
Role of micafungin in the antifungal armamentarium |
10.2174/092986707780597970. |
Curr Med Chem |
Role of micafungin in the antifungal armamentarium
Abstract
- Serious infections caused by opportunistic molds remain a major problem for public health. Immune deficiency following organ transplantation and aggressive cancer treatment has greatly increased the incidence of systemic mycoses, and invasive aspergillosis in patients with AIDS is associated with significant morbidity and mortality. Amphotericin B is the first-line therapy for systemic infection because of its broad-spectrum and fungicidal activity. However, considerable side effects limit its clinical utility. The echinocandins are large lipopeptide molecules that inhibit the synthesis of 1,3-beta-D-glucan, a key component of the fungal cell wall. Three echinocandins have reached the market, and some others are in early clinical development. Caspofungin was the first echinocandin to be licensed for clinical use in most countries. Micafungin is licensed for clinical use in Japan, China, Taiwan, Jordan, Korea, Hong-Kong and the US, and anidulafungin is currently licensed in the US. The novel class of echinocandins represents a milestone in antifungal drug research that has further expanded our therapeutic options. Studies to date have shown that micafungin exhibits extremely potent antifungal activity against clinically important fungi, including Aspergillus and azole-resistant strains of Candida. In animal studies, micafungin is as efficacious as amphotericin B with respect to improvement of survival rate. Micafungin is also characterized by a linear pharmacokinetic profile and substantially fewer toxic effects. Micafungin is a poor substrate for the cytochrome P450 enzymes, and compared to azoles, fewer drug interactions are described. No dose adjustments of the drug are required in the presence of mycophenolate mofetil, cyclosporin, tacrolimus, prednisolone, or sirolimus. Strategies using this new echinocandin agent will benefit a large number of patients with severe immune dysfunction.
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| 17514692 |
Synthesis and structure-activity relationship of cytotoxic marine cyclodepsipeptide IB-01212 analogues |
10.1002/cmdc.200700025. |
ChemMedChem |
Synthesis and structure-activity relationship of cytotoxic marine cyclodepsipeptide IB-01212 analogues
Abstract
- Several recently discovered marine products have remarkable in vitro and in vivo anticancer profiles against a wide range of tumor cell lines. Some of these compounds are currently in clinical trials. These compounds show complex structures and mechanisms of action of interest. Herein, we describe the preparation of a series of totally synthetic molecules that are structurally related to the natural marine product IB-01212 and evaluated them as antitumor agents. For this, total solid-phase syntheses of the products were performed in parallel by two distinct routes: linear synthesis and convergent synthesis. Structural modifications were introduced in several residue positions to afford 21 IB-01212 analogues for structure-relationship studies. An increase in the number of methyl groups in the macrocycle enhanced cytotoxic activity. Also, the replacement of an ester bond by an amide bond favored antitumor activity against several human cell lines. In addition, the L configuration analogues were more active against all the tumor cell lines than those containing the D configuration. A significant increase in the size and asymmetry of the macrocycle diminished biological activity with respect to that of IB-01212. These results are of great value for the discovery of new and more effective anticancer agents.
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| 17516032 |
Mutations in SIRT2 deacetylase which regulate enzymatic activity but not its interaction with HDAC6 and tubulin. |
10.1007/s11010-007-9478-6 |
Mol. Cell. Biochem. |
Mutations in SIRT2 deacetylase which regulate enzymatic activity but not its interaction with HDAC6 and tubulin.
Abstract
- Human SIRT2 is a cytoplasmic NAD-dependent deacetylase implicated in the mitotic regulation of microtubule dynamics by its association with the class II histone deacetylase 6 (HDAC6). We have previously reported that SIRT2 is multiply phosphorylated in a cell cycle dependent pattern. Here, we demonstrate that HDAC6 binds to both phosphorylated and unphosphorylated forms of SIRT2 and that tubulin binds only to the SIRT2-HDAC6 complex. Tubulin does not bind to either HDAC6 or SIRT2 individually. In addition, we show that replacement of specific serines with alanines in either isoform of SIRT2 regulates its enzymatic activity. We also found that overexpression of isoform2 was deleterious to cell survival. SIRT2 was found to be phosphorylated at serines 368 and 372, outside the conserved core domain of the Sir2 protein family. Double replacement of S368A and S372A reduced SIRT2 deacetylase activity by 44% compared to wildtype activity. Replacements of other serine, threonine, and tyrosine residues, which did not alter the phosphorylation pattern, had varying effects on SIRT2 deacetylase activity but no effect on tubulin/HDAC6 binding.
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| 17524361 |
The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis |
10.1016/j.bbrc.2007.05.062. |
Biochem Biophys Res Commun |
The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis
Abstract
- Several type-1 membrane proteins undergo regulated intramembrane proteolysis resulting in the generation of biologically active protein fragments. Presenilin-dependant gamma-secretase activity is central to this event and includes amyloid precursor protein (APP), Notch and ErbB4 as substrates. Here we show that the insulin-like growth factor 1 receptor (IGF-IR) undergoes regulated intramembrane proteolysis. A metalloprotease-dependant ectodomain-shedding event generates a approximately 52 kDa IGF-IR-carboxyl terminal domain (CTD). The IGF-IR-CTD is consequentially a substrate for gamma-secretase cleavage, liberating a approximately 50 kDa intracellular domain (ICD) that can be inhibited by a specific gamma-secretase inhibitor. This study suggests that the IGF-IR is a substrate for gamma-secretase and may mediate a function independent of its role as a receptor tyrosine kinase.
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| 17526063 |
Immobilized protease-assisted synthesis of engineered cysteine-knot microproteins |
10.1002/cbic.200700187. |
Chembiochem |
Immobilized protease-assisted synthesis of engineered cysteine-knot microproteins
Abstract
|
| 17534989 |
The cyclotide fingerprint in oldenlandia affinis: elucidation of chemically modified, linear and novel macrocyclic peptides |
10.1002/cbic.200700097. |
Chembiochem |
The cyclotide fingerprint in oldenlandia affinis: elucidation of chemically modified, linear and novel macrocyclic peptides
Abstract
- The complete suite of cyclotides present in Oldenlandia affinis (Rubiaceae), the plant that was originally found to contain this unique family of circular proteins, has been characterised. This study expands the number of known cyclotides in this plant to 17, of which nine new sequences (kalata B9-B17) were characterised in this work. In addition, five derivatives that contain oxidation products of the conserved tryptophan were identified, and it was shown that the formation of these derivatives is catalysed by exposure to sunlight. Furthermore, we describe two "linear" cyclotide analogues. These acyclic peptides have three intact disulfide bonds, and their N and C termini coincide with the hypothesised cleavage sites from the precursor protein. This work increases our knowledge about the sequence variation that is accommodated by the cyclic cystine knot scaffold, confirms its applicability as a template for drug design, and also shows the first natural degradation pathways for cyclotides. These pathways have important implications for the persistence and environmental fate of the cyclotides if used as crop-protection agents.
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| 17548428 |
Mechanisms of ceramide-mediated repression of the human telomerase reverse transcriptase promoter via deacetylation of Sp3 by histone deacetylase 1 |
10.1096/fj.07-8621com. |
FASEB J |
Mechanisms of ceramide-mediated repression of the human telomerase reverse transcriptase promoter via deacetylation of Sp3 by histone deacetylase 1
Abstract
- In this study, distinct roles of de novo-generated endogenous ceramides and mechanisms by which deacetylated Sp3 regulates the hTERT promoter activity in response to ceramide signaling were explored. The generation of C18-ceramide via the expression of ceramide synthase 1 (CerS1), and not C16-ceramide by CerS5 or CerS6 expression, resulted in repression of the hTERT promoter via deacetylation of Sp3 by histone deacetylase 1 (HDAC1) in A549 human lung adenocarcinoma cells. Then roles and mechanisms of action of ceramide-mediated deacetylation of Sp3 in inhibiting the hTERT promoter were determined using constitutively deacetylated or acetylated Sp3 mutants at lysine (K) 551. Expression of the deacetylated Sp3 mutant resulted in repression, whereas its acetylated mutant induced basal hTERT promoter activity in Drosophila S2 cells, which do not express any endogenous Sp3, and in A549 cells. Remarkably, chromatin immunoprecipitation data revealed that acetylated Sp3 mutant (K551Q-Sp3) did not bind whereas deacetylated Sp3 (K551R-Sp3) mutant bound strongly to the promoter DNA, resulting in the recruitment of histone deacetylase 1 (HDAC1) and inhibition of the association of RNA polymerase II with the promoter. Mechanistically, increased generation of C18-ceramide by hCerS1 expression, but not by its catalytically inactive mutant, mediated the association and recruitment of the deacetylated Sp3/HDAC1 complex to the hTERT promoter DNA, resulting in the local histone H3 deacetylation and repression of the promoter.
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