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1.  Novel Mechanism of Glycopeptide Resistance in the A40926 Producer Nonomuraea sp. ATCC 39727▿  
In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d-alanyl-d-alanine of lipid II with d-alanyl-d-lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide A40926, which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward A40926 during vegetative growth was 4 μg/ml, but this increased to ca. 20 μg/ml during A40926 production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for A40926 production contains a gene, vanY (ORF7), potentially encoding an enzyme capable of removing the terminal d-Ala residue of pentapeptide peptidoglycan precursors. Analysis of UDP-linked peptidoglycan precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala and only traces of the pentapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala-d-Ala. This suggested a novel mechanism of glycopeptide resistance in Nonomuraea sp. ATCC 39727 that was based on the d,d-carboxypeptidase activity of vanY. Consistent with this, a vanY-null mutant of Nonomuraea sp. ATCC 39727 demonstrated a reduced level of glycopeptide resistance, without affecting A40926 productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae, resulted in higher levels of glycopeptide resistance.
PMCID: PMC2876364  PMID: 20308385
2.  Streptomyces spp. as efficient expression system for a d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance 
BMC Biotechnology  2013;13:24.
VanYn, encoded by the dbv7 gene (also known as vanYn) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727. This filamentous actinomycete is an uncommon microorganism, difficult-to-handle but biotechnologically valuable since it produces the glycopeptide antibiotic A40926, which is the precursor of the second-generation dalbavancin in phase III of clinical development. In order to investigate VanYn role in glycopeptide resistance in the producer actinomycete an appropriate host-vector expression system is required.
The cloning strategy of vanYn gene (G-C ratio 73.3%) in the expression vector pIJ86 yielded a recombinant protein with a tag encoding for a histidine hexamer added at the C-terminus (C-His6-vanYn) or at the N-terminus (N-His6-vanYn). These plasmids were used to transform three Streptomyces spp., which are genetically-treatable high G-C content Gram-positive bacteria taxonomically related to the homologous producer Nonomuraea sp.. Highest yield of protein expression and purification (12 mg of protein per liter of culture at 3 L bioreactor-scale) was achieved in Streptomyces venezuelae ATCC 10595, that is a fast growing streptomyces susceptible to glycopeptides. VanYn is a transmembrane protein which was easily detached and recovered from the cell wall fraction. Purified C-His6-VanYn showed d,d-carboxypeptidase and d,d-dipeptidase activities on synthetic analogs of bacterial peptidoglycan (PG) precursors. C-His6-VanYn over-expression conferred glycopeptide resistance to S. venezuelae. On the contrary, the addition of His6-tag at the N-terminus of the protein abolished its biological activity either in vitro or in vivo assays.
Heterologous expression of vanYn from Nonomuraea sp. ATCC 39727 in S. venezuelae was successfully achieved and conferred the host an increased level of glycopeptide resistance. Cellular localization of recombinant VanYn together with its enzymatic activity as a d,d-peptidase/d,d-carboxypeptidase agree with its role in removing the last d-Ala from the pentapeptide PG precursors and reprogramming cell wall biosynthesis, as previously reported in glycopeptide resistant pathogens.
PMCID: PMC3610138  PMID: 23497129
Streptomyces; Heterologous protein production; d,d-carboxypeptidases; Glycopeptide production; Glycopeptide resistance; Dalbavancin
3.  Biosynthesis of Chloro-β-Hydroxytyrosine, a Nonproteinogenic Amino Acid of the Peptidic Backbone of Glycopeptide Antibiotics 
Journal of Bacteriology  2004;186(18):6093-6100.
The role of the putative P450 monooxygenase OxyD and the chlorination time point in the biosynthesis of the glycopeptide antibiotic balhimycin produced by Amycolatopsis balhimycina were analyzed. The oxyD gene is located directly downstream of the bhp (perhydrolase) and bpsD (nonribosomal peptide synthetase D) genes, which are involved in the synthesis of the balhimycin building block β-hydroxytyrosine (β-HT). Reverse transcriptase experiments revealed that bhp, bpsD, and oxyD form an operon. oxyD was inactivated by an in-frame deletion, and the resulting mutant was unable to produce an active compound. Balhimycin production could be restored (i) by complementation with an oxyD gene, (ii) in cross-feeding studies using A. balhimycina JR1 (a null mutant with a block in the biosynthesis pathway of the building blocks hydroxy- and dihydroxyphenylglycine) as an excretor of the missing precursor, and (iii) by supplementation of β-HT in the growth medium. These data demonstrated an essential role of OxyD in the formation pathway of this amino acid. Liquid chromatography-electrospray ionization-mass spectrometry analysis indicated the biosynthesis of completely chlorinated balhimycin by the oxyD mutant when culture filtrates were supplemented with nonchlorinated β-HT. In contrast, supplementation with 3-chloro-β-HT did not restore balhimycin production. These results indicated that the chlorination time point was later than the stage of free β-HT, most likely during heptapeptide synthesis.
PMCID: PMC515157  PMID: 15342578
4.  Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations 
Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted.
Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38 ± 0.02 and 0.33 ± 0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA).
In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement.
PMCID: PMC3004843  PMID: 21110849
5.  Activation of Spinal α2-Adrenoceptors Using Diluted Bee Venom Stimulation Reduces Cold Allodynia in Neuropathic Pain Rats 
Cold allodynia is an important distinctive feature of neuropathic pain. The present study examined whether single or repetitive treatment of diluted bee venom (DBV) reduced cold allodynia in sciatic nerve chronic constriction injury (CCI) rats and whether these effects were mediated by spinal adrenergic receptors. Single injection of DBV (0.25 or 2.5 mg/kg) was performed into Zusanli acupoint 2 weeks post CCI, and repetitive DBV (0.25 mg/kg) was injected for 2 weeks beginning on day 15 after CCI surgery. Single treatment of DBV at a low dose (0.25 mg/kg) did not produce any anticold allodynic effect, while a high dose of DBV (2.5 mg/kg) significantly reduced cold allodynia. Moreover, this effect of high-dose DBV was completely blocked by intrathecal pretreatment of idazoxan (α2-adrenoceptor antagonist), but not prazosin (α1-adrenoceptor antagonist) or propranolol (nonselective β-adrenoceptor antagonist). In addition, coadministration of low-dose DBV (0.25 mg/kg) and intrathecal clonidine (α2-adrenoceptor agonist) synergically reduced cold allodynia. On the other hand, repetitive treatments of low-dose DBV showing no motor deficit remarkably suppressed cold allodynia from 7 days after DBV treatment. This effect was also reversed by intrathecal idazoxan injection. These findings demonstrated that single or repetitive stimulation of DBV could alleviate CCI-induced cold allodynia via activation of spinal α2-adrenoceptor.
PMCID: PMC3434467  PMID: 22969830
6.  Blockade of Adrenal Medulla-Derived Epinephrine Potentiates Bee Venom-Induced Antinociception in the Mouse Formalin Test: Involvement of Peripheral β-Adrenoceptors 
The injection of diluted bee venom (DBV) into an acupoint has been used traditionally in eastern medicine to treat a variety of inflammatory chronic pain conditions. We have previously shown that DBV had a potent antinociceptive efficacy in several rodent pain models. However, the peripheral mechanisms underlying DBV-induced antinociception remain unclear. The present study was designed to investigate the role of peripheral epinephrine on the DBV-induced antinociceptive effect in the mouse formalin assay. Adrenalectomy significantly enhanced the antinociceptive effect of DBV during the late phase of the formalin test, while chemical sympathectomy had no effect. Intraperitoneal injection of epinephrine blocked this adrenalectomy-induced enhancement of the DBV-induced antinociceptive effect. Moreover, injection of a phenylethanolamine N-methyltransferase (PNMT) inhibitor enhanced the DBV-induced antinociceptive effect. Administration of nonselective β-adrenergic antagonists also significantly potentiated this DBV-induced antinociception, in a manner similar to adrenalectomy. These results demonstrate that the antinociceptive effect of DBV treatment can be significantly enhanced by modulation of adrenal medulla-derived epinephrine and this effect is mediated by peripheral β-adrenoceptors. Thus, DBV acupoint stimulation in combination with inhibition of peripheral β-adrenoceptors could be a potentially novel strategy for the management of inflammatory pain.
PMCID: PMC3781998  PMID: 24089621
7.  Height, adiposity and body fat distribution and breast density in young women 
Breast Cancer Research : BCR  2012;14(4):R107.
Breast density is one of the strongest risk factors for breast cancer, but determinants of breast density in young women remain largely unknown.
Associations of height, adiposity and body fat distribution with percentage dense breast volume (%DBV) and absolute dense breast volume (ADBV) were evaluated in a cross-sectional study of 174 healthy women, 25 to 29 years old. Adiposity and body fat distribution were measured by anthropometry and dual-energy X-ray absorptiometry (DXA), while %DBV and ADBV were measured by magnetic resonance imaging. Associations were evaluated using linear mixed-effects models. All tests of statistical significance are two-sided.
Height was significantly positively associated with %DBV but not ADBV; for each standard deviation (SD) increase in height, %DBV increased by 18.7% in adjusted models. In contrast, all measures of adiposity and body fat distribution were significantly inversely associated with %DBV; a SD increase in body mass index (BMI), percentage fat mass, waist circumference and the android:gynoid fat mass ratio (A:G ratio) was each associated significantly with a 44.4 to 47.0% decrease in %DBV after adjustment for childhood BMI and other covariates. Although associations were weaker than for %DBV, all measures of adiposity and body fat distribution also were significantly inversely associated with ADBV before adjustment for childhood BMI. After adjustment for childhood BMI, however, only the DXA measures of percentage fat mass and A:G ratio remained significant; a SD increase in each was associated with a 13.8 to 19.6% decrease in ADBV. In mutually adjusted analysis, the percentage fat mass and the A:G ratio remained significantly inversely associated with %DBV, but only the A:G ratio was significantly associated with ADBV; a SD increase in the A:G ratio was associated with an 18.5% decrease in ADBV.
Total adiposity and body fat distribution are independently inversely associated with %DBV, whereas in mutually adjusted analysis only body fat distribution (A:G ratio) remained significantly inversely associated with ADBV in young women. Research is needed to identify biological mechanisms underlying these associations.
PMCID: PMC3680938  PMID: 22800711
8.  Self-Resistance and Cell Wall Composition in the Glycopeptide Producer Amycolatopsis balhimycina▿ 
The prevailing resistance mechanism against glycopeptides in Gram-positive pathogens involves reprogramming the biosynthesis of peptidoglycan precursors, resulting in d-alanyl-d-lactate depsipeptide termini. Amycolatopsis balhimycina produces the vancomycin-like glycopeptide balhimycin and therefore has to protect itself from the action of the glycopeptide. We studied the roles of the accessory resistance gene orthologs vanYb, vnlRb, and vnlSb, which are part of the balhimycin biosynthetic gene cluster (represented by the subscript “b”). The VanYb carboxypeptidase cleaved the terminal d-Ala from peptidoglycan precursors, and its heterologous expression enhanced glycopeptide resistance in Streptomyces coelicolor. The VanRS-like two component system VnlRSb was not involved in glycopeptide resistance or in the expression of the vanHAX glycopeptide resistance genes. Mature A. balhimycina peptidoglycan contained mainly tri- and tetrapeptides, with only traces of the d-Ala-d-Ala-ending pentapeptides that are binding sites for the antibiotic produced. The structure of the peptidoglycan precursor is consistent with the presence of vanHAX genes, which were identified outside the balhimycin synthesis cluster. Both wild-type and non-antibiotic-producing mutant strains synthesized peptidoglycan precursors ending mainly with d-Lac, indicating constitutive synthesis of a resistant cell wall. A. balhimycina could provide a model for an ancestral glycopeptide producer with constitutively expressed resistance genes.
PMCID: PMC3165290  PMID: 21690280
9.  A40926, a new glycopeptide antibiotic with anti-Neisseria activity. 
Antimicrobial Agents and Chemotherapy  1987;31(12):1961-1966.
In the course of a search for glycopeptide antibiotics having novel biological properties, we isolated A40926. Produced by an actinomycete of the genus Actinomadura, A40926 is a complex of four main factors which contain a fatty acid as part of a glycolipid attached to the peptide backbone. Its activity was, in most respects, similar to that of other glycopeptides, such as vancomycin and teicoplanin. However, in addition to inhibiting gram-positive bacteria, A40926 was very active against Neisseria gonorrhoeae. A40926 was rapidly bactericidal for N. gonorrhoeae clinical isolates at concentrations equal to or slightly higher than the MIC. In mice, levels in serum were higher and more prolonged than those of an equivalent subcutaneous dose of teicoplanin. These properties suggest that A40926 may have potential in the therapy of gonorrhea.
PMCID: PMC175835  PMID: 2964225
10.  Pharmacokinetics of A40926 in rats after single intravenous and subcutaneous doses. 
A40926 is a new glycopeptide antibiotic with unique activity against Neisseria gonorrhoeae and high and prolonged levels in mouse blood (B. P. Goldstein, E. Selva, L. Gastaldo, M. Berti, R. Pallanza, F. Ripamonti, P. Ferrari, M. Denaro, V. Arioli, and G. Cassani, Antimicrob. Agents Chemother., 31:1961-1966, 1987). We studied the pharmacokinetics of A40926 in rats after single intravenous and subcutaneous 10-mg/kg (body weight) doses. Concentrations in plasma and urine were determined by microbiological assay. After intravenous administration, high concentrations of A40926, ranging from 132 mg/liter at 3 min to 0.7 mg/liter at 96 h, were found in plasma. Concentrations declined with a three-exponential decay correlated with a prolonged, biphasic distribution and a slow elimination (terminal half-life, 61.22 h). After completion of the distribution, the compound was widely distributed to the extravascular space. The rate-limiting step in the elimination of A40926 from the body appears to be the slow return from the deep compartment into the central one. A40926 was rapidly absorbed from the injection site after subcutaneous administration, and its availability was close to 90%. The percentage of the dose excreted in urine in 120 h was 35.9%.
PMCID: PMC172143  PMID: 3364946
11.  Novel Hybrid Virtual Screening Protocol Based on Molecular Docking and Structure-Based Pharmacophore for Discovery of Methionyl-tRNA Synthetase Inhibitors as Antibacterial Agents 
Methione tRNA synthetase (MetRS) is an essential enzyme involved in protein biosynthesis in all living organisms and is a potential antibacterial target. In the current study, the structure-based pharmacophore (SBP)-guided method has been suggested to generate a comprehensive pharmacophore of MetRS based on fourteen crystal structures of MetRS-inhibitor complexes. In this investigation, a hybrid protocol of a virtual screening method, comprised of pharmacophore model-based virtual screening (PBVS), rigid and flexible docking-based virtual screenings (DBVS), is used for retrieving new MetRS inhibitors from commercially available chemical databases. This hybrid virtual screening approach was then applied to screen the Specs (202,408 compounds) database, a structurally diverse chemical database. Fifteen hit compounds were selected from the final hits and shifted to experimental studies. These results may provide important information for further research of novel MetRS inhibitors as antibacterial agents.
PMCID: PMC3742241  PMID: 23839093
pharmacophore; molecular docking; methionyl-tRNA synthetase; virtual screening
12.  Validation of Oxygen Extraction Fraction Measurement by qBOLD Technique 
Measurement of brain tissue oxygen extraction fraction (OEF) in both baseline and functionally activated states can provide important information on brain functioning in health and disease. The recently proposed quantitative BOLD (qBOLD) technique is MRI-based and provides a regional in vivo OEF measurement (He and Yablonskiy, MRM 2007, 57:115–126). It is based on a previously developed analytical BOLD model and incorporates prior knowledge about the brain tissue composition including the contributions from grey matter, white matter, cerebrospinal fluid, interstitial fluid and intravascular blood. The qBOLD model also allows for the separation of contributions to the BOLD signal from OEF and the deoxyhemoglobin containing blood volume (DBV). The objective of this study is to validate OEF measurements provided by the qBOLD approach. To this end we use a rat model and compare qBOLD OEF measurements against direct measurements of the blood oxygenation level obtained from venous blood drawn directly from the superior sagittal sinus. The cerebral venous oxygenation level of the rat was manipulated by utilizing different anestheisa methods. The study demonstrates a very good agreement between qBOLD approach and direct measurements.
PMCID: PMC2812065  PMID: 18816808
OEF; BOLD; qBOLD; brain metabolism; brain hemodynamics; fMRI
13.  Identification and Analysis of the Balhimycin Biosynthetic Gene Cluster and Its Use for Manipulating Glycopeptide Biosynthesis in Amycolatopsis mediterranei DSM5908 
Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genes bgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography–mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.
PMCID: PMC89325  PMID: 10390204
14.  Hemorheology and Microvascular Disorders 
Korean Circulation Journal  2011;41(6):287-295.
The present review presents basic concepts of blood rheology related to vascular diseases. Blood flow in large arteries is dominated by inertial forces exhibited at high flow velocities, while viscous forces (i.e., blood rheology) play an almost negligible role. When high flow velocity is compromised by sudden deceleration as at a bifurcation, endothelial cell dysfunction can occur along the outer wall of the bifurcation, initiating inflammatory gene expression and, through mechanotransduction, the cascade of events associated with atherosclerosis. In sharp contrast, the flow of blood in microvessels is dominated by viscous shear forces since the inertial forces are negligible due to low flow velocities. Shear stress is a critical parameter in microvascular flow, and a force-balance approach is proposed for determining microvascular shear stress, accounting for the low Reynolds numbers and the dominance of viscous forces over inertial forces. Accordingly, when the attractive forces between erythrocytes (represented by the yield stress of blood) are greater than the shear force produced by microvascular flow, tissue perfusion itself cannot be sustained, leading to capillary loss. The yield stress parameter is presented as a diagnostic candidate for future clinical research, specifically, as a fluid dynamic biomarker for microvascular disorders. The relation between the yield stress and diastolic blood viscosity (DBV) is described using the Casson model for viscosity, from which one may be able determine thresholds of DBV where the risk of microvascular disorders is high.
PMCID: PMC3132688  PMID: 21779279
Blood viscosity; Hemorheology; Angina, microvascular
15.  Cloning and Analysis of the Planosporicin Lantibiotic Biosynthetic Gene Cluster of Planomonospora alba 
Journal of Bacteriology  2013;195(10):2309-2321.
The increasing prevalence of antibiotic resistance in bacterial pathogens has renewed focus on natural products with antimicrobial properties. Lantibiotics are ribosomally synthesized peptide antibiotics that are posttranslationally modified to introduce (methyl)lanthionine bridges. Actinomycetes are renowned for their ability to produce a large variety of antibiotics, many with clinical applications, but are known to make only a few lantibiotics. One such compound is planosporicin produced by Planomonospora alba, which inhibits cell wall biosynthesis in Gram-positive pathogens. Planosporicin is a type AI lantibiotic structurally similar to those which bind lipid II, the immediate precursor for cell wall biosynthesis. The gene cluster responsible for planosporicin biosynthesis was identified by genome mining and subsequently isolated from a P. alba cosmid library. A minimal cluster of 15 genes sufficient for planosporicin production was defined by heterologous expression in Nonomuraea sp. strain ATCC 39727, while deletion of the gene encoding the precursor peptide from P. alba, which abolished planosporicin production, was also used to confirm the identity of the gene cluster. Deletion of genes encoding likely biosynthetic enzymes identified through bioinformatic analysis revealed that they, too, are essential for planosporicin production in the native host. Reverse transcription-PCR (RT-PCR) analysis indicated that the planosporicin gene cluster is transcribed in three operons. Expression of one of these, pspEF, which encodes an ABC transporter, in Streptomyces coelicolor A3(2) conferred some degree of planosporicin resistance on the heterologous host. The inability to delete these genes from P. alba suggests that they play an essential role in immunity in the natural producer.
PMCID: PMC3650528  PMID: 23475977
16.  Anti-cooperative ligand binding and dimerisation in the glycopeptide antibiotic dalbavancin† †Electronic supplementary information (ESI) available. See DOI: 10.1039/C3OB42428F Click here for additional data file.  
Organic & Biomolecular Chemistry  2014;12(16):2568-2575.
Dalbavancin, a semi-synthetic glycopeptide with enhanced antibiotic activity compared to vancomycin and teicoplanin, dimerises strongly in an anti-cooperative manner with ligand binding.
Dalbavancin, a semi-synthetic glycopeptide with enhanced antibiotic activity compared to vancomycin and teicoplanin, binds to the C-terminal lysyl-d-alanyl-d-alanine subunit of Lipid II, inhibiting peptidoglycan biosynthesis. In this study, micro-calorimetry and electrospray ionization (ESI)-MS have been used to investigate the relationship between oligomerisation of dalbavancin and binding of a Lipid II peptide mimic, diacetyl-Lys-d-Ala-d-Ala (Ac2-Kaa). Dalbavancin dimerised strongly in an anti-cooperative manner with ligand-binding, as was the case for ristocetin A, but not for vancomycin and teicoplanin. Dalbavancin and ristocetin A both adopt an ‘closed’ conformation upon ligand binding, suggesting anti-cooperative dimerisation with ligand-binding may be a general feature of dalbavancin/ristocetin A-like glycopeptides. Understanding these effects may provide insight into design of novel dalbavancin derivatives with cooperative ligand-binding and dimerisation characteristics that could enhance antibiotic activity.
PMCID: PMC4082399  PMID: 24608916
17.  Hormonal control by A-factor of morphological development and secondary metabolism in Streptomyces 
Streptomyces griseus, a well-known industrial producer of streptomycin, is a member of the genus Streptomyces, which shows a complex life cycle resembling that of fungi. A-factor, a C13γ-butyrolactone compound, was discovered as a self-regulatory factor or a bacterial hormone to induce morphological differentiation and production of secondary metabolites, including streptomycin, in this organism. Accumulating evidence has revealed an A-factor-triggered signal cascade, which is composed of several key steps or components. These include: (i) AfsA catalyzing a crucial step of A-factor biosynthesis, (ii) the A-factor-specific receptor (ArpA), which acts as a transcriptional repressor for adpA, (iii) adpA, a sole target of ArpA, which encodes a global transcriptional activator AdpA, and (iv) a variety of members of the AdpA regulon, a set of the genes regulated by AdpA. A-factor is biosynthesized via five reaction steps, in which AfsA catalyzes acyl transfer between a β-ketoacyl-acyl carrier protein and the hydroxyl group of dihydroxyacetone phosphate. The receptor ArpA, belonging to the TetR family, is a homodimer, each subunit of which contains a helix-turn-helix DNA-binding motif and an A-factor-binding pocket. The three-dimensional structure and conformational change upon binding A-factor are elucidated, on the basis of X-ray crystallography of CprB, an ArpA homologue. AdpA, belonging to the AraC/XylS transcriptional activator family, binds operators upstream from the promoters of a variety of the target genes and activates their transcription, thus forming the AdpA regulon. Members of the AdpA regulon includes the pathway-specific transcriptional activator gene strR that activates the whole streptomycin biosynthesis gene cluster, in addition to a number of genes that direct the multiple cellular functions required for cellular differentiation in a concerted manner. A variety of A-factor homologues as well as homologues of afsA/arpA are distributed widely among Streptomyces, indicating the significant role of this type of molecular signaling in the ecosystem and evolutional processes.
PMCID: PMC3859367  PMID: 24367152
A-factor; Streptomyces; morphological differentiation; secondary metabolism; A-factor receptor; streptomycin biosynthesis
18.  Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance. 
Journal of Bacteriology  1985;164(1):85-94.
An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.
PMCID: PMC214214  PMID: 2995326
19.  Transcriptional Control by A-Factor of strR, the Pathway-Specific Transcriptional Activator for Streptomycin Biosynthesis in Streptomyces griseus 
Journal of Bacteriology  2005;187(16):5595-5604.
A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions −270 and −50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin.
PMCID: PMC1196073  PMID: 16077104
20.  Crystal Structures of the Glycopeptide Sulfotransferase Teg12 Complexed with the Teicoplanin Aglycone 
Biochemistry  2010;49(19):4159-4168.
The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, 13, and 14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures including: an apo structure of Teg12, Teg12 bound to the desulfated co-substrate 3'-phosphoadenosine 5'-phosphate and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions.
PMCID: PMC2888265  PMID: 20361791
21.  Antimicrobial activity of MDL 63,246, a new semisynthetic glycopeptide antibiotic. 
MDL 63,246 is a semisynthetic derivative of the naturally occurring glycopeptide antibiotic MDL 62,476 (A40926). It was more active in vitro against Staphylococcus aureus and coagulase-negative staphylococci than MDL 62,476, teicoplanin, and vancomycin and was more active than mideplanin (MDL 62,873) against some isolates. MDL 63,246 had excellent activity against streptococci and teicoplanin-susceptible enterococci, and it also had in vitro activity against some VanA enterococcal isolates. It was more active than teicoplanin and vancomycin against acute staphylococcal, streptococcal, and enterococcal septicemia in immunocompetent and neutropenic mice. It was highly efficacious in reducing the bacterial load in the hearts of rats in staphylococcal endocarditis experiments and the bacterial load of Staphylococcus epidermis in a high infection model in neutropenic mice. The excellent in vivo activity of MDL 63,246 appears to correlate both with its in vitro antibacterial activity and with its long half-life in rodents.
PMCID: PMC162785  PMID: 7492108
22.  Implementation of the CMOS MEMS Condenser Microphone with Corrugated Metal Diaphragm and Silicon Back-Plate 
Sensors (Basel, Switzerland)  2011;11(6):6257-6269.
This study reports a CMOS-MEMS condenser microphone implemented using the standard thin film stacking of 0.35 μm UMC CMOS 3.3/5.0 V logic process, and followed by post-CMOS micromachining steps without introducing any special materials. The corrugated diaphragm for the microphone is designed and implemented using the metal layer to reduce the influence of thin film residual stresses. Moreover, a silicon substrate is employed to increase the stiffness of the back-plate. Measurements show the sensitivity of microphone is −42 ± 3 dBV/Pa at 1 kHz (the reference sound-level is 94 dB) under 6 V pumping voltage, the frequency response is 100 Hz–10 kHz, and the S/N ratio >55 dB. It also has low power consumption of less than 200 μA, and low distortion of less than 1% (referred to 100 dB).
PMCID: PMC3231452  PMID: 22163953
CMOS-MEMS; condenser microphone; corrugated; sensitivity; diaphragm
23.  Intact skin and not stripped skin is crucial for the safety and efficacy of peanut epicutaneous immunotherapy (EPIT) in mice 
Epicutaneous immunotherapy (EPIT) on intact skin with an epicutaneous delivery system has already been used in preclinical and clinical studies. In epicutaneous vaccination and immunotherapy, the stripping of skin before application of the allergen is suggested to facilitate the passage of allergen through immune cells.
The aim of this study was to compare the immunological response induced by EPIT performed on intact and stripped skin in a mouse model of peanut allergy.
After oral sensitization with peanut and cholera toxin, BALB/c mice were epicutaneously treated using an epicutaneous delivery system (Viaskin® (DBV Technologies, Paris) applied either on intact skin or on stripped skin. Following EPIT, mice received an exclusive oral peanut regimen, aimed at triggering esophageal and jejunal lesions. We assessed eosinophil infiltration by histology, mRNA expression in the esophagus, antibody levels and peripheral T-cell response.
EPIT on intact skin significantly reduced Th2 immunological response (IgE response and splenocyte secretion of Th2 cytokines) as well as esophageal eosinophilia (2.7 ± 0.9, compared to Sham 19.9 ± 1.5, p < 0.01), mRNA expression of Th2 cytokines in tissue and intestinal villus sub-atrophia (2.9 ± 0.2 vs Sham, 2.1 ± 0.2, p < 0.05). By contrast, EPIT on stripped skin reinforced Th2 systemic immunological response as well as eosinophil infiltration (26.8 ± 15.1), mRNA expression of Th2 cytokines and duodenal villus/crypt-ratio (2.4 ± 0.3).
Epicutaneous allergen-specific immunotherapy needs the integrity of superficial layers of the stratum corneum to warranty safety of treatment and to induce a tolerogenic profile of the immune response.
PMCID: PMC3542018  PMID: 23140259
Food allergy; Immunotherapy; Epicutaneous; Peanut
24.  Genome-wide matching of genes to cellular roles using guilt-by-association models derived from single sample analysis 
BMC Research Notes  2012;5:370.
High-throughput methods that ascribe a cellular or physiological function for each gene product are useful to understand the roles of genes that have not been extensively characterized by molecular or genetic approaches. One method to infer gene function is "guilt-by-association", in which the expression pattern of a poorly characterized gene is shown to co-vary with the expression of better-characterized genes. The function of the poorly characterized gene is inferred from the known function(s) of the well-described genes. For example, genes co-expressed with transcripts that vary during the cell cycle, development, environmental stresses, and with oncogenesis have been implicated in those processes.
While examining the expression characteristics of several poorly characterized genes, we noted that we could associate each of the genes with a cellular phenotype by correlating individual gene expression changes with gene set enrichment scores from individual samples. We evaluated the effectiveness of this approach using a modest sized gene expression data set (expO) and a compendium of gene expression phenotypes (MSigDBv3.0). We found the transcripts that correlated best with enrichment in mitochondrial and lysosomal gene sets were mostly related to those processes (89/100 and 44/50, respectively). The reciprocal evaluation, ranking gene sets according to correlation of enrichment with an individual gene’s expression, also reflected known associations for prominent genes in the biomedical literature (16/19). In evaluating the model, we also found that 4% of the genome encodes proteins that are associated with small molecule and small peptide signal transduction gene sets, implicating a large number of genes in both internal and external environmental sensing.
Our results show that this approach is useful to infer functions of disparate sets of genes. This method mirrors the biological experimental approaches used by others to associate individual genes with defined gene expression changes. Moreover, the approach can be used beyond discovering genes related to a cellular process to discover meaningful expression phenotypes from a compendium that are associated with a given gene. The effectiveness, versatility, and breadth of this approach make possible its application in a variety of contexts and with a variety of downstream analyses.
PMCID: PMC3599284  PMID: 22824328
Gene set enrichment analysis; Gene module; Co-regulated gene sets; Gene prediction; Protein function; Olfactory receptors; Mitochondria; Lysosome; Oxidative phosphorylation
25.  Flexural Toughness of Steel Fiber Reinforced High Performance Concrete Containing Nano-SiO2 and Fly Ash 
The Scientific World Journal  2014;2014:403743.
This paper aims to clarify the effect of steel fiber on the flexural toughness of the high performance concrete containing fly ash and nano-SiO2. The flexural toughness was evaluated by two methods, which are based on ASTM C1018 and DBV-1998, respectively. By means of three-point bending method, the flexural toughness indices, variation coefficients of bearing capacity, deformation energy, and equivalent flexural strength of the specimen were measured, respectively, and the relational curves between the vertical load and the midspan deflection (PV-δ) were obtained. The results indicate that steel fiber has great effect on the flexural toughness parameters and relational curves (PV-δ) of the three-point bending beam specimen. When the content of steel fiber increases from 0.5% to 2%, the flexural toughness parameters increase gradually and the curves are becoming plumper and plumper with the increase of steel fiber content, respectively. However these flexural toughness parameters begin to decrease and the curves become thinner and thinner after the steel fiber content exceeds 2%. It seems that the contribution of steel fiber to the improvement of flexural toughness of the high performance concrete containing fly ash and nano-SiO2 is well performed only when the steel fiber content is less than 2%.
PMCID: PMC4030472  PMID: 24883395

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