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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.
doi:10.1128/AAC.00106-10
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.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1472-6750-13-24
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.
doi:10.1128/JB.186.18.6093-6100.2004
PMCID: PMC515157  PMID: 15342578
4.  Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations 
Background
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.
Results
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 http://www.unipa.it/ampuglia/Abal-proteome-maps 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).
Conclusion
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.
doi:10.1186/1475-2859-9-95
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.
doi:10.1155/2012/784713
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.
doi:10.1155/2013/809062
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.
Introduction
Breast density is one of the strongest risk factors for breast cancer, but determinants of breast density in young women remain largely unknown.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/bcr3228
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.
doi:10.1128/AAC.01372-10
PMCID: PMC3165290  PMID: 21690280
9.  Pharmacophore-based virtual screening versus docking-based virtual screening: a benchmark comparison against eight targets 
Acta Pharmacologica Sinica  2009;30(12):1694-1708.
Aim:
This study was conducted to compare the efficiencies of two virtual screening approaches, pharmacophore-based virtual screening (PBVS) and docking-based virtual screening (DBVS) methods.
Methods:
All virtual screens were performed on two data sets of small molecules with both actives and decoys against eight structurally diverse protein targets, namely angiotensin converting enzyme (ACE), acetylcholinesterase (AChE), androgen receptor (AR), D-alanyl-D-alanine carboxypeptidase (DacA), dihydrofolate reductase (DHFR), estrogen receptors α (ERα), HIV-1 protease (HIV-pr), and thymidine kinase (TK). Each pharmacophore model was constructed based on several X-ray structures of protein-ligand complexes. Virtual screens were performed using four screening standards, the program Catalyst for PBVS and three docking programs (DOCK, GOLD and Glide) for DBVS.
Results:
Of the sixteen sets of virtual screens (one target versus two testing databases), the enrichment factors of fourteen cases using the PBVS method were higher than those using DBVS methods. The average hit rates over the eight targets at 2% and 5% of the highest ranks of the entire databases for PBVS are much higher than those for DBVS.
Conclusion:
The PBVS method outperformed DBVS methods in retrieving actives from the databases in our tested targets, and is a powerful method in drug discovery.
doi:10.1038/aps.2009.159
PMCID: PMC4007494  PMID: 19935678
pharmacophore; docking; LigandScout; enrichment; hit rate
10.  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
11.  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
12.  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.
doi:10.3390/ijms140714225
PMCID: PMC3742241  PMID: 23839093
pharmacophore; molecular docking; methionyl-tRNA synthetase; virtual screening
13.  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.
doi:10.1002/mrm.21719
PMCID: PMC2812065  PMID: 18816808
OEF; BOLD; qBOLD; brain metabolism; brain hemodynamics; fMRI
14.  Adolescent and Young Adult Exposure to Physical Activity and Breast Density 
Purpose
To examine the role of early lifetime exposure to physical activity on magnetic resonance imaging (MRI) determined breast density measures.
Methods
Associations of adolescent [high school (ages 14–17 years) and early adulthood [post high school (ages 18–21 years) and past year] leisure-time physical activity, as well as a principal component score including all three estimates, were examined with percent dense breast volume (%DBV) and absolute dense breast volume (ADBV) in a cross-sectional analysis of 182 healthy women, aged 25–29 years enrolled in the Dietary Intervention Study in Children Follow-up Study (DISC06). Generalized linear mixed (GLM) models were used to examine associations after adjustment for relevant covariates for the entire analytic sample. Analyses were repeated in nulliparous women and hormonal contraceptive non-users.
Results
Physical activity during high school and post high school were not statistically significantly related to %DBV or ADBV in multivariable models. Past year physical activity was positively related to %DBV in the unadjusted and partially adjusted models (p<0.001 and p=0.01, respectively) that did not adjust for body mass index (BMI). After additional adjustment for childhood and early adulthood BMI, this association became non-statistically significant. The relation between past year physical activity and ADBV was not statistically significant. These findings were similar in non-users of hormonal contraceptives. No statistically significant relationships were found in nulliparous women or between the principal component score and %DBV or ADBV.
Conclusion
Results from this study are consistent with previous research suggesting that physical activity during adolescence and early adulthood is unrelated to breast density.
doi:10.1249/MSS.0b013e318289a7f8
PMCID: PMC3897238  PMID: 23377838
motor activity; breast composition; young adult; childhood
15.  Menstrual and reproductive characteristics and breast density in young women 
Cancer causes & control : CCC  2013;24(11):1973-1983.
Purpose
Breast density is strongly related to breast cancer risk, but determinants of breast density in young women remain largely unknown.
Methods
Associations of reproductive and menstrual characteristics with breast density measured by magnetic resonance imaging were evaluated in a cross-sectional study of 176 healthy women, 25–29 years old, using linear mixed effects models.
Results
Parity was significantly inversely associated with breast density. In multivariable adjusted models that included non-reproductive variables, mean percent dense breast volume (%DBV) decreased from 20.5 % in nulliparous women to 16.0 % in parous women, while mean absolute dense breast volume (ADBV) decreased from 85.3 to 62.5 cm3. Breast density also was significantly inversely associated with the age women started using hormonal contraceptives, whereas it was significantly positively associated with duration of hormonal contraceptive use. In adjusted models, mean %DBV decreased from 21.7 % in women who started using hormones at 12–17 years of age to 14.7 % in those who started using hormones at 22–28 years of age, while mean ADBV decreased from 86.2 to 53.7 cm3. The age at which women started using hormonal contraceptives and duration of hormone use were inversely correlated, and mean %DBV increased from 15.8 % in women who used hormones for not more than 2.0 years to 22.0 % in women who used hormones for more than 8 years, while mean ADBV increased from 61.9 to 90.4 cm3 over this interval.
Conclusions
Breast density in young women is inversely associated with parity and the age women started using hormonal contraceptives but positively associated with duration of hormone use.
doi:10.1007/s10552-013-0273-2
PMCID: PMC3960004  PMID: 23933948
Breast density; Parity; Breast feeding; Hormonal contraceptives; Menarche; Menstrual cycle
16.  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
17.  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.
doi:10.4070/kcj.2011.41.6.287
PMCID: PMC3132688  PMID: 21779279
Blood viscosity; Hemorheology; Angina, microvascular
18.  Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales 
Synthesis of peptidoglycan precursors ending in d-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d-Lac into cytoplasmic precursors. This was due to a d,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d-Lac for d-Ala and Gly. The contribution of l,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d,d-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l,d-transpeptidases merely act as a tolerance mechanism in this bacterium.
doi:10.1128/AAC.02329-13
PMCID: PMC3957865  PMID: 24395229
19.  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.
doi:10.1128/JB.02291-12
PMCID: PMC3650528  PMID: 23475977
20.  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.
doi:10.1039/c3ob42428f
PMCID: PMC4082399  PMID: 24608916
21.  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.
doi:10.2183/pjab/83.277
PMCID: PMC3859367  PMID: 24367152
A-factor; Streptomyces; morphological differentiation; secondary metabolism; A-factor receptor; streptomycin biosynthesis
22.  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.
Images
PMCID: PMC214214  PMID: 2995326
23.  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.
doi:10.1128/JB.187.16.5595-5604.2005
PMCID: PMC1196073  PMID: 16077104
24.  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.
doi:10.1021/bi100150v
PMCID: PMC2888265  PMID: 20361791
25.  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

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