ArelA-dependent regulatory cascade for auto-induction of microbisporicin production inMicrobispora corallina

Microbisporicin is a potent type I lantibiotic produced by the rare actinomycete M icrobispora corallina that is in preclinical trials for the treatment of infections caused by methicillin‐resistant isolates of S taphylococcus aureus (MRSA). Analysis of the gene cluster for the biosynthesis of microbisporicin, which contains two unique post‐translationally modified residues (5‐chlorotryptophan and 3, 4‐dihydroxyproline), has revealed an unusual regulatory mechanism that involves a pathway‐specific extracytoplasmic function sigma factor (MibX)/anti‐sigma factor (MibW) complex and an additional transcriptional regulator MibR. A model for the regulation of microbisporicin biosynthesis derived from transcriptional, mutational and quantitative reverse transcription polymerase chain reaction analyses suggests that MibR, which contains a C‐terminal DNA‐binding domain found in the LuxR family of transcriptional activators, functions as an essential master regulator to trigger microbisporicin production while MibX and MibW induce feed‐forward biosynthesis and producer immunity. Moreover, we demonstrate that initial expression of mib R, and thus microbisporicin production, is dependent on the ppGpp synthetase gene (relA) of M . corallina. In addition, we show that constitutive expression of either of the two positively acting regulatory genes, mib R or mib X, leads to precocious and enhanced microbisporicin production

The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase

RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria

The purification and properties of a ribonuclease from Salmonella typhimurium extract

A ribonuclease has been purified about 140-fold from extracts of Salmonella typhimurium through ammonium sulfate fractionation, gel filtration, and DEAE-cellulose chromatography. The optimum pH for the hydrolytic breakdown of RNA is 7. The enzyme hydrolyzes polyadenylic acid (poly A), polycytidylic acid (poly C), and polyuridylic acid (poly U) at much faster rates than transfer RNA. Polyinosinic acid (poly I) was not hydrolyzed at all. Higher concentrations of poly A and poly U (above 200 μg per ml) were inhibitory. A mixture of poly A and poly U in the proportion 1:2, which is known to produce maximum secondary interaction, is also inhibitory. These results indicate that the secondary structures of nucleic acids interfere with the action of the nuclease. The first product of hydrolysis is a 2',3'-cyclic nucleotide which is poorly hydrolyzed to the 3'-nucleotide. The enzyme behaves as an endonuclease. The properties of the S. typhimurium nuclease have been compared with those of RNase I of Escherichia coli. Although there are some differences, the S. typhimurium nuclease is like RNase I in its mode of action

Extraction & Spectrophotometric Determination of Rhenium with Thiocyanate & Hydroxamic Acids

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IV-VI MATERIALS & DEVICES FOR THERMOPHOTOVOLTAIC (TPV) POWER GENERATION

IV-VI semiconductors, with direct gap conduction and valence band extrema at the L-point in k-space, are characterized by four-fold L-valley degeneracy. Prior work [1] has shown that this degeneracy can be removed with quantum confinement in the [111] direction. The first half of this dissertation demonstrates the beneficial effects that this degeneracy removal can have on the performance of thermophotovoltaic (TPV) devices. PbSe quantum wells (QWs) with Pb1-xSrxSe barriers were evaluated. Using Schrodinger’s equation and the two different effective masses for charge carriers in the degeneracy split normal (or longitudinal) L-valley and the higher energy three-fold degenerate oblique L-valleys, the largest possible degeneracy splitting energies for three different normal valley subband energy gaps of 450 meV, 550 meV and 650 meV were found to be 21.7 meV, 37.1 meV, and 43.9 meV, respectively. Intrinsic carrier concentrations were calculated for these QWs using density of states functions and Fermi-Dirac statistics. It was found that thermally generated intrinsic carrier density can be reduced by more than an order of magnitude as compared to that of a bulk IV-VI semiconductor material with the same bandgap energy. Dark current and open circuit voltages for TPV devices made from IV-VI materials were also calculated. Results show that use of optimal QW designs can increase open circuit voltages by almost a factor of two as compared to a bulk material having the same band gap energy. With a doubling of open circuit voltages, the incorporation of QW designs in IV-VI semiconductor TPV devices is predicted to enable a 100% increase in optical-to-electrical power conversion efficiencies. The second half of this dissertation discusses the experimental results and analysis of data obtained from characterization of pn junction devices fabricated from a bulk Pb0.93Sr0.07Se active layer. The pn junction layer, which consisted of a 2 μm thick n-type layer doped with bismuth to give an electron density of n=4x1018 cm-3 and a 2 μm thick p-type layer with a selenium overpressure during growth to give a hole density of p=4x1017 cm-3, was grown by molecular beam epitaxy (MBE) on silicon, transferred to copper using a novel flip-chip eutectic bonding method, and fabricated into mesa-etched device structures with sizes of 200 x 200 μm2 and 400 x 400 μm2. Current-versus-voltage characterization was performed with and without broadband illumination at different heatsink temperatures to assess the performance of these devices as TPV cells. Measured reverse bias saturation current densities at -26 mV were observed to increase from 7.5 mA/cm2 to 26.9 mA/cm2 as the lattice temperature was increased from 10°C to 50°C. After accounting for band gap temperature dependence and thermal generation effects, these values were consistent with an increase in minority carrier lifetime from 10 ns at 10°C to 100 ns at 50°C. These data show a clear lifetime enhancing thermal re-excitation effect and confirm the high quality of these IV-VI semiconductor epitaxial layer materials for device fabrication applications. Moreover, these data show that high materials quality is maintained during device processing and that there are no significant surface recombination problems with the fabricated device structures. These pn junction devices were also operated at room temperature and in continuous wave (cw) mode as light emitting diodes (LEDs) and as mid-infrared detectors. Representing the first known demonstration of such mid-infrared IV-VI semiconductor device operation, LED emission spectra and detector spectral response showed a bandgap energy of 450 meV (2.76 μm cutoff wavelength) for the MBE-grown Pb0.93Sr0.07Se layer. Broadband illumination measurements were performed with spectrally-filtered thermal radiation and a parabolic optical concentrator (Winston cone). Unlike illumination with a laser, these testing conditions accurately simulate the environment that a TPV cell would experience in an actual TPV generator system. Open circuit voltages as high as 116 mV, short circuit current densities as high as 409 mA/cm2, and fill factors of 43% with an incident optical power density of 620 mW/cm2 were observed. Generated electrical power was greater than 20 mW/cm2 thus giving an optical-to-electrical power conversion efficiency of 3.3% for this non-QW device. External quantum efficiency (EQE) and an estimated internal quantum efficiency (IQE) based on an assumed 40% reflection loss were calculated to be 33% and 55%, respectively. Further optimization of materials growth, device design, and processing techniques as well as incorporation of anti-reflective coatings and use of higher incident optical power densities should ultimately enable power conversion efficiencies in the range of 10% for this bulk material single-junction device. This dissertation concludes with suggestions for how future work can be conducted to take advantage of the unique materials properties of nano-structured IV-VI semiconductors and to use QW materials within multi-junction TPV cell designs to achieve power conversion efficiencies that can be significantly larger than the good values that have already been experimentally demonstrated here. [1] H. Z. Wu, N. Dai, M. B. Johnson, P. J. McCann, and Z. S. Shi, “Unambiguous Observation of Subband Transitions from Longitudinal Valley and Oblique Valleys in IV–VI multiple Quantum Wells”, Applied Physics Letters 78, 2199 (2001)

Regulation of specialised metabolites in Actinobacteria – Expanding the paradigms

The increase in availability of actinobacterial whole genome sequences has revealed huge numbers of specialised metabolite biosynthetic gene clusters, encoding a range of bioactive molecules such as antibiotics, antifungals, immunosuppressives and anticancer agents. Yet the majority of these clusters are not expressed under standard laboratory conditions in rich media conditions. Emerging data from studies of specialised metabolite biosynthesis suggest that the diversity of regulatory mechanisms is greater than previously thought and these act at multiple levels, through a range of signals such as nutrient limitation, intercellular signalling and competition with other organisms. Understanding the regulation and environmental cues that lead to the production of these compounds allows us to identify the role which these compounds play in their natural habitat as well as providing tools to exploit this untapped source of specialised metabolites for therapeutic uses. Here we provide an overview of novel regulatory mechanisms that act in physiological, global, and cluster specific regulatory manners on biosynthetic pathways in Actinobacteria and consider these alongside their ecological and evolutionary implications

Interbody Fusion in Low Grade Lumbar Spondylolsithesis: Clinical Outcome Does Not Correalte with Slip Reduction and Neural Foraminal Dimension

Study DesignProspective nonrandomized study.PurposeTo find a possible correlation between clinical outcome and extent of lumbar spondylolisthesis reduction.Overview of LiteratureThere is no consensus in the literature concerning whether a beneficial effect of reduction on outcome can be expected following reduction and surgical fusion for low grade lumbar spondylolisthesis.MethodsForty six patients with a mean age of 37.5 years (age, 17–48 years) with isthmic spondylolisthesis underwent interbody fusion with cages with posterior instrumentation (TLIF). Clinical outcome was measured using visual analogue score (VAS) and Oswestry disability index (ODI). Foraminal dimensions and disc heights were measured in standard digital radiographs. These were analyzed at baseline and 1 year after surgery and changes were compared. Radiographic fusion was judged with computed tomography scans at 1 year.ResultsNinety percent of the patients had good or very good clinical results with fusion and instrumentation. Baseline and one-year postoperative mean VAS score was 6.33 (range, 5–8) and 0.76 (range, 0–3), respectively (p=0.004). Baseline and one-year postoperative, mean ODI score was 48 (range, 32–62) and 10 (range, 6–16), respectively (p<0.001). A mean spondylolisthesis slip of 32.1% was reduced to 6.7% at 1 year. Average anterior disc height, posterior disc height, vertical foraminal dimension), and foraminal) diameter improved from 9.8 to 11.7 mm (p=0.005), 4.5 to 5.8 mm (p=0.004), 11.3 to 12.6 mm (p=0.002), and 18.6 to 20.0 mm (p<0.001), respectively. The fusion rate was 75% with TLIF. There is no significant correlation between the improvements of ODI scores and the extent of slip reduction.ConclusionsNeural decompression and interbody fusion can significantly improve pain and disability but the clinical outcome does not correlate with radiological improvement in the neural foraminal dimension

Stm1p alters the ribosome association of eukaryotic elongation factor 3 and affects translation elongation

Stm1p is a Saccharomyces cerevisiae protein that is primarily associated with cytosolic 80S ribosomes and polysomes. Several lines of evidence suggest that Stm1p plays a role in translation under nutrient stress conditions, although its mechanism of action is not yet known. In this study, we show that yeast lacking Stm1p (stm1Δ) are hypersensitive to the translation inhibitor anisomycin, which affects the peptidyl transferase reaction in translation elongation, but show little hypersensitivity to other translation inhibitors such as paromomycin and hygromycin B, which affect translation fidelity. Ribosomes isolated from stm1Δ yeast have intrinsically elevated levels of eukaryotic elongation factor 3 (eEF3) associated with them. Overexpression of eEF3 in cells lacking Stm1p results in a growth defect phenotype and increased anisomycin sensitivity. In addition, ribosomes with increased levels of Stm1p exhibit decreased association with eEF3. Taken together, our data indicate that Stm1p plays a complementary role to eEF3 in translation

Features of 80S mammalian ribosome and its subunits

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic