Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Co(II) and Cr(III) complexes of formate-formamide mixed ligands: synthesis, structures, single crystal-to-single crystal transformation and magnetic behaviour

Solvothermal treatment of an equimolar mixture of Co(NO3)(2)center dot 6H(2)O, HCONH2 and NaN3 in MeOH at 100 degrees C yielded a three-dimensional NaCl type network Co(HCOO)(2)(HCONH2)(2) center dot HCONH2 (1a) containing formamides in the pores of the structure. Solvated pink 1a undergoes single crystal-to-single crystal (SCSC) transformation at 215 degrees C to form the desolvated dark brown product Co(HCOO)(2)-( HCONH2)(2) (1b) with the retention of the original framework. Reversible single crystal-to-single crystal transformation of 1b (brown) to 1a (pink) in the presence of excess formamide was also established at room temperature. The coordination environment around Co(II) in both 1a and 1b is octahedral with a CoN2O4 coordination composition. A similar reaction replacing Co(II) by Cr(III) produced a heterometallic 3D extended network Na[Cr(HCOO)(4)(HCONH2)(2)]center dot 2H(2)O (2a) at 100 degrees C. An increase in reaction temperature to 150 degrees C produced a simple mononuclear complex Cr(HCOO)(3)(HCONH2)(3) center dot 3H(2)O (2b). Variable temperature magnetic studies revealed the presence of a canting phenomena in both 1a and 1b, and hysteresis loop in the field dependent magnetisation plot at 2 K whereas complex 2a is simply paramagnetic in nature.

Nephrin-associated molecules in human glomerular diseases

The glomerular epithelial cells and their intercellular junctions, termed slit diaphragms, are essential components of the filtration barrier in the kidney glomerulus. Nephrin is a transmembrane adhesion protein of the slit diaphragm and a signalling molecule regulating podocyte physiology. In congenital nephrotic syndrome of the Finnish type, mutation of nephrin leads to disruption of the permeability barrier and leakage of plasma proteins into the urine. This doctoral thesis hypothesises that novel nephrin-associated molecules are involved in the function of the filtration barrier in health and disease. Bioinformatics tools were utilized to identify novel nephrin-like molecules in genomic databases, and their distribution in the kidney and other tissues was investigated. Filtrin, a novel nephrin homologue, is expressed in the glomerular podocytes and, according to immunoelectron microscopy, localizes at the slit diaphragm. Interestingly, the nephrin and filtrin genes, NPHS1 and KIRREL2, locate in a head-to-head orientation on chromosome 19q13.12. Another nephrin-like molecule, Nphs1as was cloned in mouse, however, no expression was detected in the kidney but instead in the brain and lymphoid tissue. Notably, Nphs1as is transcribed from the nephrin locus in an antisense orientation. The glomerular mRNA and protein levels of filtrin were measured in kidney biopsies of patients with proteinuric diseases, and marked reduction of filtrin mRNA levels was detected in the proteinuric samples as compared to controls. In addition, altered distribution of filtrin in injured glomeruli was observed, with the most prominent decrease of the expression in focal segmental glomerulosclerosis. The role of the slit diaphragm-associated genes for the development of diabetic nephropathy was investigated by analysing single nucleotide polymorphisms. The genes encoding filtrin, densin-180, NEPH1, podocin, and alpha-actinin-4 were analysed, and polymorphisms at the alpha-actinin-4 gene were associated with diabetic nephropathy in a gender-dependent manner. Filtrin is a novel podocyte-expressed protein with localization at the slit diaphragm, and the downregulation of filtrin seems to be characteristic for human proteinuric diseases. In the context of the crucial role of nephrin for the glomerular filter, filtrin appears to be a potential candidate molecule for proteinuria. Although not expressed in the kidney, the nephrin antisense Nphs1as may regulate the expression of nephrin in extrarenal tissues. The genetic association analysis suggested that the alpha-actinin-4 gene, encoding an actin-filament cross-linking protein of the podocytes, may contribute to susceptibility for diabetic nephropathy.

Single layer graphene film by ethanol chemical vapor deposition: Highly efficient growth and clean transfer method

The choice of ethanol (C2H5OH) as carbon source in the Chemical Vapor Deposition (CVD) of graphene on copper foils can be considered as an attractive alternative among the commonly used hydrocarbons, such as methane (CH4) [1]. Ethanol, a safe, low cost and easy handling liquid precursor, offers fast and efficient growth kinetics with the synthesis of fullyformed graphene films in just few seconds [2]. In previous studies of graphene growth from ethanol, various research groups explored temperature ranges lower than 1000 °C, usually reported for methane-assisted CVD. In particular, the 650–850 °C and 900 °C ranges were investigated, respectively for 5 and 30 min growth time [3, 4]. Recently, our group reported the growth of highly-crystalline, few-layer graphene by ethanol-CVD in hydrogen flow (1– 100 sccm) at high temperatures (1000–1070 °C) using growth times typical of CH4-assisted synthesis (10–30 min) [5]. Furthermore, a synthesis time between 20 and 60 s in the same conditions was explored too. In such fast growth we demonstrated that fully-formed graphene films can be grown by exposing copper foils to a low partial pressure of ethanol (up to 2 Pa) in just 20 s [6] and we proposed that the rapid growth is related to an increase of the Cu catalyst efficiency due weak oxidizing nature of ethanol. Thus, the employment of such liquid precursor, in small concentrations, together with a reduced time of growth and very low pressure leads to highly efficient graphene synthesis. By this way, the complete coverage of a copper catalyst surface with high spatial uniformity can be obtained in a considerably lower time than when using methane.

Semi-active vibration isolation using a near-zero-spring-rate device:steady state analysis of a single-degree-of-freedom model

A vibration isolator is described which incorporates a near-zero-spring-rate device within its operating range. The device is an assembly of a vertical spring in parallel with two inclined springs. A low spring rate is achieved by combining the equivalent stiffness in the vertical direction of the inclined springs with the stiffness of the vertical central spring. It is shown that there is a relation between the geometry and the stiffness of the individual springs that results in a low spring rate. Computer simulation studies of a single-degree-of-freedom model for harmonic base input show that the performance of the proposed scheme is superior to that of the passive schemes with linear springs and skyhook damping configuration. The response curves show that, for small to large amplitudes of base disturbance, the system goes into resonance at low frequencies of excitation. Thus, it is possible to achieve very good isolation over a wide low-frequency band. Also, the damper force requirements for the proposed scheme are much lower than for the damper force of a skyhook configuration or a conventional linear spring with a semi-active damper.

Association of the microRNA-Single Nucleotide Polymorphism rs2910164 in miR146a with sporadic breast cancer susceptibility: A case control study

Background Breast cancer (BC) is primarily considered a genetic disorder with a complex interplay of factors including age, gender, ethnicity, family history, personal history and lifestyle with associated hormonal and non-hormonal risk factors. The SNP rs2910164 in miR146a (a G to C polymorphism) was previously associated with increased risk of BC in cases with at least a single copy of the C allele in breast cancer, though results in other cancers and populations have shown significant variation. Methods In this study, we examined this SNP in an Australian sporadic breast cancer population of 160 cases and matched controls, with a replicate population of 403 breast cancer cases using High Resolution Melting. Results Our analysis indicated that the rs2910164 polymorphism is associated with breast cancer risk in both primary and replicate populations (p = 0.03 and 0.0013, respectively). In contrast to the results of familial breast cancer studies, however, we found that the presence of the G allele of rs2910164 is associated with increased cancer risk, with an OR of 1.77 (95% CI 1.40–2.23). Conclusions The microRNA miR146a has a potential role in the development of breast cancer and the effects of its SNPs require further inquiry to determine the nature of their influence on breast tissue and cancer.

Ratios of T-cell immune-effectors with tumour associated macrophages and PD-1/PD-L1 axis immune-checkpoint molecules, as prognosticators in diffuse large B cell lymphoma: A population-based study

Background Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured despite initial R-CHOP. Although the prognostic importance of the tumour microenvironment (TME) is established, the optimal strategy to quantify it is unknown. Methods The relationship between immune-effector and inhibitory (checkpoint) genes was assessed by NanoString™ in 252 paraffin-embedded DLBCL tissues. A model to quantify net anti-tumoural immunity as an outcome predictor was tested in 158 R-CHOP treated patients, and validated in tissue/blood from two independent R-CHOP treated cohorts of 233 and 140 patients respectively. Findings T and NK-cell immune-effector molecule expression correlated with tumour associated macrophage and PD-1/PD-L1 axis markers consistent with malignant B-cells triggering a dynamic checkpoint response to adapt to and evade immune-surveillance. A tree-based survival model was performed to test if immune-effector to checkpoint ratios were prognostic. The CD4*CD8:(CD163/CD68)*PD-L1 ratio was better able to stratify overall survival than any single or combination of immune markers, distinguishing groups with disparate 4-year survivals (92% versus 47%). The immune ratio was independent of and added to the revised international prognostic index (R-IPI) and cell-of-origin (COO). Tissue findings were validated in 233 DLBCL R-CHOP treated patients. Furthermore, within the blood of 140 R-CHOP treated patients immune-effector:checkpoint ratios were associated with differential interim-PET/CT+ve/-ve expression.

An investigation of first-order transition across charge ordered and ferromagnetic phases in Gd0.5Sr0.5MnO3 single crystals by magnetic and magnetotransport studies

Gadolinium strontium manganite single crystals of the composition Gd0.5Sr0.5MnO3 were grown using the optical float zone method. We report here the magnetic and magnetotransport properties of these crystals. A large magnetoresistance similar to 10(9)% was observed at 45 K under the application of a 110 kOe field. We have observed notable thermomagnetic anomalies such as open hysteresis loops across the broadened first-order transition between the charge order insulator and the ferromagnetic metallic phase while traversing the magnetic field-temperature (H-T) plane isothermally or isomagnetically. In order to discern the cause of these observed anomalies, the H-T phase diagram for Gd0.5Sr0.5MnO3 is formulated using the magnetization-field (M-H), magnetization-temperature (M-T) and resistance-temperature (R-T) measurements. The temperature dependence of the critical field (i.e. H-up, the field required for transformation to the ferromagnetic metallic phase) is non-monotonic. We note that the non-monotonic variation of the supercooling limit is anomalous according to the classical concepts of the first-order phase transition. Accordingly, H-up values below similar to 20 K are unsuitable to represent the supercooling limit. It is possible that the nature of the metastable states responsible for the observed open hysteresis loops is different from that of the supercooled ones.

Selective detection of single-enantiomer spectrum of chiral molecules aligned in the polypeptide liquid crystalline solvent: Transition selective one-dimensional H-1-H-1 COSY

One-dimensional (1D) proton NMR spectra of enantiomers are generally undecipherable in chiral orienting poly-gamma-benzyl-L-glutamate (PBLG)/CDCl3 solvent. This arises due to large number of couplings, in addition to superposition of spectra from both the enantiomers, severely hindering the H-1 detection. On the other hand in the present study the benefit is derived front the presence of several couplings among the entire network of interacting protons. Transition selective 1D H-1-H-1 correlation experiment (1D-COSY) which utilizes the Coupling assisted transfer of magnetization not only for unraveling the overlap but also for the selective detection of enantiopure spectrum is reported. The experiment is simple, easy to implement and provides accurate eanantiomeric excess in addition to the determination of the proton-proton couplings of an enantiomer within a short experimental time (few minutes). (C) 2009 Elsevier Inc. All rights reserved.

Separation of Metallic and Semiconducting Single-Walled Carbon Nanotubes Through Fluorous Chemistry

Separation of metallic from semiconducting single-walled carbon nanotubes has been a major challenge for some time and some previous efforts have resulted in partial success. We have accomplished the separation effectively by employing fluorous chemistry wherein the diazonium salt of 4-heptadecafluorooc tylaniline selectively reacts with the metallic nanotubes present in the mixture of nanotubes. The resulting fluoroderivative was extracted in perfluorohexane leaving the semiconducting nanotubes in the aqueous layer. The products have been characterized by both Raman and electronic absorption spectroscopy. The method avoids the cumbersome centrifugation step required by some other procedures.

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.

Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.

Homologous pairing between nucleosome cores on a linear duplex DNA and nucleoprotein filaments of RecA protein-single stranded DNA

The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination.