Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.     

Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.     

Serum paraoxonase-1 (PON1) activities (PONase/AREase) and polymorphisms in patients with type 2 diabetes mellitus in a North-West Indian population

Objective

Paraoxonase-1 (PON1), an HDL-C associated enzyme, protects lipoproteins from oxidation. There is evidence that PON1 enzyme activity is reduced in the patients with type 2 diabetes mellitus (T2DM). North-West Indian Punjabis, a distinct ethnic group has high incidence of T2DM. However till date there is no information regarding PON1 enzyme activities and PON1 polymorphisms in T2DM patients of this ethnic group.

Methods

We identified polymorphisms in the coding Q192R, L55M and promoter − 909G/C, − 162A/G, − 108C/T of the PON1 gene by using PCR-RFLP, multiplex PCR and allele specific oligonucleotide PCR assays in 250 T2DM patients and 300 healthy controls. We also assessed paraoxonase (PONase) and arylesterase (AREase) activities of PON1 enzyme.

Results

The serum PONase (114.2 vs. 178.0 nmol/min/ml) and AREase (62.7 vs. 82.5 μmol/min/ml) activities were significantly lower (p < 0.0001) in patients as compared to controls. PONase activity was affected by all the studied PON1 polymorphisms. However, AREase activity was not affected by any of these polymorphisms. Coding Q192R and promoter − 909G/C polymorphisms showed significant differences in genotypic distribution. QR, RR (Q192R) and GC, CC (− 909G/C) genotypes and L-C-A-R-G, L-T-A-R-G, L-T-G-Q-C haplotypes showed significant association with type 2 diabetes. No significant linkage disequilibrium was observed among the five polymorphisms.

Conclusion

Both PONase and AREase activities are lower in patients and this could lead to increased lipid peroxidation and accelerated atherosclerosis in them. PONase activity, but not AREase activity is influenced by PON1 polymorphisms. QR, RR, GC, CC genotypes and L-C-A-R-G, L-T-A-R-G, L-T-G-Q-C haplotypes are commoner in diabetics as compared to controls and may be related to genetic susceptibility to type 2 diabetes.     

Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CSV86

Pseudomonas putida CSV86 utilizes benzyl alcohol via catechol and methylnaphthalenes through detoxification pathway via hydroxymethylnaphthalenes and naphthaldehydes. Based on metabolic studies, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) were hypothesized to be involved in the detoxification pathway. BADH and BZDH were purified to apparent homogeneity and were (1) homodimers with subunit molecular mass of 38 and 57 kDa, respectively, (2) NAD⁺ dependent, (3) broad substrate specific accepting mono- and di-aromatic alcohols and aldehydes but not aliphatic compounds, and (4) BADH contained iron and magnesium, while BZDH contained magnesium. BADH in the forward reaction converted alcohol to aldehyde and required NAD⁺, while in the reverse reaction it reduced aldehyde to alcohol in NADH-dependent manner. BZDH showed low K _m value for benzaldehyde as compared to BADH reverse reaction. Chemical cross-linking studies revealed that BADH and BZDH do not form multi-enzyme complex. Thus, the conversion of aromatic alcohol to acid is due to low K _m and high catalytic efficiency of BZDH. Phylogenetic analysis revealed that BADH is a novel enzyme and diverged during the evolution to gain the ability to utilize mono- and di-aromatic compounds. The wide substrate specificity of these enzymes enables strain to detoxify methylnaphthalenes to naphthoic acids efficiently.     

Protective effects of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia

In the present study, the newly synthesized TRH analog (l-pGlu-(2-propyl)-l-His-l-ProNH₂; NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H₂O₂-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6 h. Exposure of NP-647 was given before and during OGD. In glutamate and H₂O₂ induced injury, exposure of NP-647 was given 1, 6 and 24 h prior to exposure of glutamate and H₂O₂ exposure. NP-647, per se found to be non-toxic in 1-100 μM concentrations. NP-647 showed protection against OGD at the 1 and 10 μM. The concentration-dependent protection was observed in H₂O₂- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.     

Backbones of folded proteins reveal novel invariant amino acid neighborhoods

Folding of naturally occurring proteins has eluded a universal molecular level explanation till date. Rather, there is an abundance of diverse views on dominant factors governing protein folding. Through rigorous analyses of several thousand crystal structures, we observe that backbones of folded proteins display some remarkable invariant features. Folded proteins are characterized by spatially well-defined, distance dependent, and universal, neighborhoods of amino acids which defy any of the conventionally prevalent views. These findings present a compelling case for a newer view of protein folding which takes into account solvent mediated and amino acid shape and size assisted optimization of the tertiary structure of the polypeptide chain to make a functional protein.     

Biophysical Variability and Pastoral Rights to Resources: West African Transhumance Revisited

This paper focuses on a conundrum that has dominated the literature on pastoral mobility and institutions in dryland regions of the world, where livestock production is the main livelihood system. High spatiotemporal variability of rainfall and forage resources are seen to require flexible rules and porous social boundaries to facilitate pastoral mobility—characteristics that run counter to conventional views of the requirements for effective common property institutions. We seek to explore this paradox by investigating the spatiotemporal variability of forage availability (using satellite derived vegetation indices as a proxy for green forage) in four transhumance zones (“transhumance sheds”) in Mali, West Africa. For each transhumance shed, three characteristics with important institutional implications are evaluated over an eleven-year period between 2000 and 2010: the inter-annual variability of forage phenology, seasonal changes in connectivity of green forage patches, and the degree to which key forage locations exist in the form of consistently early green-up and/or late senescence. Periods of vegetation green-up and senescence, which determine the timing of transhumant livestock movements, are found to be sufficiently regular from year to year to be governed by conventional institutions. Seasonal changes in the north-south connectivity of green patches are sufficiently rapid for customary systems of sharing of pasture information to be effective (rather than more technologically sophisticated systems of pasture information). Moreover, transhumance sheds contain key pastoral forage sites, which because of their consistently early greening or late senescence, are strong candidates for territorial protection from alternative land uses such as agriculture. These findings support local herders’ views of transhumance as composed of regular patterns of herd movements along prescribed corridors between key pastoral sites. The seasonal regularity of key pastoral resources has been obscured by an overemphasis on environmental unpredictability that characterizes dryland systems at certain spatial and temporal scales. This paper suggests that policies directed at improving pastoral resource governance must focus instead on securing pastoralists’ access rights to movement corridors, specific pastures and water points.     

An Appraisal of Life History Features of Kiefferulus calligaster (Kieffer, 1911) (Diptera: Chironomidae) from Kolkata,India

Life history parameters of the freshwater chironomid species Kiefferulus calligaster (Kieffer, 1911) were investigated under laboratory conditions, with the use of larval development time and wing length as key features. An index of fitness was derived using these two parameters to represent the fitness of adults as a function of the larval development. Survivorship, deduced from the data on the mortality of larval stages, was related to developmental time as—(survivorship, l_x) y = 1.16 ? 0.04 × (days). The larval development time varied between males and females with a minimum of 8 and a maximum of 12 days from first instar larva to eclosion of imagine. The average wing length of adult females was larger than males (3.9 mm ± 0.03 S.E. vs. 3.36 mm ± 0.02 S.E.), for both early and late emerging individuals. The degree of dimorphism between the sexes was prominent for wing length and larval development time. The index of fitness for the early and late emerging adults differed significantly (P < 0.05) in both the sexes.     

Screening of commercial cyclic peptide as inhibitor NS5 methyltransferase of Dengue virus through Molecular Docking and Molecular Dynamics Simulation

Dengue has become a major global health threat, especially in tropical and subtropical regions. The development of antiviral agent targeting viral replication is really needed at this time. NS5 methyltransferase presents as a novel antiviral target. This enzyme plays an important role in the methylation of 5''-cap mRNA. Inhibition of the NS5 methyltransferase could inhibit dengue virus replication. In this research, two sites of NS5 methyltransferase (S-Adenosyl methionine/SAM binding site and RNA-cap site) were used as targets for inhibition. As much as 300 commercial cyclic peptides were screened to these target sites by means of molecular docking. Analysis of ligand-enzyme binding free energy and pharmacological prediction revealed two best ligands, namely [Tyr123] Prepro Endothelin (110-130), amide, human and Urotensin II, human. According to molecular dynamic simulation, both ligands maintain a stable complex conformation between enzyme and ligand at temperature 310 K and 312 K. Hence, Urotensin II, human is more reactive at 312 K than at 310 K. However, both ligands can be used as potential inhibitor candidates against NS5 methyltransferase of dengue virus with Urotensin II, human exposes more promising activity at 312 K.     

Epidermal Growth Factor Signalling Controls Myosin II Planar Polarity to Orchestrate Convergent Extension Movements during Drosophila Tubulogenesis

Most epithelial tubes arise as small buds and elongate by regulated morphogenetic processes including oriented cell division, cell rearrangements, and changes in cell shape. Through live analysis of Drosophila renal tubule morphogenesis we show that tissue elongation results from polarised cell intercalations around the tubule circumference, producing convergent-extension tissue movements. Using genetic techniques, we demonstrate that the vector of cell movement is regulated by localised epidermal growth factor (EGF) signalling from the distally placed tip cell lineage, which sets up a distal-to-proximal gradient of pathway activation to planar polarise cells, without the involvement for PCP gene activity. Time-lapse imaging at subcellular resolution shows that the acquisition of planar polarity leads to asymmetric pulsatile Myosin II accumulation in the basal, proximal cortex of tubule cells, resulting in repeated, transient shortening of their circumferential length. This repeated bias in the polarity of cell contraction allows cells to move relative to each other, leading to a reduction in cell number around the lumen and an increase in tubule length. Physiological analysis demonstrates that animals whose tubules fail to elongate exhibit abnormal excretory function, defective osmoregulation, and lethality.