Role of an ABC Importer in Mycobacterial Drug Resistance

Phosphate specific transporter (Pst) in bacteria is involved in phosphate transport. Pst is a multisubunit system which belongs to the ABC family of transporters. The import function of this transporter is known to be operative at media phosphate concentrations below the millimolar range. However, we found amplification of this transporter in a laboratory generated ciprofloxacin resistant Mycobacterium smegmatis colony (CIP^r) which was grown in a condition when phosphate scavenging function of this operon was inoperative. Our results therefore argue the role of this ABC importer in conferring high level of fluoroquinolone resistance in CIP^r.     

Increased expression and activity of 11beta-HSD-1 in diabetic islets and prevention with troglitazone

To determine if increased local production of glucocorticoids by the pancreatic islets might play a role in the spontaneous noninsulin-dependent diabetes mellitus of obesity, we compared islet 11beta-HSD-1 mRNA and activity in islets of obese prediabetic and diabetic Zucker Diabetic Fatty (ZDF) (fa/fa) rats and lean wild-type (+/+) controls. In diabetic rat islets, both mRNA and enzymatic activity of the enzyme were increased in proportion to the hyperglycemia. Troglitazone (TGZ) treatment, beginning at 6 weeks of age, prevented the hyperglycemia, the hyperlipidemia, and the increase in 11beta-HSD-1. To determine if the metabolic abnormalities had caused the 11beta-HSD-1 increase, prediabetic islets were cultured in high or low glucose or in 2:1 oleate:palmitate for 3 days. Neither nutrient enhanced the expression of 11beta-HSD-1. We conclude that 11beta-HSD-1 expression and activity are increased in islets of diabetic, but not prediabetic ZDF rats, and that TGZ prevents both the increase in 11beta-HSD-1 and the diabetes.     

Structure of a quinobenzoxazine--G-quadruplex complex by REDOR NMR

Rotational-echo double resonance solid-state (31)P[(19)F] and (13)C[(19)F] NMR spectra have been used to locate the binding of a fluoroquinobenzoxazine to a DNA G-quadruplex labeled by phosphorothioation and [methyl-(13)C]thymidine.     

Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India

Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.     

Utility of acetyldithio-CoA in detecting the influence of active site residues on substrate enolization by 3-hydroxyl-3-methylglutaryl-CoA synthase

Hydroxymethylglutaryl-CoA synthase-catalyzed condensation of acetyl-CoA with acetoacetyl-CoA requires enolization/carbanion formation from the acetyl C-2 methyl group prior to formation of a new carbon-carbon bond. Acetyldithio-CoA, a readily enolizable analog of acetyl-CoA, was an effective competitive inhibitor of avian hydroxymethylglutaryl-CoA synthase (Ki = 28 microm). In the absence of cosubstrate, enzyme catalyzed the enolization/proton exchange from the C-2 methyl group of acetyldithio-CoA. Mutant enzymes that exhibited impaired formation of the covalent acetyl-S-enzyme reaction intermediate exhibited diminished (D159A and D203A) or undetectable (C129S) rates of enolization of acetyldithio-CoA. The results suggest that covalent thioacetylation of protein, which has not been detected previously for other enzymes that enolize this analog, occurs with hydroxymethylglutaryl-CoA synthase. Enzyme catalyzed the transfer of the thioacetyl group of this analog to 3'-dephospho-CoA suggesting the intermediacy of a covalent thioacetyl-S-enzyme species, which appears to be important for proton abstraction from C-2 of the thioacetyl group. Avian enzyme glutamate 95 is crucial to substrate condensation to form a new carboncarbon bond. Mutations of this invariant residue (avian enzyme E95A and E95Q; Staphylococcus aureus enzyme E79Q) correlated with diminished ability to catalyze enolization of acetyldithio-CoA. Enolization by E95Q was not stimulated in the presence of acetoacetyl-CoA. These observations suggest either a direct (proton abstraction) or indirect (solvent polarization) role for this active site glutamate.     

Effect of side chain length on the aggregation of amphiphilic 5,10,15-tris (1-methylpyridinium-4-yl)-20-[4-(alkoxy) phenyl] 21H, 23H porphyrin tritosylates

Self-aggregation of cationic amphiphilic 5, 10, 15-tris-(1-methylpyridinium-4-yl)-20-[4-(alkoxy)phenyl]-21H, 23H porphyrin tritosylates (2a-e) with different alkoxy chain length in aqueous and binary solvent systems has been studied by UV-visible, fluorescence and (I)H NMR spectroscopy. Binary solvent, concentration, ionic strength, presence of surfactants, and temperature govern the aggregations of 2a-e. Porphyrins having side chain length more than ten carbon atoms (2c-e) form higher aggregates, such as vesicles by sonication of dimers formed initially, whereas porphyrins with lesser side chain length (2a & b) form lower aggregates only. Further, the size and the formation of vesicles have been confirmed by transmission electron microscopy (TEM) and dye entrapment experiments for 2e.     

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin-dependent kinases regulates binding to PCNA

Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr⁵⁰⁶ in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr⁵⁰⁶→Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr⁵⁰⁶ in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr⁵⁰⁶ is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.