Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes.     

Queuosine Formation in Eukaryotic tRNA Occurs via a Mitochondria-localized Heteromeric Transglycosylase

tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNA^Asp, tRNA^Asn, tRNA^His, and tRNA^Tyr; an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis suggest that TGT weakly interacts with the outer mitochondrial membrane possibly through association with Qv1, which was found to be stably associated with the organelle.Queuosine (Q³; (7-{[(4,5-cis-dihydroxy-2-cyclo-penten-1-yl)-amino]methyl}-7-deazaguanosine) is a modified 7-deazaguanosine molecule found at the wobble position of transfer RNA that contains a GUN anticodon sequence: tRNA^Tyr, tRNA^Asn, tRNA^His, and tRNA^Asp (1). The Q-modification is widely distributed in nature in the tRNA of eubacteria, plants, and animals; a notable exception being yeast and plant leaf cells (2, 3). Interestingly, Q-modification has also been detected in aspartyl tRNA from mitochondria of rat (4) and opossum (5). In most eukaryotes, the Q molecule can be further modified by the addition of a mannosyl group to Q-tRNA^Asp and a galactosyl group to Q-tRNA^Tyr (1).Eubacteria are unique in their ability to synthesize Q. As part of this biosynthetic process, the eubacterial tRNA guanine transglycosylase (TGT) enzyme inserts the Q precursor molecule, 7-aminomethyl-7-deazaguanine (preQ₁) into tRNA, which is then converted to Q by two further enzymatic steps at the tRNA level (6). Eukaryotes by contrast salvage queuosine from food and enteric bacteria either as the free base (referred to as queuine) or as queuosine 5′-phosphate subsequent to normal tRNA turnover (7). A Q-related molecule, archaeosine, is found at position 15 of the D loop of most archaeal tRNA, where it functions to stabilize the tRNA structure (8). The enzyme involved in archaeosine biosynthesis is structurally and mechanistically related to the eubacterial TGT but with adaptations necessitated by the differences imposed by its unique substrate and tRNA specificity (9, 10).The crystal structure of the Zymononas mobilis (Z. mobilis) TGT has been determined and revealed the enzyme to be an irregular (β/α)₈ TIM barrel with a C-terminal zinc-binding subdomain (11). Insight into the residues involved in catalysis came from mutational and kinetic analysis of the recombinant Escherichia coli enzyme (12) and from the Z. mobilis TGT structure as an RNA-bound intermediate complexed to the final preQ₁-modified RNA product (13). This work showed the essential role of Asp-280 (Z. mobilis numbering) as the active site nucleophile. Asp-102, which was originally ascribed the role of active site nucleophile, functions as a general acid/base during catalysis (12, 10). Although, the E. coli and Z. mobilis TGT enzymes are monomeric in solution (14), at high protein concentrations the E. coli enzyme can oligomerize (15), and structural data from the Z. mobilis TGT has shown the formation of a 2:1 complex with tRNA; a possible functional requirement for catalysis (10).In contrast to the eubacterial enzyme, which is a single protein species, purification of the eukaryotic TGT suggested that the catalytically active enzyme is a heterodimeric molecule: subunits of 60 and 43 kDa in rabbit erythrocytes (16), 66 and 32 kDa in bovine liver (17), 60 and 34.5 kDa in rat liver (18), and a homodimer of two 68-kDa proteins in wheat germ (16, 19). A partial amino acid sequence was recovered from two of these active enzyme preparations. The identity of the proteins from bovine liver (17) could not be assigned at the time of publication. However, our searches show that the peptides from the larger 65-kDa subunit are identical to asparaginyl tRNA synthetase, and those of the smaller 32-kDa subunit correspond to 2,4-dienoyl CoA reductase. A highly pure preparation from rabbit reticulocytes (20) gave peptides with homology to the immunophilin p59, human elongation factor 2 (EF2), and a deubiquitinating enzyme, USP14. It is noteworthy that none of the peptide sequences obtained showed similarity to the eubacterial TGT. The results do suggest, however, that in eukaryotes the TGT activity could be embedded in a multisubunit complex.Most recently, Deshpande and Katze (21) identified a cDNA clone encoding a putative TGT catalytic subunit. Cloning the cDNA into a mammalian expression plasmid reconstituted TGT activity in GC₃/c1 cells, which are known to be naturally deficient in Q-containing tRNA (22). In this study, we identify for the first time the composition of the eukaryotic tRNA guanine transglycosylase, reconstitute the catalytic activity in vitro, and examine the intracellular distribution of the active subunits.     

Can human papillomavirus DNA testing of self-collected vaginal samples compare with physician-collected cervical samples and cytology for cervical cancer screening in developing countries?

Background: To determine human papillomavirus (HPV) types by polymerase chain reaction (PCR)-reverse line blot assay and examine the concordance between HPV by Hybrid Capture 2 (HC2) and PCR on self-collected vaginal and physician-collected cervical samples and cytology. Methods: This was a cross-sectional study of 546 sexually active women aged ≥30 years with persistent vaginal discharge, intermenstrual or postcoital bleeding or an unhealthy cervix. Participants self-collected vaginal samples (HPV-S) and physicians collected cervical samples for conventional Pap smear and HPV DNA (HPV-P) testing and performed colposcopy, with directed biopsy, if indicated. HPV testing and genotyping was done by HC2 and PCR reverse line blot assay. Concordance between HC2 and PCR results of self- and physician-collected samples was determined using a Kappa statistic (κ) and Chi-square test. Results: Complete data were available for 512 sets with 98% of women providing a satisfactory self-sample. PCR detected oncogenic HPV in 12.3% of self- and 13.0% of physician-collected samples. Overall, there was 93.8% agreement between physician-collected and self-samples (κ = 76.31%, 95% confidence interval [CI]: 64.97–82.29%, p = 0.04)—complete concordance in 473 cases (57 positive, 416 negative), partial concordance in seven pairs and discordance in 32 pairs. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of self-sampling for detection of cervical intraepithelial neoplasia (CIN)2+ disease were 82.5%, 93.6%, 52.4% and 98.4%, respectively; for physician-sampling they were 87.5%, 93.2%, 52.2% and 98.9%, respectively; and for cytology they were 77.5%, 87.3%, 34.1% and 97.9%, respectively. Concordance between HC2 and PCR was 90.9% for self-samples (κ = 63.7%, 95% CI: 55.2–72.2%) and 95.3% for physician-collected samples (κ = 80.4%, 95% CI: 71.8–89.0%). Conclusions: Self-HPV sampling compares favourably with physician-sampling and cytology. A rapid, affordable, HPV self-test kit can be used as the primary method of cervical cancer screening in low-resource situations.     

Serum Bile Acids Are Higher in Humans With Prior Gastric Bypass: Potential Contribution to Improved Glucose and Lipid Metabolism

The multifactorial mechanisms promoting weight loss and improved metabolism following Roux‐en‐Y gastric bypass (GB) surgery remain incompletely understood. Recent rodent studies suggest that bile acids can mediate energy homeostasis by activating the G‐protein coupled receptor TGR5 and the type 2 thyroid hormone deiodinase. Altered gastrointestinal anatomy following GB could affect enterohepatic recirculation of bile acids. We assessed whether circulating bile acid concentrations differ in patients who previously underwent GB, which might then contribute to improved metabolic homeostasis. We performed cross‐sectional analysis of fasting serum bile acid composition and both fasting and post‐meal metabolic variables, in three subject groups: (i) post‐GB surgery (n = 9), (ii) without GB matched to preoperative BMI of the index cohort (n = 5), and (iii) without GB matched to current BMI of the index cohort (n = 10). Total serum bile acid concentrations were higher in GB (8.90 ± 4.84 µmol/l) than in both overweight (3.59 ± 1.95, P = 0.005, Ov) and severely obese (3.86 ± 1.51, P = 0.045, MOb). Bile acid subfractions taurochenodeoxycholic, taurodeoxycholic, glycocholic, glycochenodeoxycholic, and glycodeoxycholic acids were all significantly higher in GB compared to Ov (P < 0.05). Total bile acids were inversely correlated with 2‐h post‐meal glucose (r = ?0.59, P < 0.003) and fasting triglycerides (r = ?0.40, P = 0.05), and positively correlated with adiponectin (r = ?0.48, P < 0.02) and peak glucagon‐like peptide‐1 (GLP‐1) (r = 0.58, P < 0.003). Total bile acids strongly correlated inversely with thyrotropic hormone (TSH) (r = ?0.57, P = 0.004). Together, our data suggest that altered bile acid levels and composition may contribute to improved glucose and lipid metabolism in patients who have had GB.     

Design,synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2′-β-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10 nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2′-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.     

Design,synthesis and evaluation of (E)-α-benzylthio chalcones as novel inhibitors of BCR-ABL kinase

Novel (E)-α-benzylthio chalcones are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phosphorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leukemic cells.     

Functional biogeography as evidence of gene transfer in hypersaline microbial communities

Background

Horizontal gene transfer (HGT) plays a major role in speciation and evolution of bacteria and archaea by controlling gene distribution within an environment. However, information that links HGT to a natural community using relevant population-genetics parameters and spatial considerations is scarce. The Great Salt Lake (Utah, USA) provides an excellent model for studying HGT in the context of biogeography because it is a contiguous system with dispersal limitations due to a strong selective salinity gradient. We hypothesize that in spite of the barrier to phylogenetic dispersal, functional characteristics—in the form of HGT—expand beyond phylogenetic limitations due to selective pressure.

Methodology and Results

To assay the functional genes and microorganisms throughout the GSL, we used a 16S rRNA oligonucleotide microarray (Phylochip) and a functional gene array (GeoChip) to measure biogeographic patterns of nine microbial communities. We found a significant difference in biogeography based on microarray analyses when comparing Sørensen similarity values for presence/absence of function and phylogeny (Student''s t-test; p = 0.005).

Conclusion and Significance

Biogeographic patterns exhibit behavior associated with horizontal gene transfer in that informational genes (16S rRNA) have a lower similarity than functional genes, and functional similarity is positively correlated with lake-wide selective pressure. Specifically, high concentrations of chromium throughout GSL correspond to an average similarity of chromium resistance genes that is 22% higher than taxonomic similarity. This suggests active HGT may be measured at the population level in microbial communities and these biogeographic patterns may serve as a model to study bacteria adaptation and speciation.