Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone: kinetic and mechanistic studies

Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (1) resulted in time-dependent, irreversible inhibition of GAPDH. The kinetics of inactivation were biphasic, exhibiting an initial rapid phase and a slower second phase. Pentalenolactone methyl ester (2) also irreversibly inactivated GADPH, albeit at a slower rate and with a higher KI. The substrate glyceraldehyde-3-phosphate (G-3-P) afforded protection against inactivation by 1, whereas the presence of NAD+ in the incubation mixture stimulated the inactivation by increasing the apparent affinity of the enzyme for the inhibitor. In steady-state kinetic experiments, 1 acted as a competitive inhibitor of GAPDH with respect to G-3-P but exhibited uncompetitive inhibition with respect to NAD+. Inactivation of NAD+-free apo-GAPDH by 1 showed simple pseudo-first-order kinetics. By titrating the free thiol residues of partially inactivated GAPDH, it was found that both pentalenolactone and pentalenolactone methyl ester react with all four Cys-SH residues of the tetrameric GAPDH.     

HIF‐1α mediates the induction of IL‐8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT‐like behaviour

Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro‐angiogenic and pro‐inflammatory genes. Here, we demonstrate that exposure of cryptic‐like intestinal epithelial cells to C1845 bacteria induces HIF‐1α protein levels. This effect depends on the binding of F1845 adhesin to the membrane‐associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI‐3K decreases HIF‐1α protein levels and blocks C1845‐induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria‐induced HIF‐1α regulates the expression of IL‐8, VEGF and Twist1, thereby pointing to a role for HIF‐1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E‐cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition‐like phenotype. Since HIF‐1α silencing results in reversion of bacteria‐induced EMT markers, we speculate that HIF‐1α plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.     

Crystal structure of Escherichia coli PdxA,an enzyme involved in the pyridoxal phosphate biosynthesis pathway

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.     

Assessment of the Correlation Between Two Defining Criteria for Bidirectional Isthmic Block in the Ablation of Typical Atrial Flutter

Background

A complete, bidirectional conduction block in the cavotricuspid isthmus (CTI) represents the end-point of the typical atrial flutter ablation. We investigated the correlation between two criteria for successful ablation, one based on the atrial bipolar electrogram morphology before and after complete CTI conduction block, compared to the standard criteria of differential pacing and reversal in the right atrial depolarization sequence during coronary sinus (CS) pacing.

Method

We conducted a retrospective study in 111 patients (81 males, average age 62±10 years) who underwent an atrial flutter ablation during September 2007 - July 2009 in the Cardiology - Rehabilitation Hospital, UMF Cluj-Napoca. We assessed the presence of a bidirectional block at the end of the procedure using the standard criteria. We then analyzed the morphology of the bipolar atrial electrograms adjacent to the ablation line, before and after CTI conduction block.

Results

A change from a qRs morphology to a rSr'' morphology when pacing from the coronary sinus and from a rsr'' morphology to a QRS morphology when pacing from the low-lateral right atrium was associated with a CTI conduction block. Sensitivity (Se), specificity(Sp), positive predictive value (PPV), negative predictive value (NPV) were 96%, 89%, 99% and 67% respectively.

Conclusion

Our study suggests that the analysis of the atrial bipolar electrogram next to the ablation line before and after CTI ablation may be used as a reliable criterion to validate CTI conduction block due to its high sensitivity, specificity and positive predictive value.     

Frequent polymerase errors observed in a restricted area of clones derived from the attachment (G) protein gene of respiratory syncytial virus.

Sequence analysis of a large number of clones derived from the carboxy-terminal one-third of the attachment (G) protein gene of subgroup A respiratory syncytial viruses revealed a region very prone to polymerase errors which resulted mainly in frameshifts because of the insertion or deletion of adenosine residues in some but not all runs of such residues. Such mutations were detected in 14% of clones derived from mRNA, 58% of clones derived from genomic-sense RNA, and 50% of clones derived from in vitro-transcribed RNA. This phenomenon appears to be dependent on the template sequence.     

The synthesis of prostaglandin F by human endometrium in organ culture

Slices of human endometrium obtained from hysterectomy specimens were cultured for 48 hours in an organ culture medium supplemented with ethanol (control, vehicle), 17-β-estradiol (.5 μg/ml), or progesterone (.5 μg/ml). Uncultured endometrial tissue, cultured tissues, and the media were assayed for prostaglandin F (PGF) by a radioimmunoassay technique. Hematoxylin and eosin and periodic acid-Schiff stain histologic controls were done on all tissues. The concentrations of PGF in picograms/milligram, corrected for percent recovery, in the differently treated tissues were: preculture 298; culture control 2210; estrogen-treated 2680; progesterone-treated 1260. All differences except those between estrogen and control (p >; .10) and progesterone and control (p < .10) are significant at the p = .02 level or better. Progesterone appears to inhibit PGF synthesis which occurs during in vitro culture of human endometrium; estrogen tended to increase PGF synthesis in this system.     

Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme

Geosmin is responsible for the characteristic odor of moist soil, as well as off-flavors in drinking water and foodstuffs. Geosmin is generated from farnesyl diphosphate (FPP, 2) by an enzyme that is encoded by the SCO6073 gene in the soil organism Streptomyces coelicolor A32 (ref. 3). We have now shown that the recombinant N-terminal half of this protein catalyzes the Mg2+-dependent cyclization of FPP to germacradienol and germacrene D, while the highly homologous C-terminal domain, previously thought to be catalytically silent, catalyzes the Mg2+-dependent conversion of germacradienol to geosmin. Site-directed mutagenesis confirmed that the N- and C-terminal domains each harbor a distinct, independently functioning active site. A mutation in the N-terminal domain of germacradienol-geosmin synthase of a catalytically essential serine to alanine results in the conversion of FPP to a mixture of sesquiterpenes that includes an aberrant product identified as isolepidozene, which was previously suggested to be an enzyme-bound intermediate in the cyclization of FPP to germacradienol.     

Brushes with sage

Wherein we learn that when working with plant terpene synthases, sometimes it is not the isotopes that get scrambled.     

Functional expression and characterization of EryA,the erythritol kinase of Brucella abortus,and enzymatic synthesis of L-erythritol-4-phosphate

The eryA gene of the bacterial pathogen Brucella abortus has been functionally expressed in Escherichia coli. The resultant EryA was shown to catalyze the ATP-dependent conversion of erythritol to L-erythritol-4-phosphate (L-E4P). The steady state kinetic parameters of this reaction were determined and the enzyme was used to prepare L-E4P which was shown to be a weak inhibitor of 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP).