Inverting character of alpha-glucuronidase A from Aspergillus tubingensis

Alpha-glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-alpha-1,2-D-xylopyranosyl-beta-1,4-xylopyranosyl-beta-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D(2)O reaction mixture by (1)H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its beta-anomer from which the alpha-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial alpha-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).     

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.     

Phylogeny and divergence of the pinnipeds (Carnivora: Mammalia) assessed using a multigene dataset

Background  

Phylogenetic comparative methods are often improved by complete phylogenies with meaningful branch lengths (e.g., divergence dates). This study presents a dated molecular supertree for all 34 world pinniped species derived from a weighted matrix representation with parsimony (MRP) supertree analysis of 50 gene trees, each determined under a maximum likelihood (ML) framework. Divergence times were determined by mapping the same sequence data (plus two additional genes) on to the supertree topology and calibrating the ML branch lengths against a range of fossil calibrations. We assessed the sensitivity of our supertree topology in two ways: 1) a second supertree with all mtDNA genes combined into a single source tree, and 2) likelihood-based supermatrix analyses. Divergence dates were also calculated using a Bayesian relaxed molecular clock with rate autocorrelation to test the sensitivity of our supertree results further.     

Inducible cytochrome P450 activities in renal glomerular mesangial cells: biochemical basis for antagonistic interactions among nephrocarcinogenic polycyclic aromatic hydrocarbons

BACKGROUND: Benzo(a)pyrene (BaP), anthracene (ANTH) and chrysene (CHRY) are polynuclear aromatic hydrocarbons (PAHs) implicated in renal toxicity and carcinogenesis. These PAHs elicit cell type-specific effects that help predict toxicity outcomes in vitro and in vivo. While BaP and ANTH selectively injure glomerular mesangial cells, and CHRY targets cortico-tubular epithelial cells, binary or ternary mixtures of these hydrocarbons markedly reduce the overall cytotoxic potential of individual hydrocarbons. METHODS: To study the biochemical basis of these antagonistic interactions, renal glomerular mesangial cells were challenged with BaP alone (0.03 - 30 microM) or in the presence of ANTH (3 microM) or CHRY (3 microM) for 24 hr. Total RNA and protein will be harvested for Northern analysis and measurements of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activity, respectively, to evaluate cytochrome P450 mRNA and protein inducibility. Cellular hydrocarbon uptake and metabolic profiles of PAHs were analyzed by high performance liquid chromatography (HPLC). RESULTS: Combined hydrocarbon treatments did not influence the cellular uptake of individual hydrocarbons. ANTH or CHRY strongly repressed BaP-inducible cytochrome P450 mRNA and protein expression, and markedly inhibited oxidative BaP metabolism. CONCLUSION: These findings indicate that antagonistic interactions among nephrocarcinogenic PAHs involve altered expression of cytochrome P450s that modulate bioactivation profiles and nephrotoxic/ nephrocarcinogenic potential.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase ineukaryotes, and essential for DNA replication. By applying serial extractions to mammaliancells synchronized by release from quiescence, we reveal dynamic changes to thesub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase,identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix.The data distinguish 3 states that correspond to loose association with chromatin prior toDNA replication, transient highly stable binding to the nuclear-matrix coincident withinitiation, and a post-initiation phase when MCM2 remains tightly associated withchromatin but not the nuclear-matrix. The data suggests that functional MCM complexloading takes place at the nuclear-matrix.     

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.     

Factors influencing the release of proteins by cultured schwann cells

Cultured rat schwann cells grown in association with sensory neurons when labeled with [(3)H]leucinem, [(3)H]glucosamine, or [(35)S]methionine release labeled polypeptides into the culture medium. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of more than 20 polypeptides with molecular weights ranging from 15,000 to more than 250,000. Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for approximately 40 percent of the leucine or methionine radioactivity in medium polypeptide. Schwann cells grown in a serum-free defined medium, in which schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides tha cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides. Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE. This change in the pattern of polypeptides release by schwann cells is accompanied by changes in the association between schwann cells and axons. An early step in the establishment of normal axon-schwann cell relations appears to be an inward migration of schwann cells into axonal bundles and spreading of schwann cells along neurites. These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by schwann cells may be important for the development of normal axonal ensheathment.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.