Uniform Action Potential Repolarization within the Sarcolemma of In Situ Ventricular Cardiomyocytes

Previous studies have speculated, based on indirect evidence, that the action potential at the transverse (t)-tubules is longer than at the surface membrane in mammalian ventricular cardiomyocytes. To date, no technique has enabled recording of electrical activity selectively at the t-tubules to directly examine this hypothesis. We used confocal line-scan imaging in conjunction with the fast response voltage-sensitive dyes ANNINE-6 and ANNINE-6plus to resolve action potential-related changes in fractional dye fluorescence (ΔF/F) at the t-tubule and surface membranes of in situ mouse ventricular cardiomyocytes. Peak ΔF/F during action potential phase 0 depolarization averaged −21% for both dyes. The shape and time course of optical action potentials measured with the water-soluble ANNINE-6plus were indistinguishable from those of action potentials recorded with intracellular microelectrodes in the absence of the dye. In contrast, optical action potentials measured with the water-insoluble ANNINE-6 were significantly prolonged compared to the electrical recordings obtained from dye-free hearts, suggesting electrophysiological effects of ANNINE-6 and/or its solvents. With either dye, the kinetics of action potential-dependent changes in ΔF/F during repolarization were found to be similar at the t-tubular and surface membranes. This study provides what to our knowledge are the first direct measurements of t-tubule electrical activity in ventricular cardiomyocytes, which support the concept that action potential duration is uniform throughout the sarcolemma of individual cells.     

Functional activity of phagocyte blood cells in pulmonary tuberculosis

The characteristics of the functional activity of phagocyte blood cells in patients with destructive pulmonary tuberculosis caused by medicinal sensitive and medicinal resistant infective agents were studied. In the process of pulmonary tuberculosis, irrespective of the medicinal sensitivity of infective agents before and during treatment, a decrease in the phagocytic activity of neutrophil granulocytes and the level of the expression of Fcgamma- and C3b-receptors on monocytes with a simultaneous increase in the spontaneous production of oxygen metabolites in neutophils and a rise in the adsorptive capacity of monocytic cells were observed.     

Molecular Basis of Neurovirulence of Flury Rabies Virus Vaccine Strains: Importance of the Polymerase and the Glycoprotein R333Q Mutation

The molecular mechanisms associated with rabies virus (RV) virulence are not fully understood. In this study, the RV Flury low-egg-passage (LEP) and high-egg-passage (HEP) strains were used as models to explore the attenuation mechanism of RV. The results of our studies confirmed that the R333Q mutation in the glycoprotein (G_R333Q) is crucial for the attenuation of Flury RV in mice. The R333Q mutation is stably maintained in the HEP genome background but not in the LEP genome background during replication in mouse brain tissue or cell culture. Further investigation using chimeric viruses revealed that the polymerase L gene determines the genetic stability of the G_R333Q mutation during replication. Moreover, a recombinant RV containing the LEP G protein with the R333Q mutation and the HEP L gene showed significant attenuation, genetic stability, enhancement of apoptosis, and immunogenicity. These results indicate that attenuation of the RV Flury strain results from the coevolution of G and L elements and provide important information for the generation of safer and more effective modified live rabies vaccine.Rabies virus (RV) belongs to the genus Lyssavirus of the family Rhabdoviridae and causes a fatal neurological disease in humans and animals (6). The RV genome is a nonsegmented negative-strand (NNS) RNA encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase (L). Among these, the G protein is a major contributor to RV pathogenicity (7, 31, 33). The G protein facilitates fast virus entry and transsynaptic spread and regulates the rate of virus replication, together with other viral elements (8, 30, 39). The G protein of nonpathogenic RV strains can trigger apoptosis, while the RV G of pathogenic strains induces less or no apoptosis (35, 59). The amino acid residue at position 333 of the G protein (G333) of some fixed strains has been shown to be an important determinant of virulence in adult mice (5). Strains that have arginine or lysine at position G333 kill adult mice, whereas mutants with other amino acids at this site cause a nonlethal infection (1, 5, 25, 36, 49, 53). However, the pathogenicity of RV strains is not solely determined by substitutions at the G333 position. Other substitutions in the G protein, such as N194K, have also been shown to affect viral pathogenicity in mice (10, 21, 50). In addition, other viral elements, such as the N, P, M, and L genes, the trailer sequence in the noncoding region, and the pseudogene, were also reported to modulate RV pathogenicity (12, 46, 57, 58). How these viral elements regulate the pathogenicity of RV remains to be fully explored, and further investigation is needed to understand the molecular basis of RV pathogenicity.Attenuated Flury RV low-egg-passage (LEP) and high-egg-passage (HEP) strains were established through serial passage in chicken brain, chicken embryos, and culture cells using a Flury RV isolated from a girl who died of rabies (23, 24). LEP has Arg at position G333 and kills adult mice after intracerebral (i.c.) inoculation, while HEP has Gln at G333 and causes only mild signs in adult mice. It has been demonstrated that HEP could regain lethality in adult mice by a single amino acid change at G333 from Gln to Arg (49), which indicated that Arg at position G333 is a key determinant of pathogenicity of Flury RV in adult mice. However, whether the Arg at G333 is indispensable for the lethal phenotype of LEP has not been demonstrated.In the current study, LEP and HEP Flury RV strains were used as models to investigate the mechanism of attenuation. We found that both G and L contribute to the attenuation of Flury RV. Substitution of Arg with Gln at G333 (G_R333Q) eliminated LEP neuroinvasiveness but not the virus'' lethal phenotype in adult mice after i.c. inoculation. The G_R333Q mutation could be kept stable only in the genome background of HEP but not in that of LEP during replication. The L gene contributes to the attenuation and enhanced immunogenicity of Flury RV by promoting the stabilization of the G_R333Q mutation during virus replication in brain tissues or cells.     

Novel Glycoside Hydrolases Identified by Screening a Chinese Holstein Dairy Cow Rumen-Derived Metagenome Library

One clone encoding glycoside hydrolases was identified through functional screening of a rumen bacterial artificial chromosome (BAC) library. Of the 68 open reading frames (ORFs) predicted, one ORF encodes a novel endo-β-1,4-xylanase with two catalytic domains of family GH43 and two cellulose-binding modules (CBMs) of family IV. Partial characterization showed that this endo-xylanase has a greater specific activity than a number of other xylanases over a wide temperature range at neutral pH and could be useful in some industrial applications.The ruminal microbes possess a repertoire of hydrolases, including glycosyl hydrolases and esterases, which mediate hydrolysis and subsequent fermentation of the diets (mainly cellulose, xylan, amylopectin, amylose, pectin) (10, 18). Thus, the ruminal microbiome has been recognized as a rich source of enzymes important not only for feed and animal industries but also for the bioenergy industry (20, 26). However, this enzyme source is largely untapped because the majority of ruminal microbes remain uncultured (17). Activity-based metagenomics enables direct identification of genes and enzymes of interest by screening metagenomic libraries for desired heterologous phenotypic traits expressed in a surrogate bacterial host. Collectively, previous studies have identified 12 esterases, 10 endoglucanases, two lipases, one cyclodextrinase, one polyphenol oxidase, and one unique multifunctional glycosyl hydrolase from ruminal metagenomic libraries (2, 6, 8-9, 21, 23). These studies provided further evidence that the ruminal microbiome contains a rich source of glycosyl hydrolases possessed by as-yet-uncultured microbes. The objective of this study was to examine a bacterial artificial chromosome (BAC) library constructed previously from the ruminal samples collected from Chinese Holstein cows for hydrolases.     

Generic switch-over during ontogenesis in Dimorphacanthella gen. n. (Collembola, Isotomidae) with barcoding evidence

A new genus Dimorphacanthella is established for Tetracanthella anommatos Chen and Yin, 1984 and Dimorphacanthella mediasetasp. n. from China. The new genus exhibits an unusual metamorphosis: small juveniles, previously called Uzelia anommatos Yue & Yin, 1999 get the second pair of anal spines resulted from moulting and become "Tetracanthella". Species identity of forms with two and four anal spines is proved by barcoding analysis. The derivation of anal spines is compared among genera having four anal spines.     

Design and synthesis of boron-containing PDE4 inhibitors using soft-drug strategy for potential dermatologic anti-inflammatory application

PDE4 inhibitors are a validated approach as anti-inflammatory agents but are limited by systemic side effects including emesis. We report a soft-drug strategy incorporating a carboxylic ester group into boron-containing PDE4 inhibitors leading to the discovery of a series of benzoxaborole compounds with good potency (for example IC₅₀ = 47 nM of compound 2) and low emetic activity. These compounds are intended for dermatological use further limiting possible systemic side effects.     

Cell cycle arrest and cytochrome c-mediated apoptotic induction by MCS-5A is associated with up-regulation of p16INK4a in HL-60 cells

MCS-5A, an analog of sangivamycin, selectively inhibits the cyclin-dependent kinases CDK1 and 4 in HL-60 cells in vitro (IC₅₀: 9.6 and 8.8 μΜ, respectively), while weakly inhibiting other housekeeping protein kinases. MCS-5A effectively induces HL-60 cell cycle arrest at the G₁ and G₂/M phases through direct inhibition of CDK1 and 4 activity. In addition, elevated expression of p16^INK4a and a reduction in the level of hyperphosphorylated pRb showed that 3 μΜ MCS-5A also induces p16^INK4a-mediated cell cycle arrest at the G₁ phase. Furthermore, apoptotic induction in MCS-5A-treated HL-60 cells is associated with the release of cytochrome c from mitochondria, which, in turn, results in the activation of procaspase-8, -9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). In addition, the involvement of p16^INK4a in this apoptotic induction was demonstrated using A549 cells with a homozygous deletion of p16^INK4a. Based on these results, we conclude that MCS-5A is a candidate therapeutic agent for the treatment of human promyelocytic leukemia via the up-regulation of p16^INK4a.