Ribosomal crystallography: a flexible nucleotide anchoring tRNA translocation, facilitates peptide-bond formation, chirality discrimination and antibiotics synergism

The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA-end and P-site preference of initial tRNA. The interactions of protein L2 tail with the symmetry-related region periphery explain its conservation and its contributions to nascent chain elongation.     

Fluid mechanics,cell distribution,and environment in CellCube bioreactors

Cultivation of MRC-5 cells and attenuated hepatitis A virus (HAV) for the production of VAQTA, an inactivated HAV vaccine (1), is performed in the CellCube reactor, a laminar flow fixed-bed bioreactor with an unusual diamond-shaped, diverging-converging flow geometry. These disposable bioreactors have found some popularity for the production of cells and gene therapy vectors at intermediate scales of operation (2, 3). Early testing of the CellCube revealed that the fluid mechanical environment played a significant role in nonuniform cell distribution patterns generated during the cell growth phase. Specifically, the reactor geometry and manufacturing artifacts, in combination with certain inoculum practices and circulation flow rates, can create cell growth behavior that is not simply explained. Via experimentation and computational fluid dynamics simulations we can account for practically all of the observed cell growth behavior, which appears to be due to a complex mixture of flow distribution, particle deposition under gravity, fluid shear, and possibly nutritional microenvironment.     

Cell-induced copper ion-mediated low density lipoprotein oxidation increases during in vivo monocyte-to-macrophage differentiation

Macrophage activation is associated with the production and release of reactive oxygen species (ROS), which are capable of mediating oxidative modification of low-density lipoprotein (LDL). In the present study we questioned whether cellular capacity to oxidize LDL increases during in vivo monocyte/macrophage maturation. We developed a novel model for macrophage maturation in vivo using mouse peritoneal macrophages (MPMs) harvested at increasing intervals after intraperitoneal thioglycollate injection. Macrophage maturation was evidenced by a progressive increase in cellular size, density, granulation, and expression of cell surface markers CD11b and CD36, and by a gradual decrement in myeloperoxidase activity. Cellular capacity to stimulate copper ion-mediated oxidation of LDL increased gradually by up to 2-fold during in vivo macrophage maturation in Balb/C mice, similar to the pattern observed during 1,25-dihydroxyvitamin D3-induced in vitro differentiation of the PLB-985 cell line. These effects were attributed to a gradual increase in production of ROS by up to 9-fold. The mechanism for the increase in cellular oxidative stress during macrophage maturation could be related, at least in part, to NADPH oxidase activation, as demonstrated by a gradual increase over time in p47phox expression (mRNA and protein) and in its translocation to the plasma membrane. In conclusion, in vivo monocyte-to-macrophage differentiation is associated with increased cell capacity to oxidize LDL, which may represent a protective mechanism for rapid removal of atherogenic LDL from extracellular spaces in the arterial wall.     

Caloric restriction ameliorates cardiomyopathy in animal model of diabetes

Background

The db/db mouse is an animal model of diabetes in which leptin receptor activity is deficient resulting accelerated cardiomyopathy when exposed to angiotensin (AT). Toll-like receptors 4 and 2 (TLR4, TLR2) are pattern recognition receptors, that recognize pathogen-associated molecular patterns and exacerbate and release inflammatory cytokines. Fetuin A (Fet A) is a fatty acid carrier which affects inflammation and insulin resistance in obese humans and animals through TLRs.The aim of this study was to investigate the effect of caloric restriction (CR) on free fatty acids (FFA) level and the inflammatory response in diabetic cardiomyopathy.

Differential GC content between exons and introns establishes distinct strategies of splice-site recognition

During evolution segments of homeothermic genomes underwent a GC content increase. Our analyses reveal that two exon-intron architectures have evolved from an ancestral state of low GC content exons flanked by short introns with a lower GC content. One group underwent a GC content elevation that abolished the differential exon-intron GC content, with introns remaining short. The other group retained the overall low GC content as well as the differential exon-intron GC content, and is associated with longer introns. We show that differential exon-intron GC content regulates exon inclusion level in this group, in which disease-associated mutations often lead to exon skipping. This group's exons also display higher nucleosome occupancy compared to flanking introns and exons of the other group, thus "marking" them for spliceosomal recognition. Collectively, our results reveal that differential exon-intron GC content is a previously unidentified determinant of exon selection and argue that the two GC content architectures reflect the two mechanisms by which splicing signals are recognized: exon definition and intron definition.     

Disparate impact of butyroyloxymethyl diethylphosphate (AN-7), a histone deacetylase inhibitor, and doxorubicin in mice bearing a mammary tumor

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection.     

Genetic Characterization of Antiplasmid Immunity through a Type III-A CRISPR-Cas System

Many prokaryotes possess an adaptive immune system encoded by clustered regularly interspaced short palindromic repeats (CRISPRs). CRISPR loci produce small guide RNAs (crRNAs) that, in conjunction with flanking CRISPR-associated (cas) genes, combat viruses and block plasmid transfer by an antisense targeting mechanism. CRISPR-Cas systems have been classified into three types (I to III) that employ distinct mechanisms of crRNA biogenesis and targeting. The type III-A system in Staphylococcus epidermidis RP62a blocks the transfer of staphylococcal conjugative plasmids and harbors nine cas-csm genes. Previous biochemical analysis indicated that Cas10, Csm2, Csm3, Csm4, and Csm5 form a crRNA-containing ribonucleoprotein complex; however, the roles of these genes toward antiplasmid targeting remain unknown. Here, we determined the cas-csm genes that are required for antiplasmid immunity and used genetic and biochemical analyses to investigate the functions of predicted motifs and domains within these genes. We found that many mutations affected immunity by impacting the formation of the Cas10-Csm complex or crRNA biogenesis. Surprisingly, mutations in the predicted nuclease domains of the members of the Cas10-Csm complex had no detectable effect on antiplasmid immunity or crRNA biogenesis. In contrast, the deletion of csm6 and mutations in the cas10 Palm polymerase domain prevented CRISPR immunity without affecting either complex formation or crRNA production, suggesting their involvement in target destruction. By delineating the genetic requirements of this system, our findings further contribute to the mechanistic understanding of type III CRISPR-Cas systems.