Genomic Modifiers of Natural Killer Cells,Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection

The MHC class I D^k molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds D^k, are required to control viral spread. The extent of D^k-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust D^k-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Interactions between Sympathomimetic Amines and Antidepressant Agents in Man

Intravenous infusions of phenylephrine, noradrenaline, adrenaline, and isoprenaline were given to healthy human volunteers after five to seven days on phenelzine, tranylcypromine, or imipramine, and cardiovascular responses were compared with those observed under control conditions. With monoamine oxidase inhibitors there was a 2-2½ fold potentiation of the pressor effect of phenylephrine, but no clinically significant potentiation of cardiovascular effects of noradrenaline, adrenaline, or isoprenaline. With imipramine there was potentiation of the pressor effects of phenylephrine (2-3 fold), noradrenaline (4-8 fold), and adrenaline (2-4 fold); there were dysrhythmias during adrenaline infusions, but no noticeable or consistent changes in response to isoprenaline.Noradrenaline and adrenaline in amounts contained in local anaesthetics used in dentistry are not likely to be significantly potentiated in otherwise healthy patients receiving monoamine oxidase inhibitors. Hazardous potentiation of their cardiovascular effects might occur in patients receiving tricyclic antidepressants.Our observations do not indicate that the hazards associated with isoprenaline inhalation by bronchial asthmatics would be increased by coincident therapy with a monoamine oxidase inhibitor or tricyclic antidepressant.     

Metagenomic analysis of a stable trichloroethene-degrading microbial community

Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.     

BRCA2 protein deficiency exaggerates doxorubicin-induced cardiomyocyte apoptosis and cardiac failure

The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.     

Adaptive failure to high-fat diet characterizes steatohepatitis in Alms1 mutant mice

The biochemical differences between simple steatosis, a benign liver disease, and non-alcoholic steatohepatitis, which leads to cirrhosis, are unclear. Fat aussie is an obese mouse strain with a truncating mutation (foz) in the Alms1 gene. Chow-fed female foz/foz mice develop obesity, diabetes, and simple steatosis. We fed foz/foz and wildtype mice a high-fat diet. Foz/foz mice developed serum ALT elevation and severe steatohepatitis with hepatocyte ballooning, inflammation, and fibrosis; wildtype mice showed simple steatosis. Biochemical pathways favoring hepatocellular lipid accumulation (fatty acid uptake; lipogenesis) and lipid disposal (fatty acid beta-oxidation; triglyceride egress) were both induced by high-fat feeding in wildtype but not foz/foz mice. The resulting extremely high hepatic triglyceride levels were associated with induction of mitochondrial uncoupling protein-2 and adipocyte-specific fatty acid binding protein-2, but not cytochrome P4502e1 or lipid peroxidation. In this model of metabolic syndrome, transition of steatosis to steatohepatitis was associated with hypoadiponectinemia, a mediator of hepatic fatty acid disposal pathways.     

Design of a mechanical larynx with agarose as a soft tissue substitute for vocal fold applications

Mechanical and computational models consisting of flow channels with convergent and oscillating constrictions have been applied to study the dynamics of human vocal fold vibration. To the best of our knowledge, no mechanical model has been studied using a material substitute with similar physical properties to the human vocal fold for surgical experimentation. In this study, we design and develop a mechanical larynx with agarose as a vocal fold substitute, and assess its suitability for surgical experimentation. Agarose is selected as a substitute for the vocal fold as it exhibits similar nonlinear hyperelastic characteristics to biological soft tissue. Through uniaxial compression and extension tests, we determined that agarose of 0.375% concentration most closely resembles the vocal fold mucosa and ligament of a 20-year old male for small tensile strain with an R(2) value of 0.9634 and root mean square error of 344.05±39.84?Pa. Incisions of 10 mm lengthwise and 3 mm in depth were created parallel to the medial edge on the superior surface of agar phantom. These were subjected to vibrations of 80, 130, and 180 Hz, at constant amplitude of 0.9 mm over a period of 10 min each in the mechanical larynx model. Lateral expansion of the incision was observed to be most significant for the lower frequency of 80 Hz. This model serves as a basis for future assessments of wound closure techniques during microsurgery to the vocal fold.