Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.     

Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.     

Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGF-beta-activated kinase-1

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-alpha-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-alpha as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x(L), cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-kappaB. We found that simvastatin inhibited TNF-alpha-induced NF-kappaB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-kappaB activation. Simvastatin inhibited TNF-alpha-induced IkappaBalpha kinase activation, which led to inhibition of IkappaBalpha phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-kappaB-dependent reporter gene expression induced by TNF-alpha, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-beta-activated kinase 1, receptor-interacting protein, NF-kappaB-inducing kinase, and IkappaB kinase beta was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IkappaB kinase and NF-kappaB-regulated gene products.     

Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and inflammatory cytokine production from mast cells

The citrus unshiu peel has been used traditionally as a medicine to improve bronchial and asthmatic conditions or cardiac and blood circulation in Korea, China, and Japan. Here, we report the effects of citrus unshiu peel water extract (CPWE) on the phorbol myristate acetate (PMA) + calcium ionophore A23187-induced hypoxia-inducible factor-1α (HIF-1α) activation and inflammatory cytokine production from the human mast cell line, HMC-1 cells. We compared CPWE with hesperidin, a common constituent of citrus unshiu. CPWE and hesperidin inhibited the PMA + A23187-induced HIF-1α expression and the subsequent production of vascular endothelial growth factor (VEGF). In addition, CPWE suppressed PMA + A23187-induced phosphorylation of the extracellular signal-regulated kinase (ERK). We also show that the increased cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α level was significantly inhibited by treatment of CPWE or hesperidin. In the present study, we report that CPWE and hesperidin are inhibitors of HIF-1α and cytokines on the mast cell-mediated inflammatory responses.     

Frequency of Secondary Symbiont Infection in an Invasive Psyllid Relates to Parasitism Pressure on a Geographic Scale in California

Two endosymbionts, an obligate primary symbiont and a facultative secondary symbiont, are harbored within the invasive red gum (eucalyptus) lerp psyllid, Glycaspis brimblecombei, in California. An extensive survey of diversity and frequency of G. brimblecombei's secondary symbiont in multiple populations throughout the state of California was conducted using PCR detection, restriction enzymes, cloning, and sequencing. A total of 380 G. brimblecombei individuals in 19 populations were screened for secondary symbionts. Based on molecular screening results, only one type of secondary symbiont was present in G. brimblecombei populations in California. Overall, 40% of the 380 psyllids screened were infected with the secondary symbiont. Interestingly, secondary symbiont infection frequencies in G. brimblecombei populations varied dramatically from 0 to 75% and were significantly related to parasitism pressure by Psyllaphaegus bliteus, a solitary endoparasitoid of the psyllid.     

Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 10⁶ and 7.1 × 10⁶ transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.     

The role of peroxiredoxin III on late stage of proerythrocyte differentiation

Peroxiredoxin III (Prdx III), the mitochondrial peroxidase, was preferentially expressed in murine erythroleukemia (MEL) cells. However, the mechanisms by which Prdx III regulates erythroid differentiation are unknown. In this study, K562 cells were differentiated by Ara-C treatment, and Prdx III was dramatically increased until day 5. We also investigated Prdx III expression pattern on in vitro erythropoiesis of human CD34(+) cells. When human CD34(+) cells became proerythrocyte on day 7, Prdx III was diminished, and then augmented on day 12. We established the stable sublines of Prdx III overexpression (O/E), and dominant-negative (D/N). The intracellular ROS level of Prdx III O/E cell line was lower than D/N stable cell lines. Moreover, Prdx III O/E cell line was placed in G1-arrest, but not D/N cell lines. Finally, the expression level of beta-globin and GATA-1 was dramatically increased in Prdx III O/E cell line.