KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactis

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.     

Disturbance of lymphocyte circulation byPseudomonas aeruginosa infection in oxazolone-sensitized mice

Pseudomonas aeruginosa infection depresses contact sensitivity to oxazolone in mice. To test whether an altered lymphocyte circulation plays a role in this depression⁵¹Cr-labeled lymphocytes fromP. aeruginosa-infected and oxazolone-sensitized donors were injected intravenously into infected and sensitized recipients, and the radioactivity uptake of several organs was determine. The controls consisted of normal mice receiving labeled lymphocytes from normal donors. While the radioactivity recovered from the liver, spleen, and mesenteric lymph nodes was similar in the test and the control group, significantly more radioactivity was recovered from the draining lymph nodes of infected and sensitized recipients. The concentration of labeled lymphocytes from sensitized donors in the draining lymph nodes of sensitized recipients was 18% greater than that of the controls but 31% lower than that of infected and sensitized animals receiving cells from infected and sensitized donors.P. aeruginosa infection enhances lymphocyte entrapment within the draining lymph nodes of oxazolone-sensitized mice.     

Alpha 1 adrenergic receptor control of renal blood vessels during aging

Aging humans and rats have a reduced renal vascular constriction response to stress, change in posture, or exercise. In this study, renal interlobar arteries from 9- (intermediate age) to 15-month-old (aging) male Wistar rats constricted less to alpha-adrenergic agonists than those of 4-month-old (young adult) rats. The reduced contraction to A61603 (alpha 1 A agonist) was similar to that to norepinephrine and phenylephrine. Therefore, it appears that the reduction in constriction is primarily related to alpha 1 A receptor stimulation. GeneChip microarray hybridization analysis of the interlobar arteries with the RAE 230A GeneChip indicated that there were no significant differences in gene expression for alpha 1 A/C, 1B, or 1D receptors between 4-month-old (young adult) and 1-year-old (aging) male Wistar rats. Competitive binding experiments (prazosin) revealed that maximal binding (Bmax, fmol/mg protein) of the alpha 1 receptors of interlobar arteries was reduced 25% by 10 months of age and 50% by 18+ months of age. Alpha 1 receptor-induced arterial constriction and prazosin binding were both down-regulated. The loss of receptor-initiated constriction likely includes down-regulation of maximum agonist binding by alpha 1 adrenergic receptors.     

Bone medullary arterioles from ovariectomized rats have smaller baseline diameters but normal eNOS expression and NO-mediated dilation

This study was designed to test the hypothesis that endogenous estrogens decrease the expression of endothelial nitric oxide synthase (eNOS) in resistance-size bone arterioles, thereby reducing endothelium-dependent vasodilator function. Sexually mature female rats were ovariectomized to reduce endogenous estrogens. Age-matched female rats served as controls. Seven to ten days after ovariectomy, bone marrow tissue was collected from the femoral canal. Immuno-histochemistry was performed to detect expression of estrogen receptors, alpha and beta and eNOS. eNOS protein content in medullary bone arterioles was compared using Western blot analysis. Endothelial cell function was assessed by quantitating the dilation of isolated, pressurized bone arterioles in response to acetylcholine. The results indicate that the endothelium of bone arterioles from ovariectomized and control rats express ER-alpha, ER-beta and eNOS. eNOS protein content in the two groups of arterioles did not differ. However, the baseline diameter of arterioles from ovariectomized rats (63+/-4 microm) was significantly smaller than the diameter of arterioles from control rats (75+/-3 microm, p<0.05). The two groups of arterioles dilated equally in response to acetylcholine. L-NAME, an inhibitor of eNOS, almost completely abolished the dilator responses to acetylcholine, but not to sodium nitroprusside. L-Arginine restored acetylcholine-induced dilation after L-NAME treatment. Thus, arteriole dilation to acetylcholine appears to be mediated almost exclusively by NO. The smaller diameter of arterioles from ovariectomized rats suggests that endogenous estrogens exert a significant dilator influence on bone arterioles. However, the dilator influence does not appear to be mediated by an increase in eNOS expression or enhanced NO-dependent vasodilation. These results indicate that estrogens do not decrease eNOS expression or diminish NO-mediated dilation of bone medullary arterioles.     

Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPK(erk1/2) leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E(2) synthesis. The addition of PGE(2) to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE(2) and MMP-9 expression by elicited COX-2(-/-) macrophages is markedly reduced when compared with the response of either COX-2(+/-) or COX-2(+/+) macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.     

A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA

It has been known for almost a decade and a half that in trypanosomes all mRNAs are trans-spliced by addition to the 5' end of the spliced leader (SL) sequence. During the same time period the conviction developed that classical cis-splicing introns are not present in the trypanosome genome and that the trypanosome gene arrangement is highly compact with small intergenic regions separating one gene from the next. We have now discovered that these tenets are no longer true. Poly(A) polymerase (PAP) genes in Trypanosoma brucei and Trypanosoma cruzi are split by intervening sequences of 653 and 302 nt, respectively. The intervening sequences occur at identical positions in both organisms and obey the GT/AG rule of cis-splicing introns. PAP mRNAs are trans-spliced at the very 5' end as well as internally at the 3' splice site of the intervening sequence. Interestingly, 11 nucleotide positions past the actual 5' splice site are conserved between the T. bruceiand T. cruzi introns. Point mutations in these conserved positions, as well as in the AG dinucleotide of the 3' splice site, abolish intron removal in vivo. Our results, together with the recent discovery of cis-splicing introns in Euglena gracilis, suggest that both trans- and cis-splicing are ancient acquisitions of the eukaryotic cell.     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C4 and D4 in isolated airway smooth muscle was investigated. In rat trachea, neither LTC4 or D4 elicited a response. In contrast, LTC4 was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD4 in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC4 was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD4 in both trachea and parenchyma. As regards LTC4-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC4 in parenchyma.     

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1‐Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1‐Tg mice express high levels of the hMTH1 hydrolase that degrades 8‐oxodGTP and 8‐oxoGTP and excludes 8‐oxoguanine from both DNA and RNA. Compared to wild‐type animals, hMTH1‐overexpressing mice have significantly lower steady‐state levels of 8‐oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age‐dependent accumulation of DNA 8‐oxoguanine that occurs in wild‐type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1‐Tg animals live significantly longer than their wild‐type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1‐Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1‐Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1‐Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.