Cellular events involved in butyric acid-induced T cell apoptosis

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Element analysis of the Polysphondylium pallidum gp64 promoter

gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence.     

cAMP-dependent protein kinase regulates Polysphondylium pallidum development

In eukaryotic cells, the universal second messenger cAMP regulates various aspects of development and differentiation. The primary target for cAMP is the regulatory subunit of cAMP-dependent protein kinase A (PKA), which, upon cAMP binding, dissociates from the catalytic subunit and thus activates it. In the soil amoeba Dictyostelium discoideum, the function of PKA in growth, development and cell differentiation has been thoroughly investigated and substantial information is available. To obtain a more general view, we investigated the influence of PKA on development of the related species Polysphondylium pallidum. Cells were transformed to overexpress either a dominant negative mutant of the regulatory subunit (Rm) from Dictyostelium that cannot bind cAMP, or the catalytic subunit (PKA-C) from Dictyostelium. Cells overexpressing Rm rarely aggregated and the few multicellular structures developed slowly into very small fruiting bodies without branching of secondary sorogens, the prominent feature of Polysphondylium. Few round spores with reduced viability were formed. When mixed with wild-type cells and allowed to develop, the Rm cells were randomly distributed in aggregation streams, but were later found in the posterior region of the culminating slug or were left behind on the surface of the substratum. The PKA-C overexpressing cells exhibited precocious development and formed more aggregates of smaller size. Moreover, expression of PKA-C under the control of the prestalk-specific ecmB promoter of Dictyostelium leads to protrusions from aggregation streams. We conclude that Dictyostelium PKA subunits introduced into Polysphondylium cells are functional as signal components, indicating that a biochemically similar PKA mechanism works in Polysphondylium.     

Inhibition of angiotensin-converting enzyme protects endothelial cell against hypoxia/reoxygenation injury

Cardiovascular tissue injury in ischemia/reperfusion has been shown to be prevented by angiotensin-converting enzyme (ACE) inhibitors. However, the mechanism on endothelial cells has not been assessed in detail. Cultured human aortic endothelial cells (HAEC) were exposed to hypoxia with or without reoxygenation. Hypoxia enhanced apoptosis along with the activation of caspase-3. Reoxygenation increased lactate dehydrogenase release time-dependently, along with an increase of intracellular oxygen radicals. ACE inhibitor quinaprilat and bradykinin significantly lessened apoptosis and lactate dehydrogenase release with these effects being diminished by a kinin B2 receptor antagonist and a nitric oxide synthase inhibitor. In conclusion, hypoxia activated the suicide pathway leading to apoptosis of HAEC by enhancing caspase-3 activity, while subsequent reoxygenation induced necrosis by enhancing oxygen radical production. Quinaprilat could ameliorate both apoptosis and necrosis through the upregulation of constitutive endothelial nitric oxide synthase via an increase of bradykinin, with the resulting increase of nitric oxide.     

Therapeutic effect of anti-macrophage inflammatory protein 2 antibody on influenza virus-induced pneumonia in mice

We investigated the effect of anti-macrophage inflammatory protein 2 immunoglobulin G (aMIP-2 IgG) on the progression of influenza virus-induced pneumonia in mice. When mice were infected with a mouse lung-adapted strain of influenza A/PR/8/34 virus by intranasal inoculation, neutrophil counts in the bronchoalveolar lavage fluid (BALF) increased in parallel with the kinetics of MIP-2 production, which peaked 2 days after infection. After intracutaneous injection of a dose of 10 or 100 microg of aMIP-2 IgG once a day on days 0 and 1, neutrophil counts in BALF on day 2 were reduced to 49 or 37%, respectively, of the value in the control infected mice administered anti-protein A IgG. The antibody administration also improved lung pathology without affecting virus replication. Furthermore, by prolonged administration with a higher or lower dose for up to 5 days, body weight loss became slower and finally 40% of mice in both treatment groups survived potentially lethal pneumonia. These findings suggest that MIP-2-mediated neutrophil infiltration during the early phase of infection might play an important role in lung pathology. Thus, MIP-2 was considered to be a novel target for intervention therapy in potentially lethal influenza virus pneumonia in mice.     

Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose-response manner, with an IC50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.     

Production of ubiquinone-10 using bacteria

Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.     

Assessing chimpanzee personality and subjective well‐being in Japan

We tested whether the cultural background of raters influenced ratings of chimpanzee personality. Our study involved comparing personality and subjective well‐being ratings of 146 chimpanzees in Japan that were housed in zoos, research institutes, and a retirement sanctuary to ratings of chimpanzees in US and Australian zoos. Personality ratings were made on a translated and expanded version of a questionnaire used to rate chimpanzees in the US and Australia. Subjective well‐being ratings were made on a translated version of a questionnaire used to rate chimpanzees in the US and Australia. The mean interrater reliabilities of the 43 original adjectives did not markedly differ between the present sample and the original sample of 100 zoo chimpanzees in the US. Interrater reliabilities of these samples were highly correlated, suggesting that their rank order was preserved. Comparison of the factor structures for the Japanese sample and for the original sample of chimpanzees in US zoos indicated that the overall structure was replicated and that the Dominance, Extraversion, Conscientiousness, and Agreeableness domains clearly generalized. Consistent with earlier studies, older chimpanzees had higher Dominance and lower Extraversion and Openness scores. Correlations between the six domain scores and subjective well‐being were comparable to those for chimpanzees housed in the US and Australia. These findings suggest that chimpanzee personality ratings are not affected by the culture of the raters. Am. J. Primatol. 71:283–292, 2009. © 2009 Wiley‐Liss, Inc.     

Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.