Stat5a inhibits IL-12-induced Th1 cell differentiation through the induction of suppressor of cytokine signaling 3 expression

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a(-/-)) CD4(+) T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-gamma production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a(-/-) CD4(+) T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a(-/-) CD4(+) T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a(-/-) CD4(+) T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4(+) T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a(-/-) CD4(+) T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3.     

High correlation between the bacterial species in denture plaque and pharyngeal microflora

Objectives : The morbidity and mortality of the dependent elderly that result from aspiration pneumonia are recognized as a major geriatric health problem. Most cases of bacterial pneumonia are initiated following colonization or superinfection of the pharynx by pathogenic bacteria, followed by aspiration of pharyngeal contents. A recent study revealed that bacteria, that commonly cause respiratory infection, colonized the dentures of dependent elderly. This suggests that denture plaque may function as a reservoir of potential respiratory pathogens to facilitate colonization on the pharynx. The purpose of this study was to determine the possible correlation between denture and pharyngeal microflora. Study Design : The denture and pharyngeal bacterial flora of 50 dependent elderly were examined, and the microorganisms identified by culturing. The agreement between the bacterial species in denture plaque and pharyngeal microflora was investigated using the Kappa method. Results : The microorganism species on the dentures and pharyngeal mucosa of the subjects had an agreement rate of 68.5%. The agreement rate for each of the bacterial species of the dentures and pharynx was also demonstrated to be high. Conclusions : Dentures should be considered an important reservoir of organisations which could colonise the pharynx, and the importance of controlling denture plaque for the prevention of aspiration pneumonia cannot be overemphasized.     

Evidence for catalytic cysteine-histidine dyad in chalcone synthase

Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA, but catalyze different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosphate (DPC). DPC also inhibited malonyl-CoA decarboxylation activity of wild-type and C164S mutants of CHS and STS. Meanwhile, Idm treatment enhanced (two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and STS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS. DPC inhibition of decarboxylation activity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; however, its inhibitory effect on CHS-C164S increased as pH increased from 6.2 to 7.4 with a midpoint of 6.4. Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine residue being modified by DPC in CHS is likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS. In addition, our results showed that Cys60 in STS is not essential for the activity and only a single cysteine (Cys164) participates in the catalysis as in CHS.     

Promotion of non-rapid eye movement sleep in mice after oral administration of ornithine

We examined the effects of ornithine on the sleep-wake cycle by monitoring the electroencephalo-gram, electromyogram, and locomotor activity of freely moving mice after oral administration of it at lights-off time (18:00). Ornithine (1.0 and 3.0 g/kg of body weight) increased the amount of non-rapid eye movement (non-REM, NREM) sleep for 2 h after its administration, with a peak at 60 min post administration, to 164% and 198%, respectively, of that of the vehicle-administered mice, without changing the amount of REM sleep. The administration of ornithine at a lower dose (0.3 g/kg of body weight) did not increase the amount of NREM sleep compared with the vehicle administration. Ornithine did not affect the power spectrum density of NREM sleep but increased the number of episodes of wakefulness and NREM sleep and that of transitions between wakefulness and NREM sleep, and decreased the mean duration of wake episodes in a dose-dependent manner for 2 h after the oral administration. These results indicate that ornithine increased the amount of NREM sleep without reducing the power spectrum density of NREM sleep.

     

Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase

Nitrous oxide (N(2)O) is a stable greenhouse gas that plays a significant role in the destruction of the ozone layer. Soils are a significant source of atmospheric N(2)O. It is important to explore some innovative and effective biology-based strategies for N(2)O mitigation. The enzyme nitrous oxide reductase (N(2)OR), naturally found in soil bacteria, is responsible for catalysing the final step of the denitrification pathway, conversion of N(2)O to dintrogen gas (N(2)). To transfer this catalytic pathway from soil into plants and amplify the abundance of this essential mechanism (to reduce global warming), a mega-cassette of five coding sequences was assembled to produce transgenic plants heterologously expressing the bacterial nos operon in plant leaves. Both the single-gene transformants (nosZ) and the multi-gene transformants (nosFLZDY) produced active recombinant N(2)OR. Enzymatic activity was detected using the methyl viologen-linked enzyme assay, showing that extracts from both types of transgenic plants exhibited N(2)O-reducing activity. Remarkably, the single-gene strategy produced higher reductase capability than the whole-operon approach. The data indicate that bacterial N(2)OR expressed in plants could convert N(2)O into inert N(2) without involvement of other Nos proteins. Silencing by homologous signal sequences, or cryptic intracellular targeting are possible explanations for the low activities obtained. Expressing N(2)OR from Pseudomonas stutzeri in single-gene transgenic plants indicated that such ag-biotech solutions to climate change have the potential to be easily incorporated into existing genetically modified organism seed germplasm.     

Responses of the weed Bidens pilosa L. to exogenous application of the steroidal saponin protodioscin and plant growth regulators 24‐epibrassinolide,indol‐3‐acetic acid and abscisic acid

• The exogenous application of plant hormones and their analogues has been exploited to improve crop performance in the field. Protodioscin is a saponin whose steroidal moiety has some similarities to plant steroidal hormones, brassinosteroids. To test the possibility that protodioscin acts as an agonist or antagonist of brassinosteroids or other plant growth regulators, we compared responses of the weed species Bidens pilosa L. to treatment with protodioscin, brassinosteroids, auxins (IAA) and abscisic acid (ABA).
• Seeds were germinated and grown in agar containing protodioscin, dioscin, brassinolides, IAA and ABA. Root apex respiratory activity was measured with an oxygen electrode. Malondialdehyde (MDA) and antioxidant enzymes activities were assessed.
• Protodioscin at 48–240 μm inhibited growth of B. pilosa seedlings. The steroidal hormone 24‐epibrassinolide (0.1–5 μm ) also inhibited growth of primary roots, but brassicasterol was inactive. IAA at higher concentrations (0.5–10.0 μm ) strongly inhibited primary root length and fresh weight of stems. ABA inhibited all parameters of seedling growth and also seed germination. Respiratory activity of primary roots (KCN‐sensitive and KCN‐insensitive) was activated by protodioscin. IAA and ABA reduced KCN‐insensitive respiration. The content of MDA in primary roots increased only after protodioscin treatment. All assayed compounds increased APx and POD activity, with 24‐epibrassinolide being most active. The activity of CAT was stimulated by protodioscin and 24‐epibrassinolide.
• The results revealed that protodioscin was toxic to B. pilosa through a mechanism not related to plant growth regulator signalling. Protodioscin caused a disturbance in mitochondrial respiratory activity, which could be related to overproduction of ROS and consequent cell membrane damage.

     

Nitrosonifedipine Ameliorates the Progression of Type 2 Diabetic Nephropathy by Exerting Antioxidative Effects

Diabetic nephropathy (DN) is the major cause of end-stage renal failure. Oxidative stress is implicated in the pathogenesis of DN. Nitrosonifedipine (NO-NIF) is a weak calcium channel blocker that is converted from nifedipine under light exposure. Recently, we reported that NO-NIF has potential as a novel antioxidant with radical scavenging abilities and has the capacity to treat vascular dysfunction by exerting an endothelial protective effect. In the present study, we extended these findings by evaluating the efficacy of NO-NIF against DN and by clarifying the mechanisms of its antioxidative effect. In a model of type 2 DN (established in KKAy mice), NO-NIF administration reduced albuminuria and proteinuria as well as glomerular expansion without affecting glucose metabolism or systolic blood pressure. NO-NIF also suppressed renal and systemic oxidative stress and decreased the expression of intercellular adhesion molecule (ICAM)-1, a marker of endothelial cell injury, in the glomeruli of the KKAy mice. Similarly, NO-NIF reduced albuminuria, oxidative stress, and ICAM-1 expression in endothelial nitric oxide synthase (eNOS) knockout mice. Moreover, NO-NIF suppressed urinary angiotensinogen (AGT) excretion and intrarenal AGT protein expression in proximal tubular cells in the KKAy mice. On the other hand, hyperglycemia-induced mitochondrial superoxide production was not attenuated by NO-NIF in cultured endothelial cells. These findings suggest that NO-NIF prevents the progression of type 2 DN associated with endothelial dysfunction through selective antioxidative effects.     

Appearance of poor-fermenting variants in brewing yeast culture.

A class of yeast variants appears after cultivation of a bottom-fermenting brewing yeast strain, IFO2003. Although IFO2003 fails to grow well above 33 degrees C, the variants can grow up to 34 degrees C. Temperature-resistance and an acquired phenotype of maltose poor-fermentation ability are strictly correlated in the bottom-fermenting brewing yeast, enabling us to develop easy estimation of the fermentation ability of the variants.     

The binding of nonmuscle caldesmon from brain to microtubules. Regulations by Ca(2+)-calmodulin and cdc2 kinase.

Nonmuscle caldesmon from bovine brain bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with values of Ka 4.5 x 10(5) M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. The phosphorylation of caldesmon by cdc2 kinase also eliminated the microtubule-binding activity. These results suggest that caldesmon may play a physiological role in the functions of microtubules.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.