Establishment of oral bacterial communities in germ-free mice and the influence of recipient age.

The acquisition of the oral microbiome is a complex process. We examined how the timing of microbial exposure alters bacterial colonization of the tooth surface. Germ-free mice were conventionalized by exposure to specific pathogen-free (SPF) mice to acquire a commensal microbiome over three distinct 4-week periods, 0-4 weeks of age (Conv0-4w), 4-8 weeks (Conv4-8w), or 8-12 weeks (Conv8-12w). Bacterial DNA was extracted from the tooth surface and analyzed by 16S rDNA sequencing. Total bacteria and inflammatory cytokine expression in gingiva were determined by quantitative real-time polymerase chain reaction. After co-housing with SPF mice, Conv0-4w and Conv4-8w mice had low bacterial diversity, whereas Conv8-12w mice had high bacterial diversity that was similar to that of SPF donor mice, as determined by both operational taxonomic units and the Shannon Index. Cluster analysis with unweighted Unifrac distance also supported these trends. This was surprising as the amount of maturation time, 4 weeks, was equal in all conventionalized mice and tooth eruption was largely completed by 4 weeks. This suggests that host factors that occur after tooth eruption have a significant effect on the microbial tooth colonization.

Differences in differential gene expression between young and mature Arabidopsis C58 tumours.

Tumorigenesis of plants triggered by Agrobacterium tumefaciens has been investigated for over a century, but a global study on changes in gene expression in plant tumours during growth and development has received little attention so far due to technical difficulties. Recently a great advance in 'omic' technologies, e.g. microarray, proteome and transcriptome analyses, has allowed differential expression profiling of genes for metabolic regulation during plant tumour growth and development. Deeken et al.(The Plant Cell Online, 18, 3617) and Lee C.-W. et al.(The Plant Cell Online, 21, 2948) used a fold change approach to profile genes differentially expressed (DE) between Arabidopsis inflorescence stalks infected with Agrobacterium strains C58 (carrying T-DNA) or GV3101 (without T-DNA) and control stalks at 3 hours, 6 days and 35 days after inoculation. We utilised ranking analysis of microarray data, a modified t-test approach, to further analyse these microarray data and compared DE gene functioning in photosynthesis, energy, nucleotide, RNA, DNA, protein and lipid metabolism, biological defence, cell wall and signalling pathways in young (6-day-old) and mature (35-day-old) tumours. There were large differences in differential expression of genes for these basic metabolic pathways between young and mature tumours. In young tumours, more genes were up-regulated in most metabolic functional categories than down-regulated, whereas in mature tumours, genes involved in basic and major metabolic pathways were more down-regulated than up-regulated, strongly indicating that relative to the control stalk, many metabolic pathways were enhance in young tumours but decayed or tended to be decayed in mature tumours.

[Effects of heat shock pretreatment on the translocation and expression of alpha B-crystallin in cultured neonatal rat cardiomyocytes].

UNLABELLED: The effects of heat shock pretreatment on translocation and expression of alpha B-crystallin were evaluated in neonatal rat cardiomyocytes. RESULTS: 1. Western blot analysis demonstrated that cytosolic soluble alpha B-crystallin rapidly translocated to insoluble intracellular structure at 10 or 30 minutes and returned to cytosolic soluble compartment at about 1-2 h after heat shock. 2. Double staining of immunofluorescence showed that alpha B-crystallin redistributed in cytosol after heat shock, while HSC70 translocated from cytosol to nucleus. 3. Quantitative analysis with immunoelectronic microscopy demonstrated alpha B-crystallin translocated to cytoskeletal structure such as Z-line and nuclear envelope after heat shock. 4. Northern-blot showed that heat shock induced alpha B-crystallin mRNA expression. The rapid translocation and increasing expression of alpha B-crystallin after heat shock suggests that this small heat shock protein may play an important protective roles during the early and late phases of myocardial ischemic preconditioning.

5-hydroxylation of omeprazole by human liver microsomal fractions from Chinese populations related to CYP2C19 gene dose and individual ethnicity.

It has been previously reported that omeprazole (OP) oxidation is mediated by CYP2C19 and CYP3A4 in human livers. In this study, we assessed their relative contributions with human liver microsomal fractions from Chinese populations that were genotyped by CYP2C19 and recruited from two ethnic groups, Han and Zhuang. The kinetics of 5-hydroxyomeprazole (5-OH-OP) formation was best described by the two-enzyme and single-enzyme Michaelis-Menten equations for liver microsomes from CYP2C19 extensive (EMs) and poor metabolizers, respectively. At a low substrate concentration that may be encountered in vivo, the monoclonal antibody to CYP2C8/9/19 strongly inhibited 5-OH-OP formation in EM microsomes, whereas troleandomycin (TAO) eliminated most of the formation at a high substrate concentration. In poor metabolizer microsomes, either TAO or anti-CYP3A4 could alone abolish 5-OH-OP formation. Furthermore, there were differences between homozygous and heterozygous EMs in the percentage of inhibition by TAO and the antibodies. At the low substrate concentration, OP 5-hydroxyaltion was correlated well with S-mephenytoin 4'-hydroxylation and CYP2C19 contents in liver microsomes of 34 Chinese individuals. Moreover, in these individuals, obviously genetic and somewhat ethnic differences in OP 5-hydroxylation were observed between different CYP2C19 genotypes (wt/wt > wt/m1 > m1/m1) and between Han and Zhuang (Han > Zhuang), respectively. The results indicate that CYP2C19 is a high-affinity enzyme for OP 5-hydroxylation by liver microsomes from Chinese individuals and that its contribution is CYP2C19 gene dependent and ethnically related. Similar studies indicate that OP sulfoxidation is mediated mainly by CYP3A4 and independent of CYP2C19 genotype status.

[Heat shock pretreatment increases alpha B-crystallin expression and protects cardiomyocytes against injury induced by hydrogen peroxide].

The protective effects of heat shock pretreatment against the injury induced by hydrogen peroxide(H2O2) were evaluated in neonatal rat cardiomyocytes. The results showed that heat shock pretreatment significantly reduced cell mortality rate, LDH release rate and increased total antioxidation of the cells. Western blot analysis demonstrated that heat shock pretreatment induced expression of alpha B-crystallin and immunoelectron microscopy demonstrated H2O2-mediated alpha B-crystallin translocation from cytoplasm to nucleus and microfilament in cardiomyocytes. The results suggest that the mechanism of protective effect of heat shock pretreatment might involve the induction and translocation of alpha B-crystallin which then protect cardiomyocytes against H2O2-induced injury by stabilizing cytoskeletal structure and increasing anti-oxidation capacity of the cells.

[The roles of alpha B-crystallin during early phase of myocardial ischemic preconditioning in rats].

OBJECTIVE: This study designed to observe intracellular translocation of alpha B-crystallin, a small heat shock protein, and its possible implication during the early phase of myocardial ischemic preconditioning in isolated Langendorff rat hearts. Eighteen male Wistar rats(180-250 g) were randomly divided into three groups: 1. Control group(Ctrl) was perfused with K-H solution throughout the experiment; 2. ischemia-reperfusion group (I-R) experienced 30 min of global ischemia and 120 min of reperfusion and 3. preconditioning and ischemia-reperfusion group(PC + I-R) was preconditioned with three cycles of short myocardial ischemia(5 min each, separated by 5 min of reperfusion) prior to 30 min of ischemia and 120 min of reperfusion. It was showed that cytosolic soluble alpha B-crystallin rapidly translocated to insoluble intracellular structure after ischemic preconditioning, then gradually returned to soluble cytosol pool and almost completely recovered at about 60 min after preconditioning. In the meanwhile, ischemic preconditioning markedly alleviated subsequent myocardial ischemia-reperfusion injury, as indicated by the improvement of LVP, + dp/dt max, coronary flow, heart rate, CPK release and malondialdehyde(MDA) production. The results suggest that the intracellular translocation of alpha B-crystallin might be important for myocardial protection during the early phase of ischemic preconditioning.

Probing CYP2C19 and CYP3A4 activities in Chinese liver microsomes by quantification of 5-hydroxyomeprazole and omeprazole sulphone.

AIM: To develop an analytical method for simultaneous quantification of 5-hydroxyomeprazole (5-OH-OP) and omeprazole sulfone (OPS), and explore whether omeprazole (OP) is an appropriate phenotypic probe for CYP2C19 and CYP3A4 in Chinese liver microsomes. METHODS: OP metabolism in vitro was conducted in Chinese liver microsomes, and the major metabolites 5-OH-OP and OPS were determined using high pressure liquid chromatography (HPLC). Monoclonal antibodies anti-CYP2C8/9/19 and anti-CYP3A4 were employed to conduct inhibition experiments. The protein contents of CYP2C19 and CYP3A4 were quantified using Western blot analysis and densitometric scanning. RESULTS: 5-OH-OP and OPS gave a baseline resolution in the HPLC analysis. The detection limits for both compounds were 0.01 nmol and the recovery (98%-102%) had good precision with relative standard deviation of < 9.5%. Both anti-CYP2C8/9/19 and anti-CYP3A4 had a significant inhibitory effect (P < 0.05) on the 5-OH-OP formation in a substrate concentration-dependent manner, and anti-CYP3A4 alone could almost abolish the formation of OPS (> 87%). At a substrate concentration of 2 mumol/L OP, good correlations were found between OP 5-hydroxylation and S-mephenytoin 4'-hydroxylation activities (r = 0.72, P < 0.01), OP 5-hydroxylation activities and CYP2C19 contents (r = 0.82, P < 0.01), and OP sulfoxidation activities and CYP3A4 contents (r = 0.78, P < 0.01) in Chinese liver microsomes. CONCLUSION: OP metabolism is mediated mainly by CYP2C19 and CYP3A4, and OP can be used to probe CYP2C19 and CYP3A4 activities in Chinese liver microsomes at appropriate substrate concentrations with the HPLC method presently developed.