Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

Specific Frequency Distribution of Erythrocytic X-Protein Alleles in Indigenous Sheep Populations in East Asia

The nonhemoglobin erythrocytic X-protein polymorphism consisting of two phenotypes called X-positive [X(+)] and X-negative [X(-)] was determined in 576 unrelated healthy native sheep of East Asia, using one-dimensional and horizontal starch gel electrophoresis. A striking difference in the frequency of the X allele coding dominantly for the X(+) type between the northern and southern populations of native East Asian sheep divided by the boundary of the Himalaya Mountains was seen (P < 0.0001). The X allele frequency ranged from 0 to 0.0438 with an average of 0.0323 in the northern population examined, consisting of the Bhyanglung, Baruwal, Yunnan, and Khalkhas sheep belonging to the Tibetan and Mongolian sheep groups. In contrast, the frequency of the same allele was in the range of 0.2037-0.4655 and the mean frequency was 0.2998 in the southern population tested, consisting of the Bengal, Kagi, Lampuchhre, Vietnamese, and Myanmar sheep, which belong to the Indian sheep group. This finding suggests that the X allele appears to be an Indian sheep marker and is potentially important in phylogenetic studies on native sheep populations, especially in East Asia.     

Association between a single-nucleotide polymorphism in the promoter of the human interleukin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelihood estimation of combinatorial effect that two genetic loci have on susceptibility to the disease

Genetic variants of interleukin-3 (IL-3), a well-studied cytokine, may have a role in the pathophysiology of rheumatoid arthritis (RA); but reports on this association sometimes conflict. A case-control study was designed to investigate association between RA and a single-nucleotide polymorphism (SNP) in the IL-3 promoter region. Comparison of cases of RA versus control individuals yielded a chi(2) value of 14.28 (P=.0002), with a genotype odds ratio of 2.24 (95% confidence interval [95%CI] 1.44-3.49). When female cases with earlier onset were compared with female control individuals, the SNP revealed an even more significant correlation, with chi2=21.75 (P=.000004) and a genotype odds ratio of 7.27 (95%CI 2.80-18.89). The stronger association that we observed in this clinically distinct subgroup (females with early onset), within a region where linkage disequilibrium was not significantly extended, suggested that the genuine RA locus should locate either within or close to the IL-3 gene. Combined genotype data on SNPs on eight other candidate genes were combined with our IL-3 results, to estimate relationships between pairs of loci and RA, by maximum-likelihood analysis. The utility of combining the genotype data in this way to identify possible contributions of various genes to this disease is discussed.     

Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of cyclooxygenase-2 mRNA in mouse osteoblasts

Both prostaglandin E2 (PGE2) and low intensity pulsed ultrasound (U/S) exposure have been reported to accelerate fracture repair. We hypothesized that these two pathways are interactive. To verify this hypothesis, we examined the regulation of PGE2 production by U/S exposure (sine wave of 1.5MHz repeating at 1kHz, 30mW/cm2, 20 minutes) in mouse osteoblastic cell line, MC3T3-E1. The production of PGE2 in osteoblasts was augmented by U/S, which was threefold at 60 min. in comparison with unexposed samples. Then we evaluated the expression of cyclooxygenase-2 (COX-2) mRNA, which is a critical enzyme for PGE2 production. U/S rapidly up-regulated the expression of COX-2 mRNA in a time dependent manner. In addition, PGE2 production by U/S was drastically suppressed by a selective inhibitor of COX-2. These results provide strong evidence that PGE2 production in osteoblasts is dependent upon the induction of COX-2 mRNA expression by U/S and offer a mechanistic insight into how U/S accelerates fracture repair.     

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.     

Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes

Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.     

Prebiotic Formation of ADP and ATP from AMP,Calcium Phosphates and Cyanate in Aqueous Solution

Adenosine-5-triphosphate was synthesized by the phosphorylation of adenosine-5-diphosphate in aqueous solution containing cyanate as a condensing reagent and insoluble calcium phosphate produced from phosphate and calcium chloride. In a similar manner, adenosine-5-diphosphate was synthesized from adenosine-5-monophosphate. When the experiment was carried out in the conditions of 4 °C and pH 5.75, the formation of adenosine-5-diphosphate and adenosine-5-triphosphate from adenosine-5-monophosphate was observed in the yields of 19 and 7%, respectively. The other nucleoside-5-triphosphates were also produced from their respective diphosphates.     

Nutritional Prevention on Hypertension, Cerebral Hemodynamics and Thrombosis in Stroke-Prone Spontaneously Hypertensive Rats

1. Stroke-prone spontaneously hypertensive rats (SHRSP/Izm), which become severely hypertensive and exhibit a very high incidence of stroke (cerebral hemorrhage and/or infarction), are used widely for the study of the hypertension and stroke. In the previous study, we indicated that high thrombotic tendency of cerebral microvessels in SHRSP/Izm compared with stroke-resistant SHR (SHR/Izm) and normotensive Wistar Kyoto rats (WKY/Izm) at aged period. 2. L-arginine, a substrate of nitric oxide (NO), and voluntary exercise reduced blood pressure and thrombotic tendency in cerebral microvessels caused by highly production of NO in vivo. Furthermore, antioxidants show that the effects of antihypertensive and antithrombosis in SHRSP/Izm. 3. Although SHRSP/Izm become genetically hypertensive and exhibit stroke, a number of nutritional factors, particularly antioxydative nutrient, have preventive effects on hypertension, cerebral blood flow dysfunction, thrombus formation, and neuronal cell death in SHRSP/Izm. Our results indicate that those treatments are beneficial in the prevention of hypertension and stroke and that the nutritional science is very important for "prediction and prevention medicine."     

Estrogen regulation of nitric oxide synthesis in the porcine oocyte

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 microM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17beta-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2 did not increase NOS-3 expression. In addition, estrogen receptor beta was detected in oocytes and cumulus cells, and estrogen receptor alpha was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.