Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Replication-coupled chromatin assembly generates a neuronal bilateral asymmetry in C. elegans

Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling cell differentiation during animal development remains largely unexplored. Here we report that the CAF-1 protein complex, an evolutionarily conserved histone chaperone that deposits histone H3-H4 proteins onto replicating DNA, is required to generate a bilateral asymmetry in the C. elegans nervous system. A mutation in 1 of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry by causing a defect in the formation of a histone H3-H4 tetramer and the consequent inhibition of CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is necessary to generate a bilateral asymmetry in C. elegans neuroanatomy and suggest that left-right asymmetric epigenetic regulation can establish bilateral asymmetry in the nervous system.     

Interactions of opioid and chemokine receptors: oligomerization of mu,kappa, and delta with CCR5 on immune cells

In this study, we examined the signaling pathways for extracellular signal-related protein kinase (ERK) activation by three structurally different peroxisome proliferator activated receptor-gamma (PPARgamma) agonists. In murine C2C12 myoblasts, treatment with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and GW1929 leads to ERK1/2 phosphorylation in a time- and concentration-dependent manner. Consistent with ERK phosphorylation, mitogen activated protein/ERK kinase (MEK) phosphorylation as well as Raf-1 kinase activity are also accordingly stimulated, while the constitutive Ser259 phosphorylation of Raf-1 is decreased. The ERK phosphorylation induced by PPARgamma agonists is not blocked by the PKC inhibitors GF109203X and Ro31-8220, the PI3K inhibitor wortmannin, the Ras inhibitor FPTI, the negative mutant of Ras, or the PPARgamma antagonist bisphenol A diglycidil ether. Expression of PPARgamma2 without DNA binding domain or with a nonphosphorylatable mutant (S112A) fails to change ERK phosphorylation by 15d-PGJ(2). On the contrary, the ERK phosphorylation by PPARgamma agonists is inhibited by the MEK inhibitor PD98059, GSH, and permeable SOD mimetic MnTBAP. Chemiluminescence study reveals that these three PPARgamma agonists are able to induce superoxide anion production, with an efficacy similar to their action on ERK phosphorylation. Consistent with this notion, we also show that superoxide anion donor 2,3-dimethoxy-1,4-naphoquinone elicits ERK phosphorylation. In this study, we for the first time demonstrate a novel mechanism, independent of Ras activation but initiated by superoxide anion production, for PPARgamma agonists to trigger the Raf-MEK-ERK1/2 signaling pathway.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate

Hydroxyl radical (·OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO₃) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the ·OH generation after injection of Fe-NTA or KBrO₃ in the kidneys. A significant increase in 8-hydroxy-2'-deoxyguanosine in nuclei of the kidney was detected only in the KBrO₃ treated mice, while the comet assay showed that the Fe-NTA and KBrO₃ treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by ·OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of ·OH generation in cellular injury induced by chemicals in vivo.     

Inhibitory effects of interleukin-1 on follicle-stimulating hormone induction of aromatase activity, progesterone secretion, and functional luteinizing hormone receptors in cultures of porcine granulosa cells

Effects of interleukin-1 (IL-1) on FSH-induced differentiation of immature porcine granulosa cells in vitro were examined in short-term (48-h) cultures. IL-1 inhibited FSH induction of aromatase activity and of LH-stimulated cAMP accumulation by granulosa cells. Both these inhibitory actions of IL-1 were concentration-dependent. Significant inhibitory effects were observed with as low as 0.05-0.25 ng/ml of IL-1, with maximal effects at 25 ng/ml. IL-1 also significantly inhibited increases in [125I]iodo-LH binding and progesterone secretion induced by FSH, as well as reducing basal levels of aromatase activity and LH-stimulated cAMP accumulation. Studies on the mechanisms of IL-1 actions on FSH-induced differentiation of immature porcine granulosa cells revealed that IL-1 reduced cAMP accumulation by the cells in response to FSH in a time- and concentration-dependent manner. IL-1 also inhibited induction of aromatase activity and LH-stimulated cAMP accumulation induced by dibutyryl cAMP, suggesting that IL-1 also affects the steps distal to cAMP generation. In contrast, IL-1 had no effect on progesterone secretion induced by dibutyryl cAMP, suggesting that post-cAMP steps of progesterone secretion were unaffected by IL-1.     

Molecular cloning, expression and properties of an alpha/beta-Galactoside alpha2,3-sialyltransferase from Vibrio sp. JT-FAJ-16

We cloned, expressed and characterized a novel alpha/beta-galactoside alpha2,3-sialyltransferase from Vibrio sp. bacterium JT-FAJ-16. Using a alpha2,3-sialyltransferase gene from a marine bacterium as a probe, a DNA sequence encoding a 402-amino-acid protein was identified from the JT-FAJ-16 genomic library. The protein showed 27.3-64.7% identity to the bacterial sialyltransferases classified into glycosyltransferase family 80. The protein showed sialyltransferase activity when expressed in Escherichia coli. The N-terminal truncated form of the enzyme was amplified in E. coli and its recovered activity was 215.7 unit/l culture medium. It was purified as a single band on SDS-PAGE through the three chromatographic steps. The specific activity of the purified recombinant enzyme reached 57.5 unit/mg protein. The alpha2,3sialylation was confirmed by (1)H- and (13)C-NMR analyses of the reaction products. The enzyme was optimally active at pH 5.5 and at 20 degrees C. Interestingly, the enzyme used both the alpha- and beta-anomers of galactosides as acceptors, suggesting that it can be described as an alpha/beta-galactoside alpha2,3-sialyltransferase. The enzyme had a wide range of acceptor substrate specificities. It transferred N-acetylneuraminic acid (NeuAc) to various monosaccharides and various oligosaccharides, and both N-linked and O-linked asialo-glycoprotein. These results suggest that the enzyme can be used as a powerful tool for the study for glycotechnology.     

New Rev-transport inhibitor with anti-HIV activity from Valerianae Radix

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, a HIV-1 viral regulatory protein, resulted in isolation of valtrate (1) as a new Rev-transport inhibitor from the nucleus to cytoplasm from Valerianae Radix. Valtrate (1) also inhibited the p-24 production of HIV-1 virus without showing any cytotoxicity against the host MT-4 cells.     

Alien pollen grains interfere with the reproductive success of native congener

The effects of invasive species on native species comprise important conservation issues. Determining the mechanisms by which invasives exclude natives is indispensable to efficiently control their impact, but most invasives remain poorly studied. The purpose of this study was to elucidate potentially important but neglected mechanisms, reproductive interference, in wild Taraxacum systems, in which invasive Taraxacum officinale has displaced its native congener T. japonicum in Japan. Hand-pollination of mixed pollen grains significantly reduced the native seed-set compared to conspecific-only pollination. Moreover, natives with a high ratio of invasive pollen on their stigmas suffered severe seed-set reduction, and the proportion of invasive pollen on native stigmas increased as frequencies of the alien neighbor increased. These results, combined with those of previous studies, revealed that depositing invasive pollen on native stigmas contributes to the observed alien-frequency-dependent reduction of native seed-set, and strongly suggest that reproductive interference was the primary cause of displacement in the Taraxacum systems.     

Effects of heavy ion to the primary [correction of rimary] culture of mouse brain cells.

To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.