Phosphorylation of Nucleotide Molecules in Hydrothermal Environments

Phosphorylation of AMP into ADP and ATP, that can outrun their hydrolysis, was made possible in a simulated hydrothermal environment when trimetaphosphate was used as the phosphate source. The best yields of phosphorylated products were obtained when the reaction fluids whose temperature was set at about 100 degrees centigrade was injected into the cold environment maintained at 0 degree in a recycling manner. Hydrothermal environments in the primitive ocean could also have served as prebiotic sites for phosphorylation, among others.     

Quantitative analysis of human tissue-specific differences in methylation

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.     

Directional degradation of β-chitin by chitinase A1 revealed by a novel reducing end labelling technique

A novel procedure for labelling the molecular ends of β-chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of β-chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the β-chitin microcrystals, and also to label the reducing tips of β-chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.     

Synthesis of polyrotaxane-biotin conjugates and surface plasmon resonance analysis of streptavidin recognition

A polyrotaxane-biotin conjugate was synthesized and its interaction with streptavidin measured using surface plasmon resonance (SPR) detection. A biodegradable polyrotaxane in whichca. 22 molecules of α-cyclodextrins (α-CDs) were threaded onto a poly(ethylene oxide) chain (M _n: 4,000) capped with benzyloxycarbonyl-L-phenylalanine was conjugated with a biotin hydorazide and 2-aminoethanol after activating the hydroxyl groups of α-CDs in the polyrotaxane usingN,N′-carbony diimidazole. The results of the high-resolution¹H-nuclear magnetic resonance (¹H-NMR) spectra and gel permeation chromatography of the conjugate showed thatca. 11 biotin molecules were actually introduced to the polyrotaxane scaffold. An SPR analysis showed that the binding curves of the biotin molecules in the conjugate on the streptavidin-deposited surface changed in a concentration dependent manner, indicating that the biotin in the conjugate was actually recognized by streptavidin. The association equilibrium constant (K _a) of the interaction between the conjugate and streptavidin tetramer was of the order 10⁷. These results suggest that polyrotaxane is useful for scaffolds as a polymeric ligand in biomedical fields.     

Sister-chromatid exchange induction by indirect mutagens/carcinogens, aryl hydrocarbon hydroxylase activity and benzo[alpha]pyrene metabolism in cultured human hepatoma cells

2 human hepatoma cell lines (C-HC-4 and C-HC-20), in which aryl hydrocarbon hydroxylase activity was induced with benz[alpha]anthracene in vitro to about 140- and 64-fold of the respective basal levels, yielded an increased frequency of sister-chromatid exchanges (SCEs) when exposed to benzo[alpha]pyrene (BP), 7,12-dimethylbenz[alpha]anthracene and 3-methylcholanthrene in vitro. Analysis of the metabolism of BP by these cells by high-pressure liquid chromatography revealed that both cell lines produced various BP metabolites including the proximate form BP-7,8-dihydrodiol which has been reported to be the most potent inducer of SCEs among the metabolites of BP. In addition, aflatoxin B1 and cyclophosphamide also induced SCEs in these cell lines. The above findings suggest that these cells may be capable of metabolizing a range of indirect mutagens/carcinogens into DNA-active forms. These cells may therefore serve as a useful test system in vitro for the detection of genotoxic agents, without the use of an exogenous activating system.     

Hepatic dysoxia commences during O2 supply dependence.

Hepatic O2 consumption (VO2) remains relatively constant (O2 supply independent) as O2 delivery (DO2) progressively decreases, until a critical DO2 (DO2c) is reached below which hepatic VO2 also decreases (O2 supply dependence). Whether this decrease in VO2 represents an adaptive reduction in O2 demand or a manifestation of tissue dysoxia, i.e., O2 supply that is inadequate to support O2 demand, is unknown. We tested the hypothesis that the decrease in hepatic VO2 during O2 supply dependence represents dysoxia by evaluating hepatic mitochondrial NAD redox state during O2 supply independence and dependence induced by progressive hemorrhage in six pentobarbital-anesthetized dogs. Hepatic mitochondrial NAD redox state was estimated by measuring hepatic venous beta-hydroxybutyrate-to-acetoacetate ratio (beta OHB/AcAc). The value of DO2c was 5.02 +/- 1.64 (SD) ml.100 g-1.min-1. The beta-hydroxybutyrate-to-acetoacetate ratio was constant until a DO2 value (3.03 +/- 1.08 ml.100 g-1.min-1) was reached (P = 0.05 vs. DO2c) and then increased linearly. Peak liver lactate extraction ratio was 15.2 +/- 14.1%, occurring at a DO2 of 5.48 +/- 2.54 ml.100 g-1.min-1 (P = NS vs. DO2c). Our data support the hypothesis that the decrease in VO2 during O2 supply dependence represents tissue dysoxia.     

NAD-dependent Alcohol Dehydrogenase and NADP-dependent Alcohol Dehydrogenase from Strawberry Seeds

Strawberry seeds are shown to contain at least two alcohol dehydrogenases; one is NAD specific and reacts with ethanol and allyl alcohol, and the other is NADP specific and reacts with benzyl alcohol and geraniol. These two alcohol dehydrogenases were distinguished on disc electrophoresis. Their properties were different each other in ammonium sulfate fractionation, optimum reaction pH and thermostability.     

DNA replication cycle in parthenogenetically developing eggs of the starfish Asterina pectinifera

Starfish oocytes artificially activated by a calcium ionophore will develop normally if the formation of polar bodies is suppressed. In the present paper, schedules of the DNA replication period (S phase) of these parthenogenotes were explicitly timed using 5-bromo-2'-deoxyuridine (BrdU) and anti-BrdU monoclonal antibody. Their schedule of S phase was identical to that of fertilized eggs. Consequently, an S phase regulation system is triggered even in parthenogenotes raised by dual treatment of egg activation and polar body suppression. The S phase schedule of parthenogenotes confirms the temporal pattern of chromosome duplication, observed by other researchers, leading to tetraploid parthenogenotes. The S phase determination also provides a basis for argument concerning the number of centrioles participating in parthenogenetic development. If polar body formation of activated eggs was not suppressed, the first S phase was normal, but the second S phase did not recur on time. A rigidly regulated system of DNA replication cycle, which should be an essential prerequisite for parthenogenesis, thus requires the content of polar bodies.     

High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes

Background

Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method.

Results

The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs.

Conclusions

This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.