Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel electrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5, 5, 10 and 20 Gy of (60)Co gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens.     

Development of a chemical strategy to produce rare aldohexoses from ketohexoses using 2-aminopyridine

Rare sugars are monosaccharides that are found in relatively low abundance in nature. Herein, we describe a strategy for producing rare aldohexoses from ketohexoses using the classical Lobry de Bruyn–Alberda van Ekenstein transformation. Upon Schiff-base formation of keto sugars, a fluorescence-labeling reagent, 2-aminopyridine (2-AP), was used. While acting as a base catalyst, 2-AP efficiently promoted the ketose-to-aldose transformation, and acting as a Schiff-base reagent, it effectively froze the ketose–aldose equilibrium. We could also separate a mixture of Sor, Gul, and Ido in their Schiff-base forms using a normal-phase HPLC separation system. Although Gul and Ido represent the most unstable aldohexoses, our method provides a practical way to rapidly obtain these rare aldohexoses as needed.     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

Effects of barbiturates on the inhibitory action of GABA on excitatory response of the stomach to stimulation of vagal afferent fibers in cat

Effects of barbiturates on the inhibitory action of GABA to the hexamethonium-resistant excitatory response of the stomach to stimulation of the vagal afferent fibers were studied in cats. Inhibition of the hexamethonium-resistant excitatory response by GABA were compared under alpha-chloralose, alpha-chloralose-phenobarbital (PhB), and alpha-chloralose-pentobarbital (PB)-anesthesia in cats. The ID50 of GABA on the hexamethonium-resistant excitatory response was not significantly affected by PhB, but reduced by PB. Both picrotoxin and bicuculline antagonized the effects of GABA. The present experiments demonstrated that PB potentiated the inhibitory effect of GABA on the hexamethonium-resistant excitatory response of the stomach, and suggested that the potentiation by PB may be due to activation of GABA-receptor-ionophore complex.     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads

Drosophila germline stem cells are regulated by the somatic microenvironment, or "niche," which ensures that the stem cells can both self-renew and produce functional gametes throughout adult life. However, despite its prime importance, little is known about how niche formation is regulated during gonadal development. Here, we demonstrate that a receptor tyrosine kinase, Sevenless (Sev), is required to ensure that the niche develops in the anterior region of the male embryonic gonads. Sev is expressed in somatic cells within the posterior region of the gonads. Sev is activated by a ligand, Bride of sevenless (Boss), which is expressed by the germline, to prevent ectopic niche differentiation in the posterior gonadal somatic cells. Thus, we propose that signal transduction from germline to soma restricts expansion of the germline-stem-cell niche in the gonads.     

Alternative splicing variants of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase (As3MT) catalyzes the methylation of trivalent arsenic (As(III)) to monomethylarsonate (MMA(V)) and dimethylarsinic acid (DMA(V)), and plays an important role in the detoxification of arsenicals. Here, we report the identification of two splicing variants of the human As3MT gene. One splicing variant was an exon-3 skipping (Δ3) form which produced a premature stop codon, and the other was an exon-4 and -5 skipping (Δ4,5) form which produced a 31.1 kDa As3MT protein. In addition to the full-length mRNA of As3MT, Δ4,5 mRNAs were detected in HepG2, A549, HL60, K562, and HEK293 cells. The methyltransferase activity of the recombinant Δ4,5 As3MT and wild-type As3MT proteins purified from Escherichia coli was determined. Speciation analysis by HPLC–ICP-MS showed a clear peak of MMA(V) after incubation of As(III) with the wild-type As3MT protein, but not with the Δ4,5 As3MT protein. In addition, COS-7 cells transfected with Δ4,5 As3MT cDNA did not convert As(III) to MMA(V) or DMA(V). The lack of methyltransferase activity of Δ4,5 As3MT seems to be related to the deletion of an S-adenosylmethionine-binding site and a critical cysteine residue. These data suggest that the expression pattern of splicing variants of the As3MT gene may affect the capacity for arsenic methylation in cells.