The LSH/DDM1 Homolog MUS-30 Is Required for Genome Stability,but Not for DNA Methylation in Neurospora crassa

LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to DNA damaging agents. MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued DNA damage-hypersensitivity of Δmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Δmus-30 is partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Δmus-30 strains. We found that MUS-30-deficient cells are not defective for DSB repair, and we observed a negative genetic interaction between Δmus-30 and Δmei-3, the Neurospora RAD51 homolog required for homologous recombination. Together, our findings suggest that MUS-30, an LSH/DDM1 homolog, is required to prevent DNA damage arising from toxic base excision repair intermediates. Overall, our study provides important new information about the functions of the LSH/DDM1 family of enzymes.     

Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.     

Comparative investigation of vitamins and their analogues on terminal differentiation, from preadipocytes to adipocytes, of 3T3-L1 cells

1. The effects of 20 kinds of vitamins or their analogues on the growth rate of preadipocytes and the terminal differentiation of preadipocytes to adipocytes was systematically compared in 3T3-L1 cells. 2. The addition of vitamin C markedly increased the growth rate of preadipocytes at over 50 microM. 3. The addition of vitamin K3 slowed down the growth rate at over 0.1 microM. 4. In water soluble vitamins and their analogues tested, the vitamin B6 group and vitamin C significantly stimulated the differentiation, and consequently increased the glycerophosphate dehydrogenase activity and triglyceride accumulation, to a concentration of over 10 microM. 5. Many fat soluble vitamins and their analogues (the vitamin A group, including beta-carotene, the vitamin D group, vitamin E and the vitamin K group) strongly inhibited the adipose conversion of 3T3-L1 cells at microM level.     

Three dimensional observation of the nerve fibers along the cerebral blood vessels

Summary Three dimensional observation of the nerve fibers along the cerebral blood vessels was investigated by scanning electron microscopy. Electron probe X-ray microanalysis was also performed in the cerebral blood vessels treated with peroxidase-antiperoxidase immunohistochemistry intensified by nickel ammonium sulfate.Nerve fibers (2–8 m in diameter) formed a plexus on the outer surface of the adventitia. After branching, the nerve fibers penetrated the blood vessel adventitia. Substance P-immunoreactive nerve fibers showed a meshwork pattern in the outer layer of the adventitia, and vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers revealed a spiral running pattern in the inner layer of the adventitia. Taken together with previous studies, these findings suggest that substance P nerve fibers in the cerebral arteries may not be related to arterial dilatation or constriction, but VIP nerve fibers may be vasodilative.     

Effect of sodium butyrate on induction of ornithine decarboxylase activity in phytohemagglutinin-stimulated lymphocytes

Effect of sodium butyrate on DNA synthesis and the induction of ornithine decarboxylase (EC 4.1.1.17), a rate-limiting enzyme of polyamine biosynthesis, was studied in phytohemagglutinin(PHA)-stimulated bovine lymphocytes. Millimolar concentrations of butyrate completely inhibited the incorporation of [³H] thymidine into the acid-insoluble fraction and reversibly suppressed the induction of ornithine decarboxylase. Other shortchain fatty acids were much less active than butyrate. These results suggest that the suppression of ornithine decarboxylase activity may be one of the reasons for the inhibition of DNA synthesis with butyrate in bovine lymphocytes, because our previous experimental results have shown that the induction of ornithine decarboxylase closely correlates with the DNA synthesis in growth-stimulated cells.     

Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis.

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.     

Frankixalus,a New Rhacophorid Genus of Tree Hole Breeding Frogs with Oophagous Tadpoles

Despite renewed interest in the biogeography and evolutionary history of Old World tree frogs (Rhacophoridae), this family still includes enigmatic frogs with ambiguous phylogenetic placement. During fieldwork in four northeastern states of India, we discovered several populations of tree hole breeding frogs with oophagous tadpoles. We used molecular data, consisting of two nuclear and three mitochondrial gene fragments for all known rhacophorid genera, to investigate the phylogenetic position of these new frogs. Our analyses identify a previously overlooked, yet distinct evolutionary lineage of frogs that warrants recognition as a new genus and is here described as Frankixalus gen. nov. This genus, which contains the enigmatic ‘Polypedates’ jerdonii described by Günther in 1876, forms the sister group of a clade containing Kurixalus, Pseudophilautus, Raorchestes, Mercurana and Beddomixalus. The distinctiveness of this evolutionary lineage is also corroborated by the external morphology of adults and tadpoles, adult osteology, breeding ecology, and life history features.     

A monoclonal antibody to chicken gizzard desmin that recognizes intermediate filaments and nuclear granules in BHK21/C13 cells

One hybridoma (AC54), which produces monoclonal antibody (MAb) that recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells, and two hybridomas (AC19 and AC36) which produce MAbs that recognize IFs only, were obtained by using a crude actin preparation from chicken gizzard as an antigen. In immunoblotting, both the AC54 and AC19 MAbs reacted with the 52 kD protein (desmin) and some other proteins in gizzard and BHK21/C13 cells. Indirect immunofluorescent microscopy of BHK21/C13 cells showed that the cytoplasmic filaments stained by these MAbs were IFs based on their colchicine-induced whorl formation. The ability of AC54 MAb to recognize IFs was more limited than that of AC19 MAb. The nuclear granules recognized by AC54 MAb were in a different location than the cytoplasmic IFs and sometimes were concentrated in the nucleolus. These results indicate that AC54 MAb is an anti-desmin MAb that reacts with some desmin-related proteins; that it recognizes IFs differently than AC19 MAb, another anti-desmin MAb; and that it recognizes nuclear granules in locations where desmin or desmin-related protein has not yet been reported.     

Effects of fluvoxamine on nerve growth factor-induced neurite outgrowth inhibition by dexamethasone in PC12 cells

In the present study, we examined the effects of fluvoxamine on nerve growth factor (NGF)-induced neurite outgrowth inhibition by dexamethasone (DEX) in PC12 cells. Fluvoxamine increased NGF-induced neurite outgrowth. Compared with co-treatment with NGF and fluvoxamine, p-Akt levels were higher than the values without fluvoxamine. The phosphorylated extracellular regulated kinase 1/2 levels were slightly increased by co-treatment with NGF and fluvoxamine. Fluvoxamine concentration-dependently improved NGF-induced neurite outgrowth inhibition by DEX. Fluvoxamine also improved the decrease in the NGF-induced p-Akt level caused by DEX. Interestingly, the sigma-1 receptor antagonist NE-100 blocked the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX. The selective sigma-1 receptor agonist PRE-084 also improved NGF-induced neurite outgrowth inhibition by DEX, which is blocked by NE-100. These results indicate that the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX may be attributable to the phosphorylation of Akt and the sigma-1 receptor.