Characterization of chitinase genes from an alkaliphilic actinomycete,Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Production of fertile somatic hybrid plants between Oriental hybrid lily and<Emphasis Type="Italic"> Lilium</Emphasis>×<Emphasis Type="Italic">formolongi</Emphasis>

Somatic hybridizations via electrofusion were performed in combinations of Oriental hybrid lilies (cvs. Acapulco and Shirotae) and Liliumxformolongi hort. (cv. Hakucho). Electrofusion-treated protoplasts divided only under nurse culture. The divided protoplasts grew into calli on the culture medium containing picloram, and the calli were then transferred to the hormone-free culture medium for induction of plant regeneration. The regenerants were transferred to a greenhouse, and were grown until the flower stage. In the fusion combinations of Acapulco + Hakucho and Shirotae + Hakucho, four regenerants apparently showed different morphological features compared with their parents. The results of molecular analyses by means of cleaved amplified polymorphic sequences markers and flow cytometry confirmed that these regenerants were somatic hybrid plants. Furthermore, we examined the stability of the morphological features of the hybrids in the next generations. This is the first report to describe the successful realization of Lilium somatic hybridization via protoplast fusion.     

Production of hydrogen peroxide by polyphenols and polyphenol-rich beverages under quasi-physiological conditions

To investigate the ability of the production of H(2)O(2) by polyphenols, we incubated various phenolic compounds and natural polyphenols under a quasi-physiological pH and temperature (pH 7.4, 37 degrees C), and then measured the formation of H(2)O(2) by the ferrous ion oxidation-xylenol orange assay. Pyrocatechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, and polyphenols such as catechins yielded a significant amount of H(2)O(2). We also examined the effects of a metal chelator, pH, and O(2) on the H(2)O(2)-generating property, and the generation of H(2)O(2) by the polyphenol-rich beverages, green tea, black tea, and coffee, was determined. The features of the H(2)O(2)-generating property of green tea, black tea, and coffee were in good agreement with that of phenolic compounds, suggesting that polyphenols are responsible for the generation of H(2)O(2) in beverages. From the results, the possible significances of the H(2)O(2)-generating property of polyphenols for biological systems are discussed.     

First investigation of ultraoligotrophic alpine Lake Puma Yumco in the pre-Himalayas,China

Lake Puma Yumco is a typical alpine lake (altitude 5030m) located in the pre-Himalayas of Tibet, China, and this study was the first limnological investigation ever conducted on it. Lake Puma Yumco (28°34N, 90°24E) has the following morphometric properties: maximum length 31km, maximum width 14km, mean width 9km, shoreline 90km, surface area 280km², and shoreline development 1.5. Transparency was approximately 10m, even in the thawing season. The extinction coefficient of the lake water was calculated as 0.15m^–1. Annual maximum transparency was estimated from the depth of the Chara zone to be 30m. Dissolved oxygen was 7mg O₂ l^–1 and showed saturated values, and salinity was 360mgl^–1. The chemical type of the lake water was Mg-Ca-HCO₃-SO₄, and it was slightly alkaline in character. Total nitrogenous nutrients (sum of ammonia, nitrite, nitrate, and urea nitrogen), phosphate, and silicate were extremely low at 1, 0.02, and 9µM, respectively. Dissolved organic carbon, nitrogen, and phosphorus concentrations were 160, 11, and 0.08µM and the molar ratio was calculated as 2100:140:1. Chlorophyll a concentration was 0.2mgm^–3. Phytoplankton and zooplankton were dominated by Aphanocapsa sp. and Diaptomidae. Both nitrogen and phosphorus appear to be the limiting parameters for phytoplankton growth. Organic carbon and nitrogen contents in lake sediments were low and the sediments contained a large amount of CaCO₃. The grain size of sediment was that of silt-sand in most cases. The present results indicate that the pre-Himalayan alpine freshwater Lake Puma Yumco is an ultraoligotrophic lake.     

Oxidative deamination by hydrogen peroxide in the presence of metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.     

An insertional mutation in the rice<Emphasis Type="Italic"> PAIR2</Emphasis> gene,the ortholog of<Emphasis Type="Italic"> Arabidopsis ASY1</Emphasis>, results in a defect in homologous chromosome pairing during meiosis

To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis. The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene. The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae. Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues. In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages. The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1. The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene.Communicated by G. Jürgens     

Multiple amino acid sensing inputs to mTORC1

The evolutionarily conserved target of rapamycin complex 1 (TORC1) is a master regulator of cell growth and metabolism. In mammals, growth factors and cellular energy stimulate mTORC1 activity through inhibition of the TSC complex (TSC1-TSC2-TBC1D7), a negative regulator of mTORC1. Amino acids signal to mTORC1 independently of the TSC complex. Here, we review recently identified regulators that link amino acid sufficiency to mTORC1 activity and how mutations affecting these regulators cause human disease.