Ribosomal DNA intergenic spacer sequence in foxtail millet, Setaria italica (L.) P. Beauv. and its characterization and application to typing of foxtail millet landraces

We determined the sequence of ribosomal DNA (rDNA) intergenic spacer (IGS) of foxtail millet isolated in our previous study, and identified subrepeats in the polymorphic region. We also developed a PCR-based method for identifying rDNA types based on sequence information and assessed 153 accessions of foxtail millet. Results were congruent with our previous works. This study provides new findings regarding the geographical distribution of rDNA variants. This new method facilitates analyses of numerous foxtail millet accessions. It is helpful for typing of foxtail millet germplasms and elucidating the evolution of this millet.     

Coenzyme Q2 induced p53-dependent apoptosis

Coenzyme Q functions as an electron carrier and reversibly changes to either an oxidized (CoQ), intermediate (CoQ.-), or reduced (CoQH2) form within a biomembrane. The CoQH2 form also acts as an antioxidant and prevents cell death, and thus has been successfully used as a supplement. On the other hand, the value of the CoQ/CoQH2 ratio has been shown to increase in a number of diseases, presumably due to an anti-proliferative effect involving CoQ. In the present study, we examined the effect of CoQ and its isoprenoid side chain length variants on the growth of cells having different p53 statuses. Treatment with CoQs having shorter isoprenoid chains, especially CoQ2, induced apoptosis in p53-point mutated BALL-1 cells, whereas treatment with longer isoprenoid chains did not. However, CoQ2 did not induce apoptosis in either a p53 wild-type cell line or a p53 null mutant cell line. These results indicated that the induction of apoptosis by CoQ2 was dependent on p53 protein levels. Moreover, CoQ2 induced reactive oxygen species (ROS) and the phosphorylation of p53. An antioxidant, l-ascorbic acid, inhibited CoQ2-induced p53 phosphorylation and further apoptotic stimuli. Overall, these results suggested that short tail CoQ induces ROS generation and further p53-dependent apoptosis.     

Effects of dimethyl sulfoxide and dexamethasone on mRNA expression of housekeeping genes in cultures of C2C12 myotubes

We used quantitative real-time RT-PCR to investigate the effects of dimethyl sulfoxide (DMSO) and dexamethasone (Dex) on the mRNA expression levels of the housekeeping genes β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), phosphoglycerate kinase 1 (PGK1), peptidylprolyl isomerase A (PPIA), and transferrin receptor (TFRC) in cultures of C2C12 myotubes. The ratios of ACTB mRNA levels to the HPRT1 mRNA level in C2C12 cells that were differentiating from myoblast cells to myotubes decreased from 0 to 120 h of culture, whereas the ratios of TFRC mRNA levels to the HPRT1 mRNA level increased from 0 to 120 h of culture. The ratios of GAPDH, GUSB, PGK1, and PPIA mRNA levels to the HPRT1 mRNA level remained constant from 0 to 120 h of culture. All housekeeping gene mRNA levels were unaffected by exposure to DMSO concentrations of 0.1% or less. The GAPDH mRNA level was increased by Dex, while the ACTB and PGK1 mRNA levels were significantly decreased by Dex. The GUSB, PPIA, and TFRC mRNA levels were unaffected by exposure to Dex. GUSB, HPRT1, and PPIA are thus suitable internal controls for evaluating mRNA expression levels in cultures of C2C12 cells.     

Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra)

The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola.     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

A method for the simultaneous determination of 3T3-L1 adipocyte metabolites by liquid chromatography/mass spectrometry using [(13)C]-stable isotopes

A useful method employing liquid chromatography mass spectrometry (LC/MS) and a stable isotope was developed for simultaneous examination of major metabolism in adipocytes, de novo fatty acid synthesis, glycerol output, and glucose uptake with high sensitivity. The addition of thiazolidinediones, potent agonists of peroxisome proliferator-activated receptors-γ, for 10 d increased glucose uptake in a dose-dependent manner. Fatty acid (FA) synthesis increased at low concentrations of thiazolidinediones (TZDs) and decreased at high concentrations. It is important to assess adipocytes from various examples of metabolism, because each example of adipocyte metabolism is directly related to obesity or metabolic syndrome in various ways. The technique makes metabolic examination easier than conventional methods by means of radioisotopes and makes it possible to identify metabolites and to apply them in biomarker screening.     

Specific Secretion of Proline-Rich Proteins by Salt-Adapted Winged Bean Cells

Six proteins, designated SAP1 through SAP6, were secreted specificallyby salt-adapted cells of winged bean (Psophocarpus tetragonolobus)in suspension cultures. The amino-terminal amino acid sequencesof SAP2 (57 kDa), SAP4 (21 kDa), SAP5 (19 kDa) and SAP6 (17kDa) were homologous to the sequences of proline-rich proteins,indicating that proline-rich proteins are secreted specificallyby these salt-adapted cells. In addition, the amino-terminalamino acid sequence of SAP2 was identical to that of SAP4, andthe amino-terminal sequence of SAP5 was identical to that ofSAP6. Secretion of SAP2 was significantly enhanced by additionof AlCl₃ but not of KCl, LiCl, CaCl₂, MgCl₂, mannnitol or sucroseto suspension cultures. Furthermore, secretion of SAP4, SAP5and SAP6 was stimulated by addition of abscisic acid to cultures,suggesting that these proteins might be secreted in responseto salt or osmotic stress. (Received September 12, 1994; Accepted January 20, 1995)     

Mariculture of kurosoi,Sebastes schlegeli

Synopsis The technology of collecting developing larvae from female kurosoiSebastes schlegeli, and raising the larvae to juveniles (100 mm total length (TL)) to be released into the oopen sea, is presented. Gravid females 40–46 cm TL were captured in May–June 1977–1980 and held in the laboratory until parturition. Fecundity of fish in this size range was 100 000–184 000. Larvae were sequentially fed rotifers,Artemia nauplii, and young sand lance,Ammodytes personatus, until reaching 25 mm; this required 35 days and yielded a survival rate of 50%. Thereafter, the fish were reared in separate size groups to avoid cannibalism. Minced or chopped sand lance and commercial food were provided until the final size of 100 mm was attained. The growth of juvenile kurosoi from 25 to 100 mm required 85 days, with a survival rate of 90%. The effect of released cultured fish on the local stock is being determined from information on the recapture of tagged fish.