Purification and characterization of feline ghrelin and its possible role

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser3, has been isolated from rat and human stomach as an endogenous ligand for the growth hormone secretagogue receptor. Here, we purified feline ghrelin and examined its possible physiological role in cats. The major active form of feline ghrelin is a 28-amino acid peptide octanoylated (C8:0) at Ser3; except for one amino acid residue replacement, this structure is identical to those of rat and human ghrelins. However, much structural divergence in peptide length and fatty acid modification was observed in feline ghrelin: peptides consisting of 27 or 26 amino acids lacking Gln14 and/or Arg28 were found, and the third serine residue was modified by octanoic acid (C8:0), decanoic acid (10:0), or unsaturated fatty acids (C8:1, C10:1 and C10:2). In agreement with the structural divergence, two kinds of cDNA with different lengths were isolated. Administration of synthetic rat ghrelin increased plasma growth hormone levels in cats, with a potency similar to that in rat or human. Plasma levels of ghrelin in cats increased approximately 2.5-fold after fasting. The present study indicates the existence of structural divergence in feline ghrelin and suggests that, as in other animals, ghrelin may play important roles in GH release and feeding in cats.     

Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)–RFLP analysis using two enzymes, Eco NI and Mva I, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR–RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops , suggesting that S. gilberti juveniles are rare in this area.     

Purification and characterization of two anionic trypsins from the hepatopancreas of carp

SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH₂-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.     

Quantitative studies on the relationship between plowing into soil of clubbed roots of preceding crops caused by Plasmodiophora brassicae and disease severity in succeeding crops

The effect of the plowing of clubbed roots of cracifers on the population of Plasmodiophora brassicae in soil was quantitatively studied by measuring the number of resting spores produced in the diseased plants. Though the mean number of resting spores per diseased plant increased with the increase of the disease severity, it remained almost identical for the disease severity classified into category 3 among host species and cultivars tested. Mean number of resting spores per diseased plant ranged from 9.3 to 10.9 (log) regardless of the value of the disease index. When the number of resting spores in soil was calculated based on these data and plant cultivation methods, the values were equivalent to 4.8-6.4 resting spores g^-1soil (log)where clubroot disease occurred severely. The value of the disease index of Chinese cabbage plants grown in the pots where clubbed roots of initially grown plants had been plowed into soil (plowing plot) was higher than that in the pots where no plants had been grown (control plot) and where the clubbed roots of initial plants had been removed (removal plot). Though the number of resting spores of P. brassicae in soil decreased by 14% of the inocoKum concentration immediately after the inoculation, the number of spores after the first cultivation in the removal plot was similar to that in the control plot. On the other hand, the number of resting spores in the plowing plot increased significantly compared with that in the control plot. The plowing of clubbed roots into soil resulted in the increase of the population of P. brassicae and disease severity of clubroot in subsequent cultivation in the field. The results corresponded to the values estimated based on the number of resting spores in soil in relation to each value of the disease index.     

Fundamental studies on wood/cellulose-plastic composites: effects of composition and cellulose dimension on the properties of cellulose/PP composite

Although wood/cellulose-plastic composites (WPC) of low wood/cellulose content have been more accepted worldwide and are promoted as low-maintenance, high-durability building products, composites containing high wood/cellulose content are not yet developed on an industrial scale. In this study, flow properties, mechanical properties, and water absorption properties of the compounds of cellulose microfiber/polypropylene (PP) and maleic anhydride-grafted polypropylene (MAPP) were investigated to understand effects of the high cellulose content and the dimensions of the cellulose microfiber. The molding processes studied included compression, injection, and extrusion. It was found that fluidity is not only dependent on resin content but also on the dimension of the filler; fluidity of the compound declined with increased fiber length with the same resin content. Dispersion of the composite was monitored by charge-coupled device (CCD) microscope. Increasing the plastic content in the cellulose-plastic formulation improved the strength of mold in addition to the bond development between resin and filler, and the tangle of fibers. The processing mode affected the physicomechanical properties of the cellulosic plastic. Compression-molded samples exhibited the lowest modulus of rupture (MOR) and modulus of elasticity (MOE) and the highest water absorption, while samples that were injection-molded exhibited the highest MOR (70 MPa) and MOE (7 GPa) and low water absorption (2%).     

Identification of an allele attributable to formation of cucumber-like flavor in wild tomato species (Solanum pennellii) that was inactivated during domestication

Carbon 6 (C6)-aldehydes formed by fatty acid 13-hydroperoxide lyase (13HPL) specific to fatty acid 13-hydroperoxides (13-HPO) are important flavor constituents in fresh tomato fruits. C9-aldehydes are usually formed by 9/13HPL showing dual specificity to 9- and 13-HPOs and are scarcely found in tomato fruits. Mature red fruits of one of the introgression lines, IL1-4, generated by hybridization of a cultivated tomato (Solanum lycopersicon) to its wild relative Solanum pennellii, form high amounts of C9-aldehydes upon homogenization. The IL1-4 fruits showed high 9/13HPL activity. One of the genes isolated from IL1-4 showed a high similarity to plant 9/13HPLs. Recombinant proteins expressed in Escherichia coli showed 9/13HPL activity. Cleaved amplified polymorphic sequence analyses indicated that the gene was specific to IL1-4 and S. pennellii. S. lycopersicon had a gene having high similarity to the S. pennellii gene. It was absent in IL1-4. Among the differences of amino acid residues found between the two genes, a Cys to Ser exchange may be responsible for the inactivation of resultant protein product of S. lycopersicon gene because the Cys is an essential amino acid residue for HPL activity. From these observations, it could be assumed that a tomato gene corresponding to S. pennellii 9/13HPL gene had been inactivated through domestication of tomatoes.     

Seasonal changes in proteolytic enzymes of yellowtail Seriola quinqueradiata (Temminck & Schlegel; Carangidae) fed extruded diets containing different protein and energy levels

Seasonal variations in pepsin, trypsin and chymotrypsin activities and protein digestibility were studied in yellowtails (Seriola quinqueradiata) reared for 1 year with extruded diets containing different levels of protein. Trypsin and chymotrypsin storage levels in the digestive tissues of starved fish were affected by seasonal changes in water temperature. Actual digestion activities of trypsin and chymotrypsin were low at lower water temperatures, but pepsin activity in the stomach tissue was not affected by low temperatures. On the other hand, trypsin and chymotrypsin activities in the intestinal contents were higher during lower water temperature months, while pepsin activity in the stomach contents was low at lower water temperatures. Apparent protein digestibility (APD) did not differ among the dietary treatments in the higher water temperature months, while in colder months it was higher in fish fed diet 1 than in fish fed diets 2 and 3. The APD values reflected pepsin activity in the stomach contents in all sampling months. Therefore, lower APD in colder months seems to be attributed to lower protease activity in the gastric digesta, implying that enhanced pepsin secretion from the stomach tissue might improve protein digestibility and growth performance in yellowtails during winter.     

Effect of taurine supplemented practical diet on growth performance and taurine contents in whole body and tissues of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effect of dietary taurine on juvenile Japanese flounder was determined by feeding three taurine-supplemented experimental diets (TAU) and a commercial diet (CD) to evaluate a practical diet for juvenile Japanese flounder. Juvenile Japanese flounder were reared on the three experimental diets supplemented with taurine at 0, 0.5, 1.0% and CD. These diets were fed to juvenile Japanese flounder of an initial mean body weight of 0.2 g for 6 weeks at 20°C and the taurine contents of the whole body and tissues were analyzed. The final average body weight of juvenile Japanese flounder fed the 1.0% TAU was significantly higher than that of the other groups. Taurine contents in the whole body and tissues increased with the increase in dietary taurine level. These results indicate that juvenile Japanese flounder require at least 15 mg/g taurine in the diet, even though a combined mix of fish, krill and squid meal was the main protein source in the experimental diets.     

Viability of maternally heat-stressed mouse zygotes in vivo and in vitro

Mammalian preimplantation embryos are susceptible to heat stress. This present study examined how maternal heat stress affects the development of mouse zygotes in vivo and in vitro. In Experiment 1, zygotes collected from female mice that were heat‐stressed for 12 h on day 1 of pregnancy were cultured in vitro. Maternally heat‐stressed zygotes developed normally to the two‐cell stage, but the majority of embryos failed to develop into morulae or blastocysts. In Experiment 2, pregnant mice were heat‐stressed on day 1 or from day 1 to day 3 of pregnancy. The number of living fetuses on day 14 of pregnancy was lower in heat‐stressed mice than in non‐stressed mice, but the difference was significant only in successively heat‐stressed mice. These results demonstrate that maternally heat‐stressed zygotes have reduced in vitro viability, but this phenomenon does not necessarily lead to embryo loss in the maternal environment.     

Evaluation of genetic variation among wild rice populations in Cambodia

A total of 448 samples in five natural populations of wild rice (Oryza rufipogon) were collected in Cambodia. They were examined using 12 SSR and two chloroplast markers to evaluate the degree of variation among populations and the genetic structure within populations. In the two annual populations, the number of plants with homozygous alleles at all 12 SSR loci were high (66.3% and 79.5%), suggesting that these plants propagate mainly through self-pollination. In the three perennial populations, no individuals had all homozygous genotypes, but redundant genotypes resulted from clonal propagation were observed. Percentages of the redundant genotypes were highly varied (3.6%, 29.2% and 86.0%). This may be due to the different stable levels of environmental conditions. As for chloroplast genome, most of the wild plants showed the same chloroplast types as most Indica-type cultivars have. However, plants with different chloroplast types were maintained, even in the same population. In tropical Asian countries, many wild rice populations were observed under similar ecological conditions examined in this study. Therefore, the present results concerning population structure will be important to further elucidate genetic features of wild rice, and will also give strong clues to utilize and conserve wild natural genetic resources.