Identification and characterization of proteoglycans in bovine neonatal skeletal muscle

In order to provide background for understanding biological roles of proteoglycans (PG) in developing skeletal muscle, we have isolated and characterized PG in bovine neonatal skeletal muscle. Two types of PG were isolated from skeletal muscle by density gradient ultracentrifugation and ion‐exchange chromatography. One was a small PG (PG‐S) with a molecular size of 100–130 kDa, another was a large PG (PG‐L) with a molecular size of 300–500 kDa. The glycosaminoglycan chains of PG‐S and PG‐L were dermatan sulfate and chondroitin sulfate, respectively, judged by cellulose acetate membrane electrophoresis. Immunoblot assays revealed that both PG bound to type I, II, III and IV collagen, laminin and fibronectin. Unlike PG‐S, PG‐L bound to type V collagen and hyaluronic acid. Small proteoglycans had a core protein of 45 kDa, which reacted with the antibody against the decorin core protein. The N‐terminal amino acid sequence of the PG‐S core protein was consistent with that of decorin from bovine bone and tendon. Thus, PG‐S from neonatal skeletal muscle was identified as decorin in bovines. Immunohistochemical analysis with antibodies against PG‐L and PG‐S demonstrated that PG‐L was located both in the perimysium and endomysium, but PG‐S was localized exclusively in the perimysium. These findings suggest that the characterized PG may have distinct roles in the ECM construction of developing skeletal muscle.     

Mechanical stretch-induced activation of skeletal muscle satellite cells is dependent on nitric oxide production in vitro

Mechanical stretch induces activation of cultured quiescent satellite cells and the activation response is owing to rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. We provide new evidence that the stretch activation is dependent on nitric oxide (NO) production. Stretch activation could be abolished by the addition of N ^G-nitro- L -arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, but not by N ^G-nitro- D -arginine methyl ester hydrochloride, a less active enantiomer of L-NAME. Adding HGF to the L-NAME culture restored the activation response, indicating that L-NAME does not directly inhibit satellite cell activation, but acts upstream from the HGF release. In addition, immunoblots of satellite cell lysate revealed the presence of nitric oxide synthase. These experiments suggest that NO is involved in linking mechanical perturbation of satellite cells to chemical signaling responsible for HGF release from its sequestration in vitro .     

Geographical distribution and seasonality of the prevalence of Leucocytozoon lovati in Japanese rock ptarmigans (Lagopus mutus japonicus) found in the alpine regions of Japan

In this study, we investigated the geographical distribution and seasonality of Leucocytozoon lovati infection in the Japanese rock ptarmigan (Lagopus mutus japonicus); this bird is one of the special natural monuments of Japan that inhabits the Japanese alpine regions. We examined blood samples from birds captured in the Kubiki, Hida, and Akaishi mountain ranges for three years from 2002 to 2005. Seventy-three blood samples from 42 males, 19 females, and 12 birds of unknown sex were used for this study. The rate of infection with L. lovati was 78.1% in the 73 birds examined. We demonstrated that the L. lovati infection was distributed across wide ranges of ptarmigan populations from the northern to the southern alpine zones. There was no sex bias in the prevalence ratio. The prevalence of L. lovati and the level of parasitization of the blood cells tended to increase from spring through summer; in contrast, a decrease was observed from summer through autumn. Although L. lovati infection was observed in a number of local populations inhabiting three mountainous regions, no infected birds were found in Mt. Johnen-dake and Mt. Maejohnen-dake. It is necessary to continue surveying the relationship between the population dynamics of the ptarmigan and the density of the arthropod vector from the perspective of in situ conservation of this endangered species.     

Phylogenetic comparison of Leucocytozoon spp. from wild birds of Japan

Eight species of Japanese birds were found to be infected with Leucocytozoon species using microscopic analysis. We used PCR and sequence analysis of the mitochondrial cytochrome b gene (cyt b) to compare the genetic background among these detected protozoa species. In 20 individuals of 22 samples, a single amplified band was detected from 6 of 8 bird species; 9 Japanese rock ptarmigans (Lagopus mutus japonicus), 4 large-billed crows (Corvus macrorhynchos), 2 carrion crows (C. corone), 2 scops owls (Otus scops), 1 Japanese grosbeak (Eophona personata), and 2 brown-eared bulbuls (Hypsipetes amaurotis), respectively. Phylogenetic analysis based on the partial cyt b sequences revealed that all Leucocytozoon isolates in Japan closely grouped with other Leucocytozoon species previously reported in the literature. Among the Japanese isolates, the phylogenetic tree suggested that L. lovati from the Japanese rock ptarmigan may be basal to the parasites found in other bird species. Our study is the first to identify the molecular relationships among Leucocytozoon parasites in the avifauna of Japan.     

Decreases in serum apolipoprotein C-III concentration in cows with ethionine-induced fatty liver

To monitor the serum concentration of apolipoprotein C-III (apoC-III), one of the functional apoproteins in lipid metabolism, in cows with ethionine-induced fatty liver, and to investigate the association of apoC-III with liver triglyceride (TG) content and serum biochemical variables, seven nonpregnant nonlactating Holstein cows (3 to 6 years old) were used. Five cows were treated with ethionine, an analogue of methionine, (days 0, 7 and 14). The remaining two controls received saline as the vehicle. Liver TG contents in the treated cows were increased markedly whenever administered, and significant increases were observed at days 14 (666.4%, 85.3 mg/g) and 21 (675.0%, 86.4 mg/g) compared with day 0. In controls, no significant changes in liver TG content and serum biochemical variables were observed during this experiment. The serum apoC-III concentration in the treated cows was decreased drastically after the first administration and fell to the lowest value at day 10 (76.2 microg/ml, 32% of day 0). The apoC-III was significantly (p<0.05) correlated with non-esterified fatty acids (r= -0.526), gamma-glutamyl transpeptidase (r= -0.407), total bilirubin (r= -0.464), positively with apolipoprotein B-100 (apoB-100, r=0.601) and cholesterol ester (r=0.449). Although apoB-100 concentrations were also reduced by the administrations, the concentrations tended to recover smoothly toward the next administration. The distinct difference in change between apoC-III and apoB-100 suggests that apoC-III may be regulated by other pathways, in addition to inhibiting the synthesis of apoproteins by ethionine.     

Synthesis and acaricidal activity of phenylpiperazine derivatives

We synthesized 33 new phenylpiperazine derivatives and assessed their acaricidal activity. These derivatives were synthesized through sequential reactions consisting of the sulfonylation of 2-substituted 4-methylaniline with chlorosulfonic acid, reduction with red phosphorus and iodine, alkylation by alkyl halide, cyclization with bis(2-chloroethyl)amine hydrochloride, and N-substitution reaction of phenylpiperazines with various reagents. All phenylpiperazines synthesized were evaluated for acaricidal activity and their structure–activity relationships discussed, it was found that 4-substituted 1-[2-fluoro-4-methyl-5-(2,2,2-trifluoroethylsulfanyl)phenyl]piperazine derivatives exhibited good acaricidal activity. Among them, 1-[2-fluoro-4-methyl-5-(2,2,2-trifluoroethylsulfanyl)phenyl]-4-(trifluoromethylsulfonyl) piperazine showed the highest level of activity against Tetranychus urticae and provided a high level of activity against Tetranychus kanzawai and Panonychus citri. In addition, studies on the effect at various stages of T. urticae exhibited that this compound showed good activity against both adults and eggs.     

Mapping and use of QTLs controlling pod dehiscence in soybean

While the cultivated soybean, Glycine max (L.) Merr., is more recalcitrant to pod dehiscence (shattering-resistant) than wild soybean, Glycine soja Sieb. & Zucc., there is also significant genetic variation in shattering resistance among cultivated soybean cultivars. To reveal the genetic basis and develop DNA markers for pod dehiscence, several research groups have conducted quantitative trait locus (QTL) analysis using segregated populations derived from crosses between G. max accessions or between a G. max and G. soja accession. In the populations of G. max, a major QTL was repeatedly identified near SSR marker Sat_366 on linkage group J (chromosome 16). Minor QTLs were also detected in several studies, although less commonality was found for the magnitudes of effect and location. In G. max × G. soja populations, only QTLs with a relatively small effect were detected. The major QTL found in G. max was further fine-mapped, leading to the development of specific markers for the shattering resistance allele at this locus. The markers were used in a breeding program, resulting in the production of near-isogenic lines with shattering resistance and genetic backgrounds of Japanese elite cultivars. The markers and lines developed will hopefully contribute to the rapid production of a variety of shattering-resistant soybean cultivars.     

粳稻品种杂交后代食味特性和产量的研究

以具有代表性的4个日本粳稻品种为母本,5个有代表性的中国粳稻品种为父本,配制20个杂交组合,并对亲本以及F4群体的产量和食味特性进行了分析.结果表明:中国亲本产量、蛋白质和直链淀粉含量的平均值显著高于日本亲本,而食味品尝试验的综合评价值显著低于日本亲本;不同组合F4群体的F测验显示,组合间在产量、蛋白质含量、直链淀粉含量、脂肪酸度和评分方面都存在显著差异;从亲本平均值和F4群体平均值的关系来看,除直链淀粉含量外,蛋白质含量、脂肪酸度和评分都呈极显著正相关.     

Effect of skeletal muscle decorin on collagen fibrillogenesis in vitro

The effect of skeletal muscle decorin on collagen fibrillogenesis was investigated, in order to provide background for understanding the functions of decorin in skeletal muscle. The self‐assembly of type I and III collagen with the addition of decorin or the core protein of decorin from bovine neonatal skeletal muscle was monitored using a spectrophotmeter. Time course changes in the absorbance of collagen solutions showed typical sigmoidal curves composed of three phases. The time of the initial phase was not different between the collagen solution with decorin and that without decorin. The increase rate of the absorbance in the second phase decreased with concentration of decorin added in collagen solutions. Similar effects on fibrillogenesis of type I and III collagens were observed when the core protein of decorin was added in collagen solutions. These results suggest that regulation of collagen fibrillogenesis by decorin depends on its core protein. The networks of reconstructed collagen fibrils with decorin were looser than those without decorin. Bovine skeletal muscle decorin could participate in the regulation of collagen fibrillogenesis and in the arrangement of collagen fibrils in the intramuscular connective tissue.     

An intact mitochondrial cox1 gene and a pseudogene with different genomic configurations are present in apple cultivars ‘Golden Delicious’ and ‘Delicious’: Evolutionary aspects

We have characterized the mitochondrial cox1 gene copies in two apple cultivars ‘Golden Delicious’ and ‘Delicious’. Both the cultivars contained an intact copy and a truncated copy of cox1. The intact ‘Golden Delicious’ and ‘Delicious’ cox1 genes, designated G-cox1 and D-cox1, respectively, were both found to be actually transcribed to give an RNA of approximately 1.7 kb. The two intact cox1 and two truncated copies (G-φcox1 and D-φcox1) shared a common 1115-bp segment flanked by four combinations of two different 5′- and 3′-sequences. PCR assay demonstrated that the configurations bearing G-cox1 and G-φcox1 existed in substoichiometric amounts within the mitochondrial genome of ‘Delicious’ whereas substoichiometric molecules carrying D-φcox1 were present in the ‘Golden Delicious’ mitochondrial genome. Although ancestor/descendant relationships cannot be inferred between the G-cox1 and D-cox1 arrangements, the results led us to hypothesize that (1) the 1115-bp segment containing part of the progenitor cox1 was duplicated, thereby generating a pseudo-cox1 copy, and (2) this was followed by homologous recombination across a portion of the 1115-bp repeats which gave rise to the descendant cox1 and pseudo-cox1 arrangements.