Detection by PCR of the Tyzzer's disease organism (Clostridium piliforme) in feces.

We examined whether the Tyzzer's disease organism, Clostridium piliforme, could be detected in feces by PCR. If the organism could be detected in feces, a diagnosis could be made without sacrifice of the animal. Using the RT strain of C. piliforme, we found that a C. piliforme band could be detected when there were > or = 1 x 10(0) bacteria present in the PCR solution, but the presence of fecal extract in the solution depressed the sensitivity 10 fold. Nevertheless, we could detect the C. piliforme-specific band in fecal extracts from rats in a naturally infected colony, and concluded that the use of PCR to detect C. piliforme DNA in fecal extracts would be a useful diagnostic technique.     

Development of an effective process for utilization of collagen from livestock and fish waste

A procedure for the extraction of protein and production of peptides by enzymic hydrolysis from bone and skin wastes containing collagen was developed. Fat and inorganic components were first removed in a pretreatment step and a high molecular weight protein extracted under acidic conditions (pH 3) using a 1 h reaction time at 60 °C. The molecular weight of extract from pig skin was greater than 100 kDa. The extract had a high water retention capacity, was beneficial for repair of rough skin, had no odor problem and was demonstrated to be safe in skin patch tests. It was thus considered acceptable for use in cosmetic materials. Pretreated fish bone and pig skin were hydrolyzed with a commercial enzyme. The hydrolysates had a high anti-radical activity (IPOX₅₀, 0.18 and 0.45 mg ml⁻¹) and a high potential for decreasing blood pressure (IC₅₀, 0.16 and 0.41 mg ml⁻¹), suggesting the hydrolysates could be a useful additive in food materials.     

Age composition of masu salmon smolts in northern Japan

Smolts of anadromous masu salmon Oncorhynchus masou aged 3+ years were found in a northern Japanese river. This is the first recording of 3+ year smolts in Japan. These fish appeared to originate in the cold upper river where 2+ year parr were found during autumn.     

CND41, a chloroplast nucleoid protein that regulates plastid development, causes reduced gibberellin content and dwarfism in tobacco

CND41, a DNA binding protein of chloroplast nucleoids, may function as a negative regulator of chloroplast gene expression. The reduction of CND41 in an antisense transformant accelerated plastid development in shoot apex cells and early young leaves, and caused a dwarf phenotype and altered leaf morphology. Plant height and leaf shape could be restored almost to those of the wild type by application of gibberellins (GAs), clearly indicating that a reduction in GA content was a prime cause of the dwarf phenotype in CND41 antisense transformants. The transformants had reduced endogenous levels of active gibberellin (GA₁), a biologically active GA, compared to those of wild-type plants. Possible relationships between chloroplast development affected by CND41 function and GA biosynthesis are discussed.     

NADH Dehydrogenases: From Basic Science to Biomedicine

This review article is concerned with two on-going research projects in our laboratory, both of which are related to the study of the NADH dehydrogenase enzyme complexes in the respiratory chain. The goal of the first project is to decipher the structure and mechanism of action of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from two bacteria, Paracoccus denitrificans and Thermus thermophilus HB-8. These microorganisms are of particular interest because of the close resemblance of the former (P. denitrificans) to a mammalian mitochondria, and because of the thermostability of the enzymes of the latter (T. thermophilus). The NDH-1 enzyme complex of these and other bacteria is composed of 13 to 14 unlike subunits and has a relatively simple structure relative to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which is composed of at least 42 different subunits. Therefore, the bacterial NDH-1 is believed to be a useful model for studying the mitochondrial complex I, which is understood to have the most intricate structure of all the membrane-associated enzyme complexes. Recently, the study of the NADH dehydrogenase complex has taken on new urgency as a result of reports that complex I defects are involved in many human mitochondrial diseases. Thus the goal of the second project is to develop possible gene therapies for mitochondrial diseases caused by complex I defects. This project involves attempting to repair complex I defects in the mammalian system using Saccharomyces cerevisiae NDI1 genes, which code for the internal, rotenone-insensitive NADH–quinone oxidoreductase. In this review, we will discuss our progress and the data generated by these two projects to date. In addition, background information and the significance of various approaches employed to pursue these research objectives will be described.     

Association between nasal allergy and a coding variant of the Fc ε RI β gene Glu237Gly in a Japanese population

The gene for the beta-chain of the high-affinity receptor for IgE (Fc epsilon RI beta) has been proposed as a candidate gene for atopy. A coding variant Glu237Gly has been studied in various populations with asthma and atopy, and the results were controversial for association of the variant with atopy/asthma. Because nasal allergy is a more common atopic disease and shows less remission than asthma, we analyzed whether the Glu237Gly variant is correlated with nasal allergy. The study enrolled 233 patients with nasal allergy and 100 control subjects. Further, three subgroups were selected: patients with perennial nasal allergy (n=149), Japanese cedar pollinosis (n=189), and allergy to multiple allergens (n=45). The allele frequency of Gly237 in the controls and patients was 0.14 and 0.20, and the frequency of Gly237-positive subjects was 0.23 and 0.356, respectively. There was a significant association between Gly237-positivity and nasal allergy, perennial nasal allergy, Japanese cedar pollinosis, and allergy to multiple allergens. Among all 333 subjects we observed a significant relationship between Gly237 and elevated levels of serum total IgE (>250 IU/ml) and very high IgE (>1000 IU/ml). Among patients positive for a specific IgE, Gly237 was significantly associated with high IgE for house dust, mite, and Japanese cedar pollen. These results suggest that the Glu237Gly variant of the Fc epsilon RI beta gene is involved in the development of nasal allergy through the process for the production of both specific and nonspecific IgE antibodies.     

Identification and structure-activity relationship of an antimicrobial peptide of the palustrin-2 family isolated from the skin of the endangered frog Odorrana ishikawae

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.     

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.