Polyphenolic constituent structures of Zanthoxylum piperitum fruit and the antibacterial effects of its polymeric procyanidin on methicillin-resistant Staphylococcus aureus

Zanthoxylum piperitum (Rutaceae) is used as a spice and a natural medicine in Japan. Our study found that ZP-CT-A, a polymeric proanthocyanidin purified from the fruit of this species, noticeably decreased the minimum inhibitory concentrations of beta-lactam antibiotics for methicillin-resistant Staphylococcus aureus (MRSA). The structure of ZP-CT-A was characterized on the basis of (13)C NMR and size exclusion chromatographic data and the results of thiolytic degradation. A mechanistic study of the effects of ZP-CT-A indicated that it suppressed the activity of beta-lactamase and largely decreased the stability of the bacterial cell membrane of MRSA, as shown by a reduction in the tolerance of MRSA to low osmotic pressure and high ionic strength solutions.     

PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance.

Agonist-induced activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPAR gamma was investigated by gene targeting. Homozygous PPAR gamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPAR gamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPAR gamma agonist treatment. Heterozygous PPAR gamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPAR gamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPAR gamma.     

Hyperosmotic stress-induced somatic embryogenesis and its continuous culture in Japanese honewort (Cryptotaenia japonica)

Japanese honewort (Cryptotaenia japonica) is consumed as a traditional vegetable and has medicinal applications. In Japan, C. japonica is mainly produced using hydroponic culture systems; however, damping-off is often caused by the adherence of pathogens to its seeds. Therefore, the use of sterile artificial seeds in hydroponic culture is likely to be effective for preventing disease. In this study, we established methods for stress-induced somatic embryogenesis and artificial seed production in Japanese honewort. Shoot apex explants from seedlings were treated with 0.7 M sucrose as a hyperosmotic stress for 3 or 6 weeks, and then transferred to stress-free conditions. Somatic embryos were formed after culture in stress-free conditions for 7 weeks. Stress-treated shoot apex explants that formed somatic embryos were cultured in Murashige and Skoog liquid medium with shaking. After 2 weeks of culture, approximately 800 somatic embryos were formed from each explant. Somatic embryos were formed continuously during 37 weeks under the same culture conditions. Thus, somatic embryogenesis was effectively induced in Japanese honewort via hyperosmotic stress, and embryogenic competence was maintained under stress- and phytohormone-free conditions. The somatic embryos produced by liquid culture were used to produce artificial seeds by enveloping the embryos in whipped alginate gel to avoid hypoxic conditions. The artificial seeds had a high germination rate (72%). This system is suitable for the sterile, highly productive hydroponic culture of Japanese honewort.     

Biochemical analysis of the bone of SHC rats with spontaneous hypercholesterolemia and renal dysfunction.

Spontaneously hypercholesterolemic (SHC) rats exhibit severe abnormalities in renal function and bone metabolism at old ages, in addition to hypercholesterolemia. SHC rats were also found to show endocrine abnormalities such as hyperthyroidism from young ages. In this study, biochemical and microdensitometric analyses were carried out using femurs to characterize further the abnormality in bone metabolism: whether biochemical markers of the bone may be affected by these abnormalities. At 6 weeks of age, the ashed weight and calcium content of the dried femurs were slightly lower in SHC rats than in age-matched Sprague-Dawley (SD) rats. None of the markers of microdensitometric analysis was changed. At 24 weeks of age, the ashed weight of dried femurs and the density of the marrow region of femurs were lower in the SHC rats. The results indicate that SHC rats exhibit severe abnormality in bone metabolism leading to biochemical changes in the bone at old ages whereas changes in bone markers were minimal at young ages before the onset of severe renal dysfunction.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

A differential microdetermination for the various forms of biopterin.

Conditions for the quantitative oxidation and destruction of tetrahydrobiopterin and quinoid dihydrobiopterin and the separation of biopterin from its reduced forms by ECTEOLA-Sephadex column chromatography are described. A procedure for the quantitation of tetrahydrobiopterin plus quinoid dihydrobiopterin, 7,8-dihydrobiopterin, and biopterin using a Crithidia bioassay is presented. Using these procedures it was found that tetrahydrobiopterin plus quinoid dihydrobiopterin are the prevalent forms in liver and blood of mice and that biopterin was the predominant form in the tails of tadpoles. In human urine, approximately half of the biopterin was found as tetrahydrobiopterin plus quinoid dihydrobiopterin and the other half was 7,8-dihydrobiopterin. The presence of tetrahydrobiopterin and quinoid dihydrobiopterin was confirmed by a coenzyme assay for the hydroxylation of phenylalanine.     

Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata

The structure of macronuclear DNA of a hypotrichous ciliate, Stylonychia pustulata, was examined by both electron microscopy and nucleotide sequence analysis. The DNA in the macronucleus consists of small linear molecules with average length of about 3400 base pairs (bp). Most, if not all, of these DNA molecules have identical inverted terminal repeat sequence of 20 nucleotide residues. This sequence is 5'-CCCCAAAACCCC-AAAACCCC.     

Basic fibroblast growth factor-like substance in nuclei of male germ cells undergoing meiosis

The presence of a basic fibroblast growth factor-like immunoreactive substance was demonstrated in the nuclei of germ cells at stages from spermatocyte to spermatid in adult rat testis by using immunohistochemistry with an antibody raised against a synthetic peptide corresponding to residues 1-10 of bovine basic fibroblast growth factor [1-146]. The fluorescence was very weak in the nuclei and cytoplasm of spermatogonia, Sertoli cells, and most of the interstitial compartments, except for capillary endothelial cells. This is the first study to demonstrate the presence of basic fibroblast growth factor-like immunoreactive material in the nuclei of haploid cells in vivo.