Quantification and characterisation of cyclo-oxygenase and lipid peroxidation inhibitory anthocyanins in fruits of Amelanchier

The levels of bioactive anthocyanins in the fruits of Amelanchier alnifolia, A. arborea and A. canadensis have been determined by HPLC. Cyanidin 3-galactoside (1) was present in the fresh fruit of the three species at concentrations of 155, 390 and 165 mg/100 g, respectively. Cyanidin 3-glucoside (2) was present only in A. alnifolia and A. canadensis at concentrations of 54 and 48 mg/100 g, respectively. The anthocyanins were confirmed by LC-ESI/MS and NMR studies. At 100 ppm, anthocyanin mixtures from the three species inhibited cyclo-oxygenase (COX)-1 and -2 enzymes at 66 and 67%, 60 and 72%, and 51 and 76%, respectively. The positive controls used in the COX assays were aspirin, Celebrex and Vioxx at 180, 1.67 and 1.67 ppm, respectively, and showed 74 and 69%, 5 and 82% and 0 and 85% COX-1 and COX-2 inhibition, respectively. Anthocyanins 1 and 2 and cyanidin (3) inhibited COX-1 enzyme 50.5, 45.62 and 96.36%, respectively, at 100 ppm, whereas COX-2 inhibition was the highest for 3 at 75%. In the lipid peroxidation inhibitory assay, anthocyanin mixtures at 10 ppm from the three species showed activities of 72, 73 and 68%, respectively, compared with 89, 87 and 98% for commercial anti-oxidants butylated hydoxyanisole, butylated hydroxytoluene, and tert-butylhydroxyquinone at 1.67, 2.2 and 1.67 ppm, respectively. At 10 ppm, compounds 1-3 inhibited lipid peroxidation by 70, 75 and 78%, respectively.     

The effect of electrical field strength on activation and development of cloned caprine embryos

The activation procedure used in nuclear transfer (NT) is one of the critical factors affecting the efficiency of animal cloning. The purpose of this study was to compare the effect of two electrical field strengths (EFS) for activation on the developmental competence of caprine NT embryos reconstructed from ear skin fibroblasts of adult Alpine does. The NT embryos were obtained by transfer of the quiescent fibroblasts at the fourth passage into the enucleated metaphase II (M II) oocytes. Four to five hours after electrical fusion, the NT-embryos were activated by EFS either at 1.67 or at 2.33 kV/cm and immediately incubated in 6-DMAP (2 mM) for 4 h. The cleavage rate of the NT-embryos activated with 2.33 kV/cm was greater than that activated with 1.67 kV/cm after in vitro culture for 18 h (65.6% versus 19.6%, p < 0.001). No pregnancy was found in 14 recipient does after transferring 51 NT embryos at 1-2 cell stages activated with 1.67 kV/cm. In contrast, two of the seven recipients were pregnant and gave birth to three kids after transferring 61 NT embryos at 1-2 cell stages activated by 2.33 kV/cm. The birth weights of three cloned kids were within the normal range of Alpine goats. However, one kid died 1h after birth while the remaining two are still healthy. DNA analysis by polymerase chain reaction (single-strand conformation polymorphism, SSCP) confirmed that the three kids were genetically identical to the nuclear donor.     

Insulin receptors in isolated mouse pancreatic acini.

Specific insulin receptors were measured in isolated mouse pancreatic acini. Scatchard analyses revealed a high affinity binding site with a K_d of 1.67 nM and a lower affinity site with a K_d of 83 nM. Binding of insulin to these receptors was rapid, one-half maximal binding occurring at 2 min and maximal binding at 30 min. Insulin stimulated the uptake of the glucose analogue 2-deoxy-D-glucose; maximum effects were detected at 1.67 μM. Insulin, in contrast, had no direct effects on alpha-aminoisobutyric acid uptake. The finding of high affinity insulin receptors in pancreatic acinar cells supports the hypothesis that insulin may directly regulate specific functions in the exocrine pancreas.     

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.     

Glucose stimulates and potentiates islet amyloid polypeptide secretion by the B-cell.

Islet amyloid polypeptide (IAPP) has been shown to be actively secreted by the pancreatic B-cell along with insulin. To determine whether the modulation of B-cell IAPP secretion is similar to that of insulin, we assessed IAPP release in response to glucose at 4 different concentrations (1.67, 5.5, 8.8 and 16.7 mM) and to non-glucose secretagogues at different glucose concentrations in a neonatal rat islet monolayer culture preparation. Glucose alone stimulated IAPP and insulin secretion in a dose dependent fashion with maximal release for both peptides occurring at 8.8 mM. B-cell secretion of IAPP in response to arginine, isobutylmethylxanthine or both together was potentiated by increasing glucose concentrations from 1.67 to 16.7 mM. This same pattern of glucose potentiation was observed for insulin secretion. The data indicate that the pattern of peptide responses of cultured neonatal B-cells to glucose is similar for both IAPP and insulin release. Furthermore, the data suggest that glucose is capable of potentiating B-cell secretion of both IAPP and insulin.     

Inhibitory effects of the macrolide antimicrobial tylosin on anaerobic treatment

A laboratory-scale anaerobic sequencing batch reactor (ASBR) was operated using a glucose-based synthetic wastewater to study the effects of tylosin, a macrolide antimicrobial commonly used in swine production, on treatment performance. The experimental period was divided into three consecutive phases with different influent tylosin concentrations (0, 1.67, and 167 mg/L). The addition of 1.67 mg/L tylosin to the reactor had negligible effects on the overall treatment performance, that is, total methane production and effluent chemical oxygen demand did not change significantly (P < 0.05), yet analyses of individual ASBR cycles revealed a decrease in the rates of both methane production and propionate uptake after tylosin was added. The addition of 167 mg/L tylosin to the reactor resulted in a gradual decrease in methane production and the accumulation of propionate and acetate. Subsequent inhibition of methanogenesis was attributed to a decrease in the pH of the reactor. After the addition of 167 mg/L tylosin to the reactor, an initial decrease in the rate of glucose uptake during the ASBR cycle followed by a gradual recovery was observed. In batch tests, the specific biogas production with the substrate butyrate was completely inhibited in the presence of tylosin. This study indicated that tylosin inhibited propionate- and butyrate-oxidizing syntrophic bacteria and fermenting bacteria resulting in unfavorable effects on methanogenesis.     

Actions of Ya-hom, a herbal drug combination, on isolated rat aortic ring and atrial contractions

The effect of the Thai popular medicine Ya-hom on cardiovascular function was studied in isolated rat aortic ring and atrium by comparison with norepinephrine (NE). Water extraction of Ya-hom at concentrations of 0.83, 1.67, 8.33 and 16.67 mg/ml stimulated aortic ring contraction dose-dependently. The maximum contraction, at 16.67 mg/ml, was about 14% that of NE. This stimulatory effect of Ya-hom was inhibited partially by phentolamine, which indicated that the effect of Ya-hom was partially dependent on the alpha receptor, similar to NE. Administration of Ya-hom with NR decreased the force of aortic ring contraction as compared to the effect of NE alone, indicating that Ya-hom may have a partial alpha-agonist activity. Ya-hom at concentrations of 1.67, 8.33 and 16.67 mg/ml showed a dose-dependent, positive inotropic and negative chronotropic effects. Ya-hom increased the force of isolated atrial contraction with a slow onset and prolonged action. In contrast to norephinephrine, which acted on beta1 receptor, causing positive inotropic and chronotropic effects, propranolol did not alter the effect of Ya-hom on the atrial contraction. This shows that the action of Ya-hom on atrial contraction does not involve beta receptor.     

广西石灰岩地区蜈蚣蕨居群的遗传多样性研究

采用等位酶分析方法 ,研究了广西石灰岩地区蜈蚣蕨居群的遗传多样性 ,分析了其空间变化趋势。检测了 8个酶系统 ,1 5个酶位点。分析结果表明 :广西石灰岩地区蜈蚣蕨居群遗传多样性程度较高 ,每个位点的等位基因平均数为 1 .6 7,多态位点为 5 7.78% ,平均期望杂合度为 0 .2 4 9。蜈蚣蕨居群的遗传组成在居群间有一定的差异 ,但差异的程度并不与空间距离成正比     

Genetic mapping of sporulation operons in Bacillus subtilis using a thermosensitive sporulation mutant.

A thermosensitive sporulation mutant was used to determine the order of sporulation operonsin the urs region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-SPO-96(SpoII)-spo-85(SpoV)-spo-279(SpoII)-furA-ura-cysC-spo-NG1.67(SpoIII). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972).     

雅氏瓦螨对氟胺氰菊酯的抗性机理初探

用有效成分为1000μg/L的氟胺氰菊酯药液对雅氏瓦螨Varroa jacobsoniOudemans敏感个体的腹部表皮进行穿透性测定,结果表明药液无法通过表皮起毒杀作用。通过添加酶抑制剂多功能氧化酶(PBO)和酯酶(DEF)的增效测定,结果显示PBO在抗性和敏感螨中分别增加毒效为3.27和1.80倍;而DEF为3.23和1.67倍。反映在抗性蜂螨的抗药性与多功能氧化酶的活性有密切相关,也与酯酶活性有关。对抗性螨和敏感螨的羧酸酯酶的活力测定,显示出在抗性螨中酶活指数高140%以上。同时对酯酶电泳进行扫描,也发现抗性螨与敏感螨的峰值存在差异。     

Resemblance for age at menarche in female twins reared apart and together

Menarche is a significant developmental event in the lives of young females. Genetic and family environmental influences on the timing of its occurrence are explored in the first formal analysis using reared-apart and reared-together monozygotic (MZA, MZT) and dizygotic (DZA, DZT) twin pairs. Mean age at menarche was 12.50 years (SD = 1.67) for the reared-apart pairs and 12.86 years (SD = 1.49) for the reared-together pairs. Intraclass correlations for age at menarche were 0.56 for MZA twins, 0.16 for DZA twins, 0.70 for MZT twins, and 0.41 for DZT twins. The mean within-pair difference was 1.07 years (SD = 1.04) for MZA twins, 1.67 years (SD = 1.59) for DZA twins, 0.64 year (SD = 0.86) for MZT twins, and 1.43 years (SD = 1.34) for DZT twins. These results are consistent with genetic influence, although the lower correlations for reared-apart twins and their larger within-pair differences suggest that age at menarche is partly affected by common rearing environments. Feeling understood by one's father during the growing-up years was significantly associated with earlier age at menarche, and a comparable trend was found for feeling understood by one's mother. These findings are considered with reference to current theories of pubertal timing.     

A genetic study of immunoglobulin E

Path analysis gives evidence of genetic heritability (.425) for serum IgE levels. Complex segregation analysis indicates, in addition to significant polygenic heritability, a major regulatory locus RE, with homozygotes re/re maintaining persistently high levels of IgE. The gene frequency of re is .489, and the displacement is 1.67 standard deviations.     

Cloning of cDNA for argininosuccinate synthetase mRNA and study of enzyme overproduction in a human cell line

Previous studies of the human cell line RPMI-2650 (wild type) and its canavanine-resistant variants have demonstrated differences in argininosuccinate synthetase activity as follows: canavanine-resistant much greater than wild type grown in citrulline greater than wild type grown in arginine (Su, T.-S., Beaudet, A. L., and O'Brien, W. E. (1981) Biochemistry 20, 2956-2960). A recombinant plasmid containing a 1.55-kilobase insert complementary to the mRNA for human argininosuccinate synthetase was isolated by the combined use of differential colony hybridization and immunoprecipitation of the products of plasmid-selected mRNA translation. Both blot and dot hybridization analysis of polyadenylated RNA indicated a major mRNA species of 1.67 kilobase in all cells, and the levels of mRNA correlated well with the levels of enzyme activity: canavanine-resistant, 180; wild type grown in citrulline, 7; and wild type grown in arginine, 1. One major mRNA species of 1.67 kilobase and one minor species of 2.68 kilobase were observed in wild type and canavanine-resistant cell lines. Reassociation kinetics of pAS1 with genomic DNA from human liver, canavanine-resistant cells, and wild type cells were not significantly different. Blot hybridization of genomic DNA revealed no detectable differences between wild type cells, canavanine-resistant cells, and human leukocytes. The data demonstrated that there were multiple copies, perhaps 10 or more, of argininosuccinate synthetase-like sequences in human DNA and that the canavanine-resistant phenotype was not due to gene amplification.     

Lysine biosynthesis in Rhodotorula glutinis: properties of pipecolic acid oxidase.

Pipecolic acid oxidase from Rhodotorula glutinis, which converts pipecolic acid to alpha-aminoadipic-delta-semialdehyde, an intermediate of the biosynthetic pathway of lysine, was purified 290-fold. The enzyme from the crude extract and purified preparation exhibited a molecular weight of approximately 43,000 and was composed of a single subunit. The purified enzyme was heat labile and exhibited a pH optimum of 8.5 and an apparent Km for L-pipecolic acid of 1.67 X 10(-3) M. L-Proline acted as a competitive inhibitor for the enzyme. The enzyme was inhibited by the sulfhydryl agents p-chloromercuribenzoate and mercuric chloride. The in vitro enzyme activity required oxygen and upon oxidation of pipecolic acid, oxygen was reduced to hydrogen peroxide.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRES trans-acting factor polypyrimidine tract binding protein (PTB) to the 5′-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5′-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5′-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40–100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic β-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5′-UTR of the human insulin mRNA.     

Small mammal populations of an agroecosystem in the Atlantic Forest domain, southeastern Brazil.

This study reports 2 years of the population dynamics and reproduction of a small mammal community using the removal method. The study was conducted in a rural area of the Atlantic Forest, in Sumidouro, Rio de Janeiro State, Brazil. The population sizes, age structure and reproduction were studied for the four most common species in the study area. The overall diversity was 1.67 and ranged between 0.8 to 1.67. The species richness was 13 considering the whole study. The most abundant species were the rodents Nectomys squamipes (n = 133), Akodon cursor (n = 74), Oligoryzomys nigripes (n = 25) and the marsupials Didelphis aurita (n = 58) and Philander frenatus (n = 50). Seven other rodents were captured once: Necromys lasiurus, Akodon montensis, Sooretamys angouya, Oecomys catherine, Oxymycterus judex, Euryzygomatomys spinosus and Trinomys iheringi. There were higher peaks for diversity and species richness during the winter (dry) months, probably due to higher food availability. The marsupials had a seasonal reproduction with highest population sizes at the end of the rainy seasons. Nectomys squamipes reproduced mostly during rainy periods. Akodon cursor reproduced predominantly in the winter with the highest population peaks occurring during this season. The analysis of the population dynamics of the rodent species indicated that no species behaved as an agricultural pest, probably due to the heterogeneous landscape of high rotativity of vegetable cultivation. Rodent populations were more susceptible to the removal procedure than marsupial ones.     

Rheology of hyaluronic acid

The dynamic viscoelastic properties of hyaluronic acid solutions have been measured over the frequency range 0.02–1.67 cps. The effects of varying temperature, hyaluronic acid concentration, pH, and ionic strength on the dynamic shear moduli were studied. The solutions exhibited a sharp transition from viscous to elastic behavior as the strain frequency increased. No entanglement coupling of the hyaluronic acid molecules was evident over the concentration range 2.0–4.0 mg./ml. Solutions at pH 2.5 showed a pronounced elastic behavior relative to both higher and lower pH's. This effect was attributed to a stiffening of the hyaluronic acid molecule at this pH.     

Effect of hyperthermia on the radioprotective action of cysteamine and isoproterenol

Heating of mouse bone marrow cells up to 42 degrees C was shown to increase their radiosensitivity (DMF = 0.80 +/- 0.12). At this temperature, the radioprotective efficiency of cysteamine was lost completely (DMF = 0.78 +/- 0.09), and radioprotective activity of d,l-isoproterenol significantly decreased (DMF declined from 2.41 +/- 0.23 to 1.67 +/- 0.16). It is assumed that the radioprotective effect of cysteamine on mammalian cells is associated with the processes of the postirradiation DNA repair for just these processes are inhibited by heating. The mechanism of action of a beta-agonist of isoproterenol is perhaps only partially associated with DNA repair.     

Production of an EDRF-like activity in the cytosol of N1E-115 neuroblastoma cells

Accumulation of cyclic GMP in cultured rat lung fibroblasts was used to test the hypothesis that N1E-115 neuroblastoma cells produce an endothelium-derived relaxing factor (EDRF)-like activity. By using this assay, the production of an EDRF-like activity in homogenates and cytosolic fractions of N1E-115 neuroblastoma cells was observed. Detection of the activity required the presence of superoxide dismutase and was inhibited by hemoglobin. Production of the EDRF-like factor was dependent on L-arginine and NADPH. The apparent Km for L-arginine was 1.25 microM and the apparent Km for NADPH was 1.67 microM. The production of the EDRF-like activity was inhibited by the L-arginine analogs, NG-monomethyl-L-arginine and NG-nitro-L-arginine, with apparent Ki values of 1.0 and 0.3 microM, respectively.     

Amino acid conjugated sophorolipids: A new family of biologically active functionalized glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).