Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.     

N-Deacetylation of 2-acetamido-2-deoxy-hexosecontaining oligosaccharides and polysaccharides by calcium in liquid ammonia

N-Deacetylation of 2-acetamido-2-deoxy-hexose residues is accomplished in liquid ammonia containing calcium. Oligosaccharides, lacto-N-fucopentaose II and lacto-N-difucohexaose I, containing 3,4-disubstitutedN-acetylhexosamine residues are quantitativelyN-deacetylated. When applied to polysaccharides, however, only partialN-deacetylation was achieved.Author for correspondence. AXRD     

人体单臂间歇运动对发汗调定点的影响

本工作系在微小气候相对恒定条件下,对10名健康男青年每人进行四项实验。实验 Ⅰ 为测定双侧腿足浸入43℃水中,诱发左前臂屈侧显现定量汗点时的口腔温度(舌下)阈值,作为发汗调定点参考值(ToSSP);实验 Ⅱ 为 Ⅰ 附加右臂间歇轻负荷运动(77W)时测定 ToSSP,部分对象还记录了皮肤电反应;实验 Ⅲ、Ⅳ 为 Ⅰ、Ⅱ 均附加4.5m/s 气流(22—25℃)直吹头面部,再分别测定 ToSSP。实验 Ⅰ 与 Ⅱ 同体对照22人次,Ⅲ 与 Ⅳ 同体对照24人次。结果表明,实验 Ⅱ、Ⅳ 的 ToSSP 均值及其潜伏期均值分别较 Ⅰ、Ⅲ 者降低(P<0.01)或缩短(P<0.001);Ⅰ、Ⅱ间的 ToSSP 均值差、潜伏期均值差,分别与 Ⅲ、Ⅳ 之间者无显著差异(P>0.2);Ⅱ、Ⅳ 的ToSSP 均值各与其实验开始前的口温均值亦无明显差异(P>0.5)。此结果支持运动时体温调定点下降的论点,并提示在研究体温调定点活动时,以 ToSSP 为指标较用发汗速率为优越,因 ToSSP 不为许多干扰因素所影响。     

Microcalorimetric measurements of tissue cells in vitro

A culture flask was designed for the microcalorimetric measurements of tissue cells by an MS 80 standard calvet microcalorimeter. Tissue cells cultured in this flask behaved in the same manner as in the common culture flask used in cytobiological studies. The thermograms of human adenocarcinoma gastric cells (SGc 7901) and HeLa cells were obtained. The heat output power of SGc 7901 cells continuously increased for 70 h with an initial cell number of 3.0 X 10(5). The thermogram was reproducible under strictly controlled conditions. The relationship between the heat output power and the number of SGc 7901 cells within 48 h was obtained. The heat output power was 40 pW/cell to 49 pW/cell when the cell number was in the range 4.5 X 10(5) to 10.4 X 10(5). It was 62.3 +/- 2.9 pW/cell for HeLa cells when the cell number was 6 X 10(5).     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats

With the techniques of specific radioimmunoassay and gel filtration it was found that CGRP was distributed in various tissues of normotensive (WKY) and spontaneously hypertensive rats (SHR) with the highest concentration in the lumbar spinal cord (1197 +/- 94.8 pg/mg tissue) and the lowest in the auricle (15.0 +/- 2.1 pg/mg tissue). In comparison with WKY, CGRP concentration in the plasma was decreased and in the abdominal aorta and hypothalamus was increased in SHR. Gel filtration revealed only one major CGRP molecular form in the tissues. In addition, CGRP reduced the mean arterial pressure (MAP) in SHR in a dose-dependent manner. These data suggest that CGRP may play an important role in the pathogenesis of hypertension and its possible therapy.     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

Biosynthesis of fusarochromanone and its monoacetyl derivative by Fusarium equiseti.

One fluorescent compound previously named TDP-2 was isolated and purified from a rice culture of Fusarium equiseti (Alaska 2-2). Mass spectral and nuclear magnetic resonance data indicated that it is a C-3'-N-acetyl derivative of fusarochromanone, a newly discovered mycotoxin. Time course studies of synthesis of these two compounds on autoclaved rice and Czapek-Dox medium enriched with soybean peptone indicated that fusarochromanone was converted to TDP-2 in the cultures. A high concentration of peptone in the liquid medium may stimulate both fusarochromanone synthesis and its conversion to TDP-2.