一株地衣芽孢杆菌碱性蛋白酶的研究I.

从皖北盐碱地土样中分离到 0 1 1菌株 ,对其进行了菌种鉴定以及胞外碱性蛋白酶的初步研究。结果表明 :0 1 1菌株符合地衣芽孢杆菌种的特征 ,但该菌芽孢端生、孢囊膨大、中度耐盐、高度耐碱 ,可在NaCl浓度为 1 3%和pH1 1的培养基中生长 ,这些特征又不同于该种几个模式株 ,因而将 0 1 1菌株鉴定为地衣芽孢杆菌的一个亚种 ,命名为地衣芽孢杆菌砀山亚种(Bacilluslicheniformissubsp .dangshanensis)。该菌在发酵培养基中能产生较高产量的胞外碱性蛋白酶 ( 72 5u/mL)。酶的最适作用条件 :60℃、pH9.0 ,该酶在pH6.0～ 1 1范围内稳定     

Repair process and enzymatic activity associated with induction of shell regeneration in the invasive species Pomacea canaliculata

ABSTRACT

This study investigated the repair process and enzymatic activity responses after induction of shell regeneration in Pomacea canaliculata. The survival rates, height of regenerated shell, and activities of alkaline phosphatase (ALP) and carbonic anhydrase (CA) were investigated and recorded for 15 days. A triangular piece of shell about 8?mm wide was excised from the ventral region of the shell. On the basis of photographs and enzyme assay, it was concluded both yellow-shell apple snails and black-shell apple snails had high survival rates (100%). The regenerated new shell was initially thin, with the wound sealed 5 days after induction of shell regeneration; after that the regenerated shell thickened. Three snails were sampled at 0, 0.5, 1, 2, 3, 5, 10 and 15 days after shell excision for analysis of enzymatic activity of ALP and CA. Trends of the activity of ALP and CA in the mantle were similar. Initially they both showed a decline on the first day, then rose on following days. Activity of ALP declined to a minimum on the first day, and an activity peak in both colour forms appeared on the fifth day (P?<?0.05) and was maintained for the rest of the experiment. The minimum value of CA occurred after 0.5 days, and a significant increase was also observed on the fifth day (P?<?0.05). This study has revealed that apple snails have good shell regeneration abilities, and that ALP and CA probably play a crucial role in shell growth and regeneration.     

Heme crevice disorder after sixth ligand displacement in the cytochrome c-551 family

1H NMR and visible absorption spectroscopy were used to monitor sixth ligand methionine displacement reactions in four members of the ferricytochrome c-551 family from Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas stutzeri substrain ZoBell, and Nitrosomonas europae. Potassium cyanide displaces the methionine ligand with very modest changes in the visible spectra, but profound changes in the NMR spectra. The initial product formed kinetically, designated complex I, changes with time and/or heating to a more thermodynamically favored product termed complex II. Spectra indicate that both I and II are actually a family of closely related conformational isomers. Low temperature NMR spectra of complex II indicate that some of the isomers are in chemical exchange on the NMR time scale. High pH also displaces the methionine ligand in a manner similar to the well-known alkaline transition of mitochondrial cytochrome c. However, the reaction occurs at higher pH values and over a narrower pH range for the c-551 family, and the transition pH range is different for the different proteins studied. The final alkaline forms also show peak widths and a number of peaks indicative of multiple conformational isomers.     

FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

 Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse, bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D₂O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54  °C in horse and at 57  °C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35  °C, followed by a second amide II transition at 50  °C revealing a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50  °C (tuna) and 54  °C (horse) are also observed during the thermal titration of the CN^–-ligated cytochromes (where CN^– displaces the Met80 ligand), but a well-defined 35  °C amide II transition is absent from the titration curve of the CN^–adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5  °C range with T _m values at 74  °C (bovine c), 70  °C (horse c) and 65  °C (tuna c), as monitored by changes in the amide I′ bands. The FTIR spectra were also used to compare the secondary structures of the ferricytochromes c at 25  °C. Curve fitting of the amide I (H₂O) and amide I′ (D₂O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the α-helical absorption of tuna c indicates the presence of less-stable helical structures. CN^– adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D₂O is a powerful tool in protein conformational analysis. Received: 1 April 1999 / Accepted: 24 August 1999     

A comparative evaluation of aflatoxin B1 genotoxicity in fish models using the Comet assay

Aflatoxin B₁ (AFB₁) is classified as a Group I hepatocarcinogen in humans by the International Agency for Research on Cancer (IARC). The alkaline Comet assay is a simple and rapid method by which DNA damage can be demonstrated as a function of tail moment. The present work is the first to evaluate the genotoxicity of AFB₁ in fish using the Comet assay. Two different species of fish were selected as models due to previously established sensitivity to AFB₁: rainbow trout (sensitive) and channel catfish (resistant). Fish were i.p. injected with 0.5 mg AFB₁/1 ml DMSO/1 kg body weight. The Comet assay was performed after 4 and 24 h on whole blood, liver, and kidney cells of both species. Trout blood and kidney tissue tested displayed significant (p<0.05) and extensive DNA damage (shown by increased tail moment) after 4 h which then decreased by 24 h. In liver cells, damage progressively increased over time. Conversely, similarly treated catfish showed no elevation in DNA damage over controls at the same doses. These results suggest that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB₁ and for evaluating organ specific effects of these agents in different species.     

假单胞菌碱性木聚糖酶的纯化及性质

假单胞菌(Pseudomonas)G62可产生两种胞外木聚糖酶,即XynA和XynB。经过硫酸铵沉淀、阴离子和阳离子交换层析、分子筛色谱,最终得到 两种电泳纯酶。XynA的分子量及等电点分别为42kD和91,XynB的分子量和等电点分别是 20kD和88。经薄层色谱分析证明,两酶以不同的方式水解木聚糖,但都不产生木糖,即 两酶都为内切酶,它们的最适作用温度均为50℃。XynA的最适作用pH为7.0～9.8,而XynB的为7.0～7.5。在65℃时的半寿期XynA为6 min,XynB为140 min。XynA的Km和Vmax分别是5.56 mg·ml-1和543μmol·min-1·mg-1,XynB的Km和Vmax分别是7.72 mg·ml-1和819μmol·min-1·mg-1。两酶受Cu2+、Fe3+、Pb2+、Zn2+和Hg2+强烈抑制。化学修饰的初步结果表明,两酶的活性位点氨基酸均含有色氨酸和羧基氨基酸。     

松嫩平原碱化草地植物群落分布的空间和环境因素分析

在１９９６年野外实地采样的基础上,运用ＤＣＡ消势对应分析（ｄｅｔｒｅｎｄｅｄｃｏｒｅｓｐｏｎｄｅｎｃｅａｎａｌｙｓｉｓ）对群落主要变化趋势及其与土壤环境因子之间的关系进行了分析。运用ＤＣＣＡ消势典范对应分析（ｄｅｔｒｅｎｄｅｄｃａｎｏｎｉｃａｌｃｏｒｅｓｐｏｎｄｅｎｃｅａｎａｌｙｓｉｓ）对影响松嫩平原碱化草地群落结构季节动态的空间和环境因素进行了定量的分解。结果表明：在影响群落分布的各因子中,环境因子独立约占４０％,而环境＿空间耦合因子占３５％,空间因子独立约占３％,其他因子约占２０％。在诸多因子中,土壤的盐渍化程度在整个生长季起着决定性作用,但土壤水分和氮素的作用因季节而变化。在干旱季节,植物生长的主要制约因子为土壤碱化度和土壤水分,而土壤氮素的作用处于次要地位。但在降雨较多、土壤湿润度较大的季节,土壤氮素的影响明显增强,成为仅次于土壤碱化度的决定性因素。     

黄鳝性腺自然逆转过程中vasa基因的表达分析

本研究采用RNA反义探针原位杂交技术,对vasa基因在黄鳝(Monopterusalbus)性腺发育过程中的表达情况进行了分析。结果表明:vasamRNA在Ⅰ、Ⅱ、Ⅲ期卵母细胞的胞质中均匀分布,在Ⅳ、Ⅴ期卵母细胞中vasamRNA有向胞质外周皮层迁移集中的趋势,但不明显;退化的卵粒也呈现vasamRNA阳性反应;在Ⅲ、Ⅳ期卵巢的被膜中检测到带有vasa阳性信号的细胞,这些细胞可能是待向精原细胞分化、迁移到卵巢被膜上的原始生殖细胞(Primordialgermcell,PGC),在性逆转过程中这些PGC可能由卵巢被膜迁移到精小叶中并发育成精子;在成熟精巢中,vasa在精原细胞和初级精母细胞中表达。进一步采用碱性磷酸酶染色法分析黄鳝卵巢及精巢后发现:在卵巢中,除了卵母细胞外,卵巢被膜中也检测到了带有碱性磷酸酶阳性信号的细胞;在成熟精巢中,只在生殖腺囊内的雄性生殖细胞中检测到碱性磷酸酶,而精巢被膜中没有检测到带有碱性磷酸酶阳性信号的细胞。本研究结果初步表明:黄鳝的雄性生殖细胞可能起源于雌性阶段卵巢被膜中的原始生殖细胞[动物学报51(3):469-475,2005]。     

Morphology and Molecular Phylogeny of Trimyema koreanum n. sp., a Ciliate from the Hypersaline Water of a Solar Saltern

ABSTRACT. A new ciliate, Trimyema koreanum n. sp., isolated from hypersaline water (salinity of 293‰) from a solar saltern in Korea, was investigated using live observation, protargol impregnation, and gene sequencing. Trimyema koreanum is about 30 × 13 μm in vivo, has usually 23 longitudinal ciliary rows forming two distinct ciliary girdles visible both in vivo and in protargol impregnation. A third indistinct ciliary girdle as well as a girdle of mucocysts is distinguishable only in impregnated cells. We suggest T. koreanum as a new species, differing from the most similar species, T. marinum, by the presence of two distinct ciliary girdles (T. marinum usually has six ciliary girdles clearly visible in living cells and three anterior spirals that encircle the cell completely). Although the number of known 18S rRNA sequences in the genus Trimyema was limited, the Trimyema group including T. koreanum forms a strong clade. The phylogenetic position confirms that the isolate belongs to the genus Trimyema and is different from previously sequenced species. Trimyema koreanum is able to consume both prokaryotes and small eukaryotes (specifically, the alga Dunaliella sp.).     

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K _cat). The enzyme was also calcium dependent and with 2 mM Ca⁺², the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca⁺², respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.