牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物,是一种重要的新型抗癌药物。然而,紫杉醇在植物中含量低且难提取,限制了高效应用。利用基因工程手段,借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径,构建含有牻牛儿基牻牛儿基焦磷酸(Geranylgeranyl diphosphate,GGPP)合酶和紫杉二烯合酶的融合基因表达载体p Bg GGTS和独立表达盒表达载体p Bg GGg TS,并分别转入灰盖鬼伞LT2菌株中,经过选择性筛选、PCR鉴定、Southern blotting杂交验证,分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株,分别提取菌丝体和发酵液分析。GC-MS分析表明,两种工程菌株与原出发菌株的菌丝提取物无明显差异峰,而与出发菌株的发酵液提取物相比,两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰,采用GC-MS特征质量离子分析方法判定为紫杉二烯,分别为44 ng/L(转化p Bg GGg TS)和30 ng/L(转化p Bg GGTS)。结果表明,通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因,可以生物合成紫杉二烯。     

Design, synthesis and biological evaluation of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors

Herein we describe the design, synthesis and biological activities of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors. The target compounds 2a–2j were designed and synthesized from the structural characteristics of N-phenylthiourea, tyrosinase inhibitor and tyrosine, and l-DOPA, the natural substrates of tyrosinase. Among them, (2R/S,4R)-2-(2,4-dimethoxyphenyl)thiazolidine-4-carboxylic acid (2g) caused the greatest inhibition 66.47% at 20 μM of l-DOPA oxidase activity of mushroom tyrosinase. Kinetic analysis of tyrosinase inhibition revealed that 2g is a competitive inhibitor. We predicted the tertiary structure of tyrosinase, and simulated the docking of mushroom tyrosinase with 2g. These results suggest that the binding affinity of 2g with tyrosinase is high. Also, 2g effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-MSH. These data strongly suggest that 2g can suppress the production of melanin via the inhibition of tyrosinase activity.     

ECTOCELLULAR GLUCOSIDASE AND PEPTIDASE ACTIVITY OF THE MIXOTROPHIC DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

The activities of the enzymes α‐ and β‐glucosidase, and leucine aminopeptidase were measured in cultures of the dinoflagellate Prorocentrum minimum (Pavill.) J. Schiller and in field samples collected during dinoflagellate blooms occurring in tributaries of the Chesapeake Bay, Maryland, USA. Activities were measured using fluorogenic artificial substrates and partitioned among the >5 μm size fraction, small microbes fraction (0.1–5 μm), and dissolved phase (<0.1 μm). P. minimum and most other photosynthetic dinoflagellates are >5 μm in size and thus can be separated from the small microbes fraction, which contains most bacteria. Little to no glucosidase activity was detected associated with the >5 μm size fraction in cultures or in field samples, with most of the activity (67% to 93% in cultures, 54% to 100% in field samples) in the small microbes size fraction for both α and β glucosidase. In contrast, 67% to 90% of the total leucine aminopeptidase (LAP) activity in cultures was measured in the >5 μm fraction. Within a culture, LAP activity in the size fraction containing P. minimum decreased in response to ammonium and urea additions, but not in response to nitrate. In field samples, LAP activity was positively correlated with dinoflagellate abundance and chl a, and negatively correlated with ammonium concentration. During blooms, up to 34% of LAP activity was associated with the >5 μm fraction, indicating that when abundant, dinoflagellates may make a substantial contribution to ectocellular LAP activity in the water column.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses

Development, abiotic and biotic stress each affect the physical architecture and chemical composition of the plant cell wall, making maintenance of cell-wall integrity an important component of many plant processes. Cellulose biosynthesis inhibition (CBI) was employed to impair the functional integrity of the cell wall, and the plant's response to this specific stress was characterized in an Arabidopsis seedling model system. CBI caused changes in the expression of genes involved in mechanoperception, the response to microbial challenge, and lignin and cell-wall polysaccharide biosynthesis. Following CBI, activation of a UDP- d -xylose 4-epimerase gene correlated with increases in arabinose and uronic acid content in seedling cell walls. Activation of pathogen response genes, lignin deposition and lesion formation were dependent on externally supplied sugars and were suppressed by osmotic support. Lignin deposition in the root elongation zone caused by CBI was reduced in atrbohd (NADPH oxidase) mutant seedlings but increased in jasmonic acid resistant1 ( jar1-1 ) mutant seedlings. Phytohormone measurements showed that CBI-induced increases in jasmonic (JA) and salicylic acids were dependent on sugar availability and prevented by osmotic support. We show that CBI activates responses commonly attributed to both abiotic and microbial challenges. Glucose/sucrose and turgor pressure are critical components in maintenance of cell-wall integrity and the regulation of induced responses, including JA biosynthesis. Lignin deposition induced by CBI is regulated by JAR1-1 and NADPH oxidase-dependent signalling processes. Our results identify components of the mechanism that mediates the response to impairment of cell-wall integrity in Arabidopsis thaliana .     

The carotenoid pigments in the juice and flavedo of a mandarin hybrid (Citrus reticulata) cv michal during ripening

The carotenoid pigments ofthe mandarin hybrid (Citrus reticulata) cv Michal, in the juice and flavedo were characterized at three ripening stages, before, during and after colour break. During ripening the characteristic mandarin pattern was formed in the juice, which contained cryptoxanthin as the principal pigment. In the flavedo the chloroplast carotenoid pattern of the green fruit changed into the characteristic pattern of C. reticulata with a high level of citraurin which, together with cryptoxanthin, imparts an intensive reddish tint to the hybrid. The flavedo contained an unusual C₃₀ apocarotenoid, β-citraurinene (8′-apo-β-caroten-3-ol). The flavedo carotenoids of this accidental hybrid were compared with the carotenoids of the presumed parents Dancy tangerine and Clementine. The hybrid resembles more the second parent, from which it inherited the ability to biosynthesize a higher amount of citraurin as well as citraurinene. Citraurinene, considered a Citrus hybrid-specific pigment, was found for the first time in a Citrus variety. A possible biosynthetic pathway of the Citrus C₃₀ -apocarotenoids is proposed.     

氨基糖苷抗生素庆大霉素： 基础研究的新进展及应用研究的新潜力

庆大霉素是临床上重要的氨基糖苷类抗生素。近年来,伴随现代生物技术水平的发展,人们对该抗生素有了更深入的了解。本文综述了近年来国内外关于庆大霉素的作用机制和耐药机制、生物合成途径和结构改造,及其新活性等方面研究成果,并对庆大霉素的应用与发展前景进行展望。     

Microalgal lipids biochemistry and biotechnological perspectives

In the last few years, there has been an intense interest in using microalgal lipids in food, chemical and pharmaceutical industries and cosmetology, while a noteworthy research has been performed focusing on all aspects of microalgal lipid production. This includes basic research on the pathways of solar energy conversion and on lipid biosynthesis and catabolism, and applied research dealing with the various biological and technical bottlenecks of the lipid production process. In here, we review the current knowledge in microalgal lipids with respect to their metabolism and various biotechnological applications, and we discuss potential future perspectives.