Purification and some properties of inducible lysine decarboxylase from Vibrio parahaemolyticus.

Inducible lysine decarboxylase from Vibrio parahaemolyticus AQ 3627 was purified to apparent homogeneity and characterized. The enzyme displayed a molecular weight of 531000 by gel filtration and 79000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required pyridoxal phosphate as a cofactor, and the pH optimum was 5.5. The Km value for L-lysine was 3.2 mM, and the enzyme was inhibited by 6-aminocaproic acid and alpha-fluoromethyl analogs of cadaverine. delta-Hydroxylysine and S-aminoethyl-L-cysteine was active as substrates to a lesser extent than L-lysine. The amino-terminal amino acid sequence was determined to be Met-Asn-Ile-Phe-Ala-Ile-Leu. These properties were compared with those of other bacterial lysine decarboxylases.     

Characteristics of the H(+)-translocating adenosine triphosphatase of Vibrio parahaemolyticus

We have characterized H(+)-translocating adenosine triphosphatase (ATPase) in membrane vesicles of Vibrio parahaemolyticus. The ATPase required high concentrations (about 0.5 M) of Na2SO4 (or other salts) for its maximum activity. Magnesium ion stimulated the ATPase activity, but Ca2+ did not. The activity of ATPase was inhibited by tetrachlorosalicylanilide, an H+ conductor, but not by another H+ conductor, carbonylcyanide-m-chlorophenylhydrazone. The activity was strongly inhibited by dicyclohexylcarbodiimide or Zn2+, and partially inhibited by azide, but not at all by vanadate.     

Purification and Partial Characterization of (6-4) Photoproduct DNA Photolyase from Xenopus laevis

Abstract— The (6-4) photoproduct DNA photolyase was detected in two vertebrate animals Crotalus atrox (rattlesnake) and Xenopus laevis (South African clawed toad). The enzyme was extensively purified from X. laevis and characterized. The highly purified enzyme is fluorescent with an excitation maximum at 420-440 nm and emission maximum at 460-480 nm. The photorepair action spectrum matches the fluoresoence excitation spectrum with a 430 nm maximum.     

Cellular Fatty Acid Composition of Vibrio parahaemolyticus by Reversed-Phase High-Performance Liquid Chromatography

Abstract

High-performance liquid chromatography was used to separate and identify cellular fatty acids isolated from Vibrio parahaemolyticus, a gram-negative estuarine microorganism associated with seafood-borne enteritis in man. Fatty acids were isolated from statically grown bacterial cultures, saponified, and derivatized with an ultraviolet tag. Aliquots of derivatized fatty acids were injected onto a reversed-phase column with water:acetonitrile gradient as the mobile phase and ultraviolet detection at 254 nm. The predominant fatty acids found for the V. parahaemolyticus strains studied were C12, C14, C16:1, C16, C18:1, and C18. In addition, previously unreported fatty acids C13, C17, C19, and C21 were identified. Comparison of HPLC with GLC fatty acid separations showed good agreement with the exception that HPLC was able to resolve previously unidentified fatty acid constituents.     

Formycin A resistant mutants due to defect in adenosine transport system in Vibrio parahaemolyticus

An antibiotic formycin A inhibited growth of Vibrio parahaemolyticus under certain conditions, which suggested that formycin A was taken up by cells under these conditions. We found that formycin A was transported via the adenosine transport system which we previously reported as a Na(+)-coupled cotransport system. We isolated many formycin A resistant mutants, and about half of them grew very poorly on adenosine as a sole source of carbon. Judging from their reversion frequencies, these mutants seemed to have single mutations. Respiration driven uptake of 14C-adenosine was not observed in such mutants; also, Na+ uptake induced by the addition of adenosine or formycin A to a cell suspension was completely abolished in them. Thus we conclude that these mutants possess a defect in the Na+/adenosine cotransport system, and have become formycin A resistant.     

表面活性剂的提纯与鉴定

简要综述了迄今使用表面活性剂纯化及鉴定方法,重点介绍了近几年以来的进展。     

Simultaneous Irradiation with Different Wavelengths of Ultraviolet Light has Synergistic Bactericidal Effect on Vibrio parahaemolyticus

Ultraviolet (UV) irradiation is an increasingly used method of water disinfection. UV rays can be classified by wavelength into UVA (320–400 nm), UVB (280‐320 nm), and UVC (<280 nm). We previously developed UVA sterilization equipment with a UVA light‐emitting diode (LED). The aim of this study was to establish a new water disinfection procedure using the combined irradiation of the UVA‐LED and another UV wavelength. An oxidative DNA product, 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG), increased after irradiation by UVA‐LED alone, and the level of cyclobutane pyrimidine dimers (CPDs) was increased by UVC alone in Vibrio parahaemolyticus. Although sequential irradiation of UVA‐LED and UVC‐induced additional bactericidal effects, simultaneous irradiation with UVA‐LED and UVC‐induced bactericidal synergistic effects. The 8‐OHdG and CPDs production showed no differences between sequential and simultaneous irradiation. Interestingly, the recovery of CPDs was delayed by simultaneous irradiation. The synergistic effect was absent in SOS response‐deficient mutants, such as the recA and lexA strains. Because recA‐ and lexA‐mediated SOS responses have crucial roles in a DNA repair pathway, the synergistic bactericidal effect produced by the simultaneous irradiation could depend on the suppression of the CPDs repair. The simultaneous irradiation of UVA‐LED and UVC is a candidate new procedure for effective water disinfection.     

广灭灵的制备、纯化及结构鉴定

合成了广灭灵农药粗品，采用柱层析分离技术，以硅胶为填料，乙酸乙脂：石油醚(沸点30℃-60℃)＝1：4为展开剂(Rt＝0．5)进行了纯化。用元素分析法(EAS)、熔点法(MDS)、紫外光谱法(UV)、红外光谱法(IR)、质谱法(MS)及核磁共振法(^1H NMR^13、^13C NMR)进行了结构鉴定及表征。     

Purification and Structure Identification of Hyaluronic Acid

Polysaccharide produced by mutated strain of Streptococcus zooepidemicus was purified by the procedures including Savage method, quaternary ammonium compound precipitation, DEAE-cellulose(DE52) chromatography and Sephadex G-75 gel filtration. The structure of the purified polysaccharide has been characterized by means of chemical composition analysis,13C NMR spectrum, infrared spectrum and circular dichroism (CD). All the results showed that the purified polysaccharide was hyaluronic acid (HA). The single helix conformation of the purified HA was determined by Congo red experiment. The molecular weight of the HA was about 1.16×106D, which was measured by viscosity method.     

Evaluation of different polymerase chain reaction methods for the identification of Vibrio parahaemolyticus strains isolated by cultural methods

Control of contamination by Vibrio parahaemolyticus in fishery products is often hampered by the lack of standardized methods and by the uncertainty associated with biochemical identification of the isolates. In this study, 5 polymerase chain reaction (PCR) methods for the identification of V. parahaemolyticus to the species level were evaluated by using 25 Vibrio reference strains and 163 isolates from fishery products, environmental sources, and clinical samples. Sequence targets of the methods were toxR, gyrB, and tlh genes (tested with 2 protocols), and the fragment pR72H. Isolate identification was confirmed by sequencing of the 16S rRNA gene and by PCR protocols for the identification of other Vibrio species. The PCR assay targeting the toxR gene achieved the highest performance (100% inclusivity and exclusivity). The 2 PCR protocols based on tlh gene detection, although showing the same inclusivity (100%), differed in the exclusivity (50 and 91%, respectively). Finally, the results provided by the PCR assays targeting the gyrB gene and pR72H fragment were less reliable and, in some cases, difficult to assess. According to the results of this study, the characteristics of accuracy expressed by the toxR identification method make it a suitable candidate as a reference method for the molecular identification of V. parahaemolyticus strains.     

Rolling circle amplification based amperometric aptamer/immuno hybrid biosensor for ultrasensitive detection of Vibrio parahaemolyticus

Microchimica Acta - The authors describe an antibody-aptamer based hetero-sandwich amperometric biosensor for the foodborne pathogen Vibrio parahaemolyticus. Antibody on the surface of a gold...     

银杏叶提取物中染料木素的分离纯化及结构鉴定

对银杏叶提取物进行酸水解,然后利用正相硅胶柱色谱及重结晶法从银杏叶水解物中分离出染料木素,经紫外光谱（UV）、质谱（MSn）、核磁共振（1H NMR、13C NMR）等波谱学方法鉴定证实了其结构。高效液相色谱分析表明,所提纯的染料木素纯度达到了98%以上。     

DNA光解酶模型研究的近期进展

用模型化合物来模拟DNA光解酶与底物的作用, 有助于认识DNA光复活作用机理. 评述了环丁烷型嘧啶二聚体光解酶和(6-4)光产物光解酶的模型研究进展, 并展望了该领域的发展前景．     

A disposable amperometric enzyme immunosensor for rapid detection of Vibrio parahaemolyticus in food based on agarose/Nano-Au membrane and screen-printed electrode

Recent outbreaks of foodborne illnesses continue to support the need for rapid and sensitive methods for detection of foodborne pathogens. A disposable electrochemical immunosensor for detection of Vibrio parahaemolyticus (VP) based on the screen-printed electrode (SPE) coated with agarose/Nano-Au membrane and horseradish peroxidase (HRP) labeled VP antibody (HRP-anti-VP) has been developed in this paper. Then, the immunosensor was characterized by cyclic voltammetry (CV) and laser scanning confocal microscope (LSCM). The immunosensor was incubated with the one-step immunoassay format involving VP for 30 min at room temperature (25 ± 0.5 °C). The access of the active center of HRP catalyzing the oxidation reaction of thionine by H₂O₂ was partly inhibited by VP, which connected on the surface of the immunosensor by immunoreaction. VP could be quantificationally detected according to the shift of reduction current while CV was used as electrochemical means to detect the products of the enzymatic reaction. Under the optimum conditions of immunoreaction and electrochemical detection, VP was rapidly detectable by sigmoidal curve method and form a linear calibration between 10⁵ and 10⁹ cfu/ml with an associated detection limit of 7.374 × 10⁴ cfu/ml (S/N = 3). The immunosensor had acceptable specificity, reproducibility, stability and accuracy, indicating that the immunosensor could satisfy the need of practical sample detection.     

莲子草假隔链格孢毒素的分离纯化与结构鉴定

从莲子草假隔链格孢(Nimbya alternantherae)中分离纯化了一种毒素物质, 并对其进行了结构鉴定, 为研究其致病机理及进一步的化学合成奠定了基础.     

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.