细胞中酪氨酸酶活性的生化酶学法测定

根据HideyaA .和WrathalJ.介绍的方法 ,建立了细胞中酪氨酸酶活性测定的改进型生化酶学方法 ,以适应美白化妆品研究开发的需要。结果显示 :改进后的方法 ,精确度测试的变异系数均值为 6 2 % ,准确度测试P值 >0 0 5,稳定性测试 ,方差分析无显著性差异。

Effect of Different Pretreatment Methods on Enzymatic Saccharification and Ethanol Production from Sugarcane Shoots and Leaves

Sugarcane shoots and leaves consist of 35.2% cellulose, 23.43% hemicellulose, 12.6% lignin and 6.59% ash on dry solid （DS） basis and have the potential to serve as low cost feedstocks for ethanol production. To improve the enzymatic digestibility of these biomass and bioethanol production, three pretreatment methods had been investigated and compared, including： （1） 2% w/v NaOH solution autoclaving pretreatment; （2） 2% w/v H2SO4 solution autoclaving pretreatment and （3） two steps of 2% w/v NaOH solution autoclaving followed by 2% w/v H2SO4 solution autoclaving pretreatment. Among them, the best result for ethanol production was obtained when 15 g DS of sugarcane shoots and leaves was pretreated by using two step of 2% w/v NaOH solution autoclaving followed by 2% w/v H2SO4 solution autoclaving. The highest ethanol concentration 30.40 g/L （92.65% in fermentation efficiency） was obtained from reducing sugar 89.25 g/L at 48 h. Moreover, the washing step of solid residue after pretreatment could reduce furfural and hydroxymethylfurfural （HMF） in all pretreatment methods when compared to unwashing solid residue after pretreatment.

Biodiesel Production by Enzymatic Catalysis Process Using Two Analytical Ways： Gas Chromatography and Total Glycerol Determination

Currently, biodiesel is presented as one of the best alternatives for gradually replacing the use of fossil fuels, but it has some factors that make it economically impractical if it does not have a government support. For this reason, research efforts focused on this area have been responsible for optimizing the process of biodiesel production by different catalytic routes to achieve greater efficiency at a lower cost. In this case, the biggest problem has been the high cost generated by an investigation, which in many occasions is the main factor to decide if an investigation could be carried out. Trying to reduce these costs, in the current study, we are using a technique of glycerol quantification by volumetric methods and comparing obtained results with the chromatographic method, which is conventionally used and comparatively much more expensive. Biodiesel employee was obtained by an enzymatic catalysis process varying one of three process variables：oil：alcohol molar ratio, temperature and proportion of catalyst. The numerical differences obtained between the two quantification methods generated relative errors lower than 10%, resulting in some occasions lower than 1%. By gas chromatography analysis the best yield was obtained at the same conditions of the volumetric method, a temperature of 45 ℃, an oil：alcohol ratio 1：4 and 8 wt.% of catalyst, but a yield of 95.5% and 97.1%, respectively. Due to the high precision of gas chromatography, this method is used to carry out a surface response analysis obtaining as ideal operating conditions a temperature of 43.5 ℃, 8.9 wt.%. of catalyst and an oil：alcohol ratio 1：4.

Phenotypic and Genotypic Comparison of Pseudomonas stutzeri in Freshwater Fish in Indonesia

Pseudomonas stutzeri caused an outbreak of freshwater fish in Luwuk Banggai （tilapia and catfish）, Bali （tilapia）, Jambi （tilapia and catfish） and Tanjung Pinang （catfish）. The study was purposed to comprehensively identify special phenotypic and genotypic characteristics of P. stutzeri isolated from several areas in Indonesia, including its morphometric and biochemical characteristics and molecular variation. Bacteria were isolated from internal organs （kidney, ulcer and eye） of fish. They were then identified using morphology and biochemical test. DNA isolates were entirely extracted, amplified and reversed on 16S rRNA region, and further then were sequenced. Phylogenetic trees of bacteria were constructed using neighbor-joining and maximum-parsimony methods. The colony were similar, such as rod shape （Jambi, Tanjung Pinang, Bali）, bacil shape （Luwuk Banggai）, transparant in tryptic soy agar （TSA） （Luwuk Banggai）, creamy beige in glutamate starch phenol red （GSP） （Bali）, gram negative, motile, no reaction in the oxidative-fermentative test, positive result in catalase and oxidase test, negative in lysine decarboxylase and ornithine decarboxylase test and positive result in indole test; gelatin was degraded （only Bali）, urea was not degraded, no color change in Methyl-red and Voges-proskaeur （MR-VP） test; acid not produce from glucose, inositol or sucrose. Citrate was utilized by some isolates： positive （Jambi, Tanjung Pinang） and negative （Bali, Luwuk Banggai）. Results showed us that isolates of Jambi, Bali and Tanjung Pinang were monophyletic species with P. stutzeri $8 and ZH-1 comparing to gen bank. However, merely phenotypic analysis among Pseudomonas sp. was confused compared to each other.

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis

AIM: To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. METHODS: uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a noncancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR)mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. RESULTS: The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. CONCLUSION: uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro , suggesting its potential as a therapeutic target.

Gold nanolabels and enzymatic recycling dual amplification-based electrochemical immunosensor for the highly sensitive detection of carcinoembryonic antigen

A sensitive electrochemical immunoassay system for the detection of a protein tumor biomarker through a dual amplified strategy was reported. Firstly, this protocol involves in the electropolymerization of o-aminobenzoic acid (o-ABA) on a glass carbon electrode (GCE). Subsequently, capture anti-CEA (Abl) is covalently linked to poly(o-ABA) (PAB) film, via N-(3-dimethylamminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimid sodium salt (NHS) activation of the carboxyl groups and surface blocking with ethanolamine. Later, the target, carcinoembryonic antigen (CEA), is sandwiched between an electrode surface confined Ab1 and the alkaline phosphatase-labeled signal anti-CEA antibodies conjugated with gold nanoparticles (Ab2-ALP-AuNP bioconjugates). The dual biocatalytic signal amplification for CEA monitoring is achieved by coupling the numerous enzymes loaded on the AuNPs with redox-recycling of the enzymatic products in the presence of the secondary enzyme and the corresponding substrate. The novel dramatic signal amplification strategy, exhibits a good linearity at the studied concentration range from 0.005 to 50 ng mL-1 towards CEA with a detection limit of 2 pg mL-1 (S/N=3). There is a 5-100-fold improvement in detection limit compared to other similar studies. The developed dual signal amplified strategy shows good selectivity, regeneration, stability and acceptable reproducibility. Therefore, the signal amplification approach holds great potential applications in detection of ultra-trace protein biomarkers.