碱-酶法与酸-酶法水解处理铬革屑的比较研究

碱-酶两步法水解铬革屑试验结果显示：在碱用量较少且水解温度较低的条件下，处理铬革屑的水解率比单独使用MgO提高62％，比碱-酶-步法提高30％。在两步法中，每次加酶前将浴液的pH值调至酶的最适pH有助于酶活力的充分释放。碱-酶两步法试验中，碱性蛋白酶D处理铬革屑的水解率最高，达到了88．29％，水解液的铬含量为242mg／kg。酸-酶两步法水解铬革屑试验结果显示：草酸预处理后再分别用不同的酶分次水解铬革屑的水解率均在40％-50％，低于碱-酶两步法；水解液的铬含量在14107-16689mg／kg，远高于碱-酶两步法。碱-酶两步法结合处理明显优于酸-酶两步法。

酸-菌-酶法处理黄姜皂素生产废渣的工艺

通过正交试验,考察了酸-菌-酶法共降解黄姜皂素生产废渣的最佳糖化工艺条件。结果表明,皂素生产废渣先进行酸水解,再利用黄孢原毛平革菌对其中的木质素进行降解,最后进行酒精酶水解,得到这种酸-菌-酶法糖化工艺的最优条件为,蒸汽处理后的废渣先经液固比20∶1、稀硫酸(w=4%)、100℃的条件下酸水解120 min;再于30℃、pH6、加菌量100 mL.L-1的培养条件下利用黄孢原毛平革菌(Phanerochaete chry-sosporium)降解4 d;最后在酶用量0.9 mL、温度为50℃、pH4.8的工艺条件下酶解100 min。黄姜皂素废渣经该条件处理后,木质素去除率达42.82%,还原糖得率最高达59.72%,比采用酸-酶法水解后的还原糖得率提高了25.62%。这种酸-菌-酶法结合工艺可以更有效地降解皂素生产废渣,得到较高的还原糖量。

高效液相色谱法与L-蛋氨酸γ-裂解酶法在血清同型半胱氨酸含量检测中的应用

目的:比较高效液相色谱法与L-蛋氨酸γ-裂解酶法对血清同型半胱氨酸含量测定效果。方法:选取2013年12月-2015年9月于本院健康体检的体检者120例作为研究对象,将其分为高效液相色谱法组和L-蛋氨酸γ-裂解酶法组,每组60例。比较两组变异系数、回收率及准确度。结果:两组不同浓度批内和日间变异系数比较,差异均无统计学意义(P>0.05);两组不同浓度回收率比较,差异均无统计学意义(P>0.05);两组检测同型半胱氨酸含量比较,差异无统计学意义(P>0.05),L-蛋氨酸γ-裂解酶法组检测准确率为99.24%,高于高效液相色谱法组的89.63%,比较差异有统计学意义(P<0.05)。结论:在同型半胱氨酸含量的测定中,两组检测稳定性无明显差异,相对高效液相色谱法而言,L-蛋氨酸γ-裂解酶法检测准确率较高。在临床检验中,可以根据检测工作需求选择合适的检测方法。

酸—酶联用液化法制备小麦淀粉麦芽糖浆的工艺研究

以小麦淀粉为原料,利用盐酸、中温α-淀粉酶共同液化进行麦芽糖的制备。采用单因素实验结合正交实验法优化工艺参数。结果表明:酸—酶联用液化法的最佳工艺条件为:淀粉浆料液比(3∶10),5%盐酸添加量12 mL,酸化时间12 min,温度100℃,浓度为0.0143g/100mL的中温α-淀粉酶加酶量8ml,酶液化时间30 min,pH值5.4,温度64℃,所得液化液DE值为8.12%。通过无机酸与酶法共同液化小麦淀粉得到的液化液DE值控制在具体数值范围内,为以小麦淀粉为原料制备麦芽糖浆的工业生产提供了理论依据,也为下一步对小麦淀粉在板框过滤时出现的困难分析奠定基础。

Enzymatic Synthesis of Fatty Acid Methyl Esters from Crude Rice Bran Oil with Immobilized Candida sp. 99-125

The non-edible crude rice bran oil was extracted from white rice bran, and then was catalyzed by immobilized lipase for biodiesel production in this study. The effects of water content, oil/methanol molar ratio, temperature, enzyme amount, solvent,number of methanol added times and two-step methanolysis by using Candida sp. 99-125 as catalyst were investigated. The optimal conditions for processing 1 g rice bran oil were: 0.2 g immobilized lipase, 2 ml n-hexane as solvent, 20% water based on the rice bran oil mass, temperature of 40 °C and two-step addition of methanol. As a result, the fatty acid methyl esters yield was 87.4%. The immobilized lipase was proved to be stable when it was used repeatedly for 7 cycles.

A New (2+1)-Dimensional Integrable Equation

A new nonlinear partial differential equation （PDE） in 2＋1 dimensions is obtained from the mKP equation by means of an asymptotically exact reduction method based on Fourier expansion and spatio-temporal resealing. In order to demonstrate integrability property of the new equation, the corresponding Lax pair is obtained by applying the reduction technique to the Lax pair of the mKP equation.

Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

Activity and Stability of Arginine Deiminase for Producing L-citrulline

A novel Enterococcus faecalis strain designated N J402 was found with high activity of arginine deiminase （ADI）. The optimum condition for catalytic activity was determined in terms of temperature （about 40℃）, thermostability （available 37℃） and pH （6-7）. The effects of substrate and product concentration were studied. The effects of various metal ions added in reaction mixtures on the biocatalyst were investigated and ADI of N J402 was found to exhibit Co^2＋ dependence, different from previous reports. Surfactant, cetyl trimethyl ammonium bromide, was one of the most important keys for producing L-citrulline. The enzyme in resting cells possessed the quality of high stability for reuse.

Biosynthesis of 4-hydroxyphenylpyruvic acid from L-tyrosine using recombinant Escherichia coli cells expressing membrane bound L-amino acid deaminase

4-Hydroxyphenylpyruvic acid （4-HPPA）, a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to prod uce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase （mL-AAD）, which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide （twin-arginine translocation pathway, Tat） and integrated pelB （the general secretory pathway, Sec）-Tat signal peptide was determined and compared firstly, using two pET systems （pET28a and pET20b）. It was found that the Tat pathway （pET28a-mlaad） resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts BI21 （DE3） and 0.05 mmol. L- 1 IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol,L 1 4-HPPA was obtained from 100 mmol.L 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.

酸-酶两段法脱除橘瓣囊衣工艺研究

采用酸-酶两段法对橘瓣进行脱囊衣试验。通过单因素实验和正交实验,得到酸-酶两段法脱囊衣的优化工艺参数为:盐酸浓度0.20%、作用时间20min、果胶酶浓度1.0%、果胶酶作用温度45℃。在此条件下,经30min处理后,橘瓣的脱囊衣率达到90%以上;与传统酸碱方法相比,此方法废水COD值降低32%,用水量节约45%。

Effect of Enzymes in Buccal Mucous Membrane on Buccal Absorption of Insulin

To evaluate the effect of proteolytic enzymes on the absorption of insulin in the buccal mucosa, the trichloroacetic acid (TCA) method was used to estimate the degradation of insulin under different conditions in the buccal mucosal homogenates. In vivo experiments estimating the enhancement of hypoglycaemic effect by enzyme inhibitors were also conducted. The results showed that proteolytic enzymes in the buccal mucosa were less active than in the intestine. Bacitracin, aprotinin and sodium deoxycholate could inhibit the degradation of insulin in the buccal mucosal homogenates. The degradation of insulin in buccal mucosal homogenates of normal hamsters was smaller than that of diabetic hamsters. In vivo experiments of hypoglycaemia supported the in vitro results. When given buccally, bacitracin, aprotinin and sodium deoxycholate could increase the relative pharmacological bioavailability of insulin. When co-administered with aprotinin(0.1%), bacitracin(0.5%) and sodium deoxycholate(5%), the relative pharmacological bioavailabilities of insulin were 4.84%, 6.60% and 14.95% respectively. The in vitro and in vivo results suggest that proteolytic enzymes are present in the buccal mucosa, which limit absorption of insulin. Co-administration with some enzyme inhibitors can improve the bioavailability of insulin via buccal delivery and sodium deoxycholte is more efficient than some enzyme inhibitors used for improving buccal absorption.

The Value of the Measurement of Urinary Hyaluronic Acid Levels for the Diagnosis of Bladder Cancer

Objective To investigate the value of the measurement of urinary hyaluronic acid (HA) levels for the diagnosis of bladder cancer and the possibility of replacing ELISA-like assay with radioimmunoassay to detect the levels of urinary HA. Methods Using the ELISA-like assay and radioimmunoassay at the same time to measure the HA levels in the urine specimens from 49 bladder cancer patients, 12 benign bladder tumor patients, 30 other genitourinary disease patients and 20 normal controls. Results There is not much difference between the consequences of the urinary HA levels whether we used the ELISA-like assay or radioimmunoassay to detect every specimen (P>0.05). When we used the results with radioimmunoassay for analysis, we found the levels of urinary HA of bladder cancer patients were 2–4 times than those of the benign bladder tumor patients, other genitourinary disease patients or normal individuals (P<0.01); With 137.5 ngHA/mg protein (113.6±23.9 ng/mg) as a minimum cutoff limit, this assay had a good sensitivity (91.8%) and specificity (91.9%) for the diagnosis of bladder cancer. Its difference in sensitivity meant a lot when compared with urine cytology (48.9%,P<0.01). Conclusion The urinary HA assay is a simple, convenient, noninvasive credible and cheap method with satisfactory sensitivity and specificity for the diagnosis of bladder carcinoma; radioimmunoassay is also a good means to measure the urinary HA levels. Key words Bladder carcinoma - Hyaluronic acid - Urine

Research on Activity and Expression of Tyrosinase in Varicorhinus macrolepis

[Objective] This study aimed to analyze tyrosinase activity and its expression in Varicorhinus macrolepis. [Method] V. macrolepis was used as experimental material for the analysis and research of tyrosinase in nine kinds of organs and tissues of male and female V. macrolepis individuals by using polyacrylamide gel electrophoresis and biochemical staining method, spectrophotometry and enzyme histochemical technology. [Result] Tyrosinase exists in the liver and pancreas, intestine and spleen of female and male V. macrolepis and in the gallbladder of male V. macrolepis. Tyrosinase activities in various tissues of V. macrolepis varied largely. Specifically, tyrosinase activities in the spleen was the maximum, which was higher in female V. macrolepis than in males. According to the enzyme histochemistry results, strong positive signals of tyrosinase existed in the spleen, intestine, liver and pancreas and gallbladder of V. macrolepis, which was the strongest in the spleen. [Conclusion] In this paper, research on tissue localization of tyrosinase in V. macrolepis had been first reported, which provided theoretical basis for further exploring the functions of tyrosinase in V. macrolepis.

A Simplified Method for Purifying Osteoclasts from Human Giant Cell Tumor of Bone

Objective: To purify and identify the osteoclasts from the tissue of humangiant cell tumor of bone. Methods: We have developed a new method that allows the purification oflarge numbers of authentic osteoclasts (OCs). The OCs were isolated from tissue of human giant celltumor of bone by 0.25% trypsin and collagenase. We characterized OCs in terms of the expression ofdifferent phenotypic markers of OCs. The phenotypic markers of OC included Tartrate-resistant acidphosphatase staining (TRAP). The expression of calcitonin receptor (CTR), cathepsin K and receptoractivator of necrosis factor κB (RANK) mRNA were examined by RT-PCR. Results: The OC cell purifiedby above method functioned normally in vitro. The purity was about 79.7%. They showed the normalosteoclast phenotypes markers of OC. Conclusion: The method provides a system for performingbiochemical and molecular studies of OCs. The study indicates that the method of purifying theosteoclasts from human GCT cell can be used for research of bone metabolism.

Role of receptor tyrosine kinases in gastric cancer: New targets for a selective therapy

Receptor tyrosine kinases （RTKs） such as the epidermal growth factor receptor family participate in several steps of tumor formation including proliferation and metastatic spread. Several known RTKs are upregulated in gastric cancer being prime targets of a tailored therapy. Only preliminary data exist, however, on the use of the currently clinically available drugs such as trastuzumab, cetuximab, bevacizumab, gefitinib, erlotinib, and imatinib in the setting of gastric cancer. Preclinical data suggest a potential benefit of their use, especially in combination with ＂conventional＂ cytostatic therapy. This review summarizes the current knowledge about their use in cancer therapy as well as new approaches and drugs to optimize treatment success.

Increased fatty acid synthase as a potential therapeutic target in multiple myeloma

Objective： To determine fatty acid synthase （FAS） expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma. Methods： FAS expression was determined by immunohistochemistry, reverse-transcription polymerase chain reaction （RT-PCR） and immunoblot analysis in bone marrow samples obtained from 27 patients with multiple myeloma （MM patients） and peripheral blood mononuclear cells （PBMCs） obtained from 12 healthy donors In parallel, additional analyses were performed on 2 human multiple myeloma cell lines, U266 and RPM18226. U266 cells were treated with cerulenin at various concentrations （5 to 320 μg/ml） for 24 h, and metabolic activity was measured by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assays. Apoptosis was evaluated by dual Annexin V/Pl （propidium iodide） labeling and flow cytometry （FCM） in U266 cells treated with 20 μg/ml cerulenin for 12 h or 24 h. Results： By immunohistochemistry, we found that 19 of 27 bone marrow samples obtained from MM patients expressed significantly high levels of FAS. Similarly, by RT-PCR, 22 of 27 bone marrow samples obtained from MM patients, U266 and RPM18226 showed FAS expression, whereas PBMC samples from 12 healthy donors did not express detectable level of FAS. FAS protein expression was confirmed by immunoblot analysis in 16 of 27 bone marrow samples obtained from MM patients, U266 and RPM18226 cell lines, and no FAS protein expression was detected in PBMC samples from 12 healthy donors. U266 cells were highly sensitive to cerulenin treatment, with a dosage-related effect on metabolic activity, as a measure for cell proliferation. U266 cells treated with 20 μg/ml cerulenin for 12 and 24 h also showed early sign of apoptosis with 56.9% and 69.3% Annexin V^＋/Pl cells, and late apoptotic and necrotic cells with 3.2% and 17.6% Annexin V^＋/Pl^＋ cells. Conclusion： Increased FAS expression existed in multiple myeloma samples and human myeloma cell lines. Cerulenin greatly inhibited metabolic activity/cell proliferation of U266 cells and induced apoptosis, suggesting that FAS is an effective target for pharmacological therapy in human multiple myeloma.

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

Advances in Research on Hepatitis B Virus DNA Integration

Since HBV DNA integration was discovered for the first time in 1980, various methods have been used to detect and study it, such as Southern Blot, in situ hybridization, polymerase chain reaction and so on. HBV DNA integration is thought to be random on the whole although some hot spots of integration were described by some researchers, one of which might be the repetitive sequences of the genomic DNA. Besides, DNA damage, especially double-strand breaks could promote HBV DNA integration into host genome. HBV DNA integration into cells may damage the stability of the genome, cause DNA rearrangement, promote DNA deletion and induce the formation of HCC.

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.

Effect of An-pressing manipulation on the serum levels of T-AOC and CK-MM in volunteers with delayed onset muscle soreness in biceps brachii

Objective：To observe the effect of An-pressing manipulation on biceps brachii with delayed onset muscle soreness （DOMS） in healthy male volunteers.Methods：A total of 30 male college student volunteers were randomly divided into a blank group,a model group and a treatment group,10 cases in each group.Subjects in the blank group did not receive any intervention;subjects in the model group received active weight-bearing eccentric exercise on the non-favored side of the upper limb to establish the models,while not receiving any treatment;subjects in the treatment group received both the same modeling and An-pressing manipulation treatment.The subjective rating of perceived exertion （RPE）,subjective soreness sensation threshold and soreness grade were evaluated before modeling,immediately after modeling,and 24,48,72,96 and 120 h after modeling.Serum total antioxidant capacity （T-AOC） was measured before modeling,immediately after modeling,and 24,48 and 72 h after modeling.Serum creatine kinase MM isoenzyme （CK-MM） was measured before modeling and 24,48 and 72 h after modeling.Results：At 24,48,72 and 120 h after treatment,the soreness grades of the treatment group were lower than those of the model group （all P〈0.05）.The RPE scores of the treatment group were lower than those of the model group （all P〈0.05） immediately after modeling,at 24,48,72,96 and 120 h after modeling.The subjective soreness sensation threshold of the treatment group was higher than that of the model group immediately after modeling,at 24,48,72 and 96 h after modeling （all P〈0.05）.Immediately after modeling,T-AOC value in the treatment group was higher than that in the model group and blank group （both P〈0.05）.CK-MM of the treatment group was lower than that of the model group at 48 h and 72 h after modeling （P〈0.05）.Conclusion：An-pressing manipulation shows a certain therapeutic effect on biceps brachii with DOMS by strengthening the body＇s antioxidant and anti-damage abilities,which can effectively reduce the pain and accelerate the recovery from fatigue damage.