白屈菜总碱对舞毒蛾离体酶活性的影响

通过对白屈菜粗提物纯化获得白屈菜总碱,对舞毒蛾3龄幼虫5种离体酶活进行测定。测定结果表明:用AB-8大孔吸附树脂纯化白屈菜粗提物,得到5种生物单碱,含量从高到低依次为:黄连碱>白屈菜碱>盐酸小檗碱>血根碱>四氢黄连碱。纯化后的白屈菜总碱对5种离体酶活均有不用程度的抑制作用,对GST抑制作用最强,IC_(50)为5.333mg/mL,对CarE的抑制作用最弱,IC_(50)为3.475×10~3 mg/mL。白屈菜总碱对舞毒蛾3龄幼虫5种离体酶的敏感性依次为GST>AChE>CAT>SOD>CarE。

磁场影响离体酶和微生物体内酶催化活性的研究(摘要)

作为一种生物催化剂,酶是促进一切代谢反应的物质,在人类生产生活和生命过程中起着非常重要的作用。因此,酶学的研究始终是生物学的重大课题。近年来,随着生物磁学的发展,人们对磁场影响生物特性和生命活动的研究日益深入,已开始把目标转向与生命活动息息相关的物质——酶的研究,而且也转向遗传物质——染色体和 DNA 的研究。研究这两种影响具有十分重要而深远的意义,不仅可以从理论上探讨磁生物效应的机制,还将使磁生物效应在工农业生产及医疗、环保等方面得到更广泛的应用。

三种微生物离体氨基酸脱羧酶催化活性的研究

氨基酸脱羧酶(AAD)是生物胺合成的关键酶。微生物AAD一般为胞内酶,但性质稳定,在脱离细胞体系中仍可保持催化活性。文章以具有产生物胺能力的3种微生物细胞超声破碎后的粗酶液为对象,探究了前体氨基酸浓度、pH和盐浓度对离体AAD催化活性的影响。结果显示,多数情况下前体氨基酸未充分转化为生物胺,但低浓度前体氨基酸显著降低了离体AAD的催化活性;Lactobacillusplantarum 1-8和Escherichia coli BA-1的AAD在pH为6时催化活性最强,Debaryomyces hansenii MB-1Y的AAD在pH为5时催化活性最强;盐浓度提升导致AAD的催化活性降低。该研究可为探讨食品发酵中AAD的催化特性及其控制提供参考。

Total plasma homocysteine and methylenetetrahydrofolate reductase C677T polymorphism in patients with colorectal carcinoma

AIM: To investigate the behaviour of total plasma homocysteine (tHcy) and its most common genetic determinant defect, the methylenetetrahydrofolate reductase C677T (C677TMTHFR) polymorphism in patients with early stage colorectal carcinoma. METHODS: tHcy was quantified by Abbott IMx immunoassay; screening for C677TMTHFR substitution was performed by PCR and restriction analysis. RESULTS: The frequency of the C/T and T/T genotypes of the C677TMTHFR gene polymorphism did not differ between the groups. The mean tHcy was statistically higher in cancer patients than in control subjects carrying the same C/C or C/T genotype, whereas there was no difference in the T/T homozygous carriers of the two groups. tHcy was significantly higher in the T/T homozygous carriers than in C/C and C/T genotype carriers. CONCLUSION: The statistically significant increase of tHcy observed in C/C and C/T genotype carriers among our cancer patients is related to substrate consumption dependent on the tumor cell proliferation rate, whereas the tHcy increase observed in T/T genotype carriers of both groups probably depends on the enzymatic deficit of the homocysteine conversion to methionine and/or on the folate deficiency.

Biorefinery of bacterial cellulose from rice straw:enhanced enzymatic saccharification by ionic liquid pretreatment

The pretreatment of rice straw is often used to enhance the hydrolysis. 1-allyl-3-methylimidazolium chloride （ [ AMIM ] C1） is a kind of low viscous, nontoxic and recyclable ionic liquid. It was used to treat rice straw and improve the enzymatic hydrolysis of rice straw in this study. The factors influencing the pretreatment were as follows： the dosage of rice straw in [ AMIM ] Cl, crush mesh of rice straw, pretreatment temperature and time. After the pretreatment with a 3 % （the weight ratio of rice straw to ionic liquid） rice straw dosage in [AMIM]Cl at 110 ℃ for 1 h, the yield of reducing sugar of regenerated rice straw by 33 U/mL cellulase hydrolysis was 53.3 %, which was two times higher than that of un-treated rice straw （23.7 % ）. More researches regarding straw biorefinery to bacterial cellulose are being performed in the lab and prospective results will be published in near future.

Self-Assembled Monolayer of Lipoic Acid on Gold and Its Application to Rapid Determination of 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin

Lipoic acid (LA) has received great attention in the area of gold surface functionalization. In this study, LA was employed as a linker for the attachment of antibody to the gold surface of surface plasmon resonance (SPR) chip. By using this chip in a homemade SPR immunosensor, low molecular weight compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) can be detected at a low level of 0.01 ng/mL. There is a good linear relationship(R2 =0.943 1) between the results of SPR biosensor and enzyme-linked immunosorbent assay(ELISA).

Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis

Voltage-dependent anion channel （VDAC）I is the main channel of the mitochondrial outer membrane （MOM） and it has been proposed to be part of the permeability transition pore （PTP）, a putative multiprotein complex candidate agent of the mitochondrial permeability transition （MPT）. Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane （MIM）. Conversely, silencing VDAC1 ex- pression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and BcI-XL, indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome e release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDACl-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

Lipase-catalyzed Synthesis of Caffeic Acid Phenethyl Ester in Ionic Liquids： Effect of Specific Ions and Reaction Parameters

Caffeic acid phenethyl ester(CAPE)is a rare,naturally occurring phenolic food additive.This work systematically reported fundamental data on conversion of caffeic acid(CA),yield of CAPE,and reactive selectivity during the lipase-catalyzed esterification process of CA and phenylethanol(PE)in ionic liquids(ILs).Sixteen ILs were selected as the reaction media,and the relative lipase-catalyzed synthesis properties of CAPE were measured in an effort to enhance the yield of CAPE with high selectivity.The results indicated that ILs containing weakly coordinating anions and cations with adequate alkyl chain length improved the synthesis of CAPE.[Emim][Tf2N]was selected as the optimal reaction media.The optimal parameters were as follows by response surface methodology(RSM):reaction temperature,84.0°C;mass ratio of Novozym 435 to CA,14︰1;and molar ratio of PE to CA,16︰1.The highest reactive selectivity of CAPE catalyzed by Novozym 435 in[Emim][Tf2N]reached 64.55%(CA conversion 98.76%and CAPE yield 63.75%,respectively).Thus,lipase-catalyzed esterification in ILs is a promising method suitable for CAPE production.

Characterization of proteases from Planomicrobium sp. L-2 isolated from the gastrointestinal tract of Octopus variabilis (Sasaki)

A crude protease produced from Planomicrobium sp. L-2 is described, and its effectiveness as an additive in liquid detergent evaluated. We isolate the protease-producing Planomicrobium sp. L-2 from the gastrointestinal tract of Octopus variabilis. At least three caseinolytic protease clear bands were observed in zymogram analysis. The crude alkaline protease was highly tolerant of a pH range from 7.0 to 9.0, and temperatures to 50℃ after incubation for 1 h. Proteolytic enzymes were stable towards three surfactants （5% Tween 80, 1% Triton X-100 and 0.05% SDS） and an oxidizing agent （1% hydrogen peroxide）, in addition to being highly stable and compatible with popular commercial laundry powered detergent brands available in China. Our study demonstrates the potential these proteases have for development into novel classes of detergent additive. This study also suggests that the gastrointestinal tract of Octopus variabilis may be a rich source of commercially valuable strains of enzvme.

Orlistat-lnduced Modulation of Plasma Metabolites Independent of Weight Loss in Overweight Females

Orlistat-induced weight loss results in amelioration in several comorbidities including obesity-related dyslipidemia. The aim of the present study was to characterize the changes in the blood plasma metabolic profile from overweight women treated with Orlistat, a lipase inhibitor. A metabonomic approach employing LHNMR was applied to the access metabolic profile in lean and overweight women after a 120 day treatment with 120 mg of Orlistat three times daily. Twenty overweight women （BMI： 32.8 ± 2.9 kg/m2） were evaluated before and after Orlistat treatment and seven normal weight women （BMh 21.8 ± 1.4 kg/m2） were taken as control. After 120 days of treatment with Orlistat, no significant weight changes were observed. However, Orlistat-induced metabolic changes in overweight subjects decreased the profile differences between lean and obese individuals, independent of weight loss. These were associated to decreasing levels of lactate and calcium. Higher levels of lactate, alanine and lipids from overweight subjects were detected in comparison to lean individuals. These results show that a lipase inhibitor shifts the metabolic profile of overweight subjects towards normality, independent of weight loss.

Enzymatic saccharification of cellulose using an ionic liquid [Amim] [COOH] pretreatment

In this study we used l-allyl-3-methyl imidazole formate （[Amim][COOH]） as ionic liquid to pre-treat the cellulose and determined the rate of polymerization and enzymatic hydrolysis. The results showed that pretreatment with （[Amim][COOH]） significantly decreased the cellulose polymerization. As the pretreatment temperature went up, the enzymatic hydrolysis rate was first increased and then decreased The maximal enzymatic hydrolysis rate was achieved when the pretreatment temperature was 90 ℃. Under the ultrasonic condition, the initial rate of enzmatic hydrolysis for the ionic liquid-treated cellulose was up to 11.10 gL-1h-1, which was 33% increase compared to the untreated cellulose. Scanning Electronic Microscopy （SEM） and Fourier Transform Infrared-Raman Spectroscop （FT-IR） analysis showed that ionic liquidtreated cellulose started to depolymerize. In addition, the cr3＇stallinity of the cellulose was significantly decreased after pretreatment with ionic liquid.

Analysis of bulked segregants to identify molecular markers linked with cocoon weight and cocoon shell weight in the silkworm Bombyx mori L

Two silkworm strains viz, B20 A (high cocoon shell ratio) and C.Nichi (low cocoon shell ratio) were sib mated for 10 generations to determine the homozygosis. Both bulked segregant analysis(BSA) and near isogenic lines (NIL) studies were done to identify the RFLP markers closely linked to cocoon shell parameters. Three hundred and fifty two random clones were identified as the low copy number sequence and used for identification of Restriction Fragment Length Polymorphic (RFLP) marker linked to cocoon weight and cocoon shell character. In the bulk segregant analysis, DNA from the parents (B20 A, C.Nichi), F 1 and F 2 progeny of high shell ratio (HSR) and low shell ratio (LSR) were screened for hybridization with the random clones. Polymorphic banding pattern achieved through southern hybridization with different probes indicated the probable correlation of polymorphism with high and low cocoon shell character which are possible landmarks in identifying the putative marker(s) for the cocoon shell character. Out of the 100 probes tried with parents, F 1, F 2 and their bulks, 10 probes were found to be closely linked to cocoon shell characters.

Optimization the Cell Wall Degrading Enzymes and Technique for Isolation of Protoplasts in Potato

Plasma membrane of plant cells is surrounded by cellulose wall and adjacent cells are joined together by a thick pectin rich matrix. Separation of plant cells and removal of the cell wall experimentally, by either a mechanical or an enzymatic process, results in the production ofprotoplast. Protoplasts are useful tools to study the uptake and transport ofmacromolecules and production of somatic hybrids. Protoplasts can be obtained from all types of actively growing young and healthy tissues. The most convenient and widely used source of plant protoplasts is leaf. Juvenile seedling tissues, cotyledons are other alternative tissues most frequently used for protoplasts isolation. All the environmental and genotypic factors, which affect the cell wall thickenings and compactness indirectly, influence the number of protoplasts recovered. Protoplasts are isolated by two methods, mechanical and enzymatic. The enzyme mixture solution of celluiose/macerozyme is used to digest the cell wall. The critical factors affecting the obtaning ofprotoplasts are the kinds of cell wall degrading enzymes, the physiological state of plant leaves, the type of osmotic stabilizers and the composition of reaction solution. With the improvement of technique and enzyme combination rate, the yield of collected protoplasts will be increased higher.

Physiological traits of rice seedlings in response to inorganic arsenic

The aim of our study was to better understand the different responses of rice seedling to different species of inorganic arsenic As203 （As（Ill）） and Na2HAsO4 （As（V））. Our results indicate that the biomass of rice seedling decreased as arsenic concentration increased, with the decrease being more significant at higher arsenic concentrations. In addition, the analysis of superoxide dimutase （SOD）, peroxidase （POD）, and catalase （CAT） in rice roots and leaves showed that the activity of these three enzymes significantly decreased in rice tissues, especially in rice roots, as arsenic concentration was increased,. Further, the uptake and utilization efficiencies of N, P, and K were found to decrease as arsenic concentration was increased. However, the uptake and utilization efficiencies of P and K were mainly affected by As（IlI）, whereas those of N were mainly affected by As（V）. Inductively coupled plasma-mass spectrometry （ICP-MS） was used to assay arsenic accumulation in rice tissues; the results indicate that the arsenic content in rice tissues was enhanced when arsenic concentration was increased, especially in rice roots after arsenic treatment.

Multiplexed cytokine detection on plasmonic gold substrates with enhanced near-infrared fluorescence

Protein microarrays based on fluorescence detection have been widely utilized for high-throughput functional proteomic analysis. However, a drawback of such assays has been low sensitivity and narrow dynamic range, limiting their capabilities, especially for detecting low abundance biological molecules such as cytokines in human samples. Here, we present fluorescence-enhancing microarrays on plasmonic gold films for multiplexed cytokine detection with up to three orders of magnitude higher sensitivity than on conventional nitrocellulose and glass substrates. Cytokine detection on the gold plasmonic substrate is about one to two orders of magnitude more sensitive than enzyme-linked immunosorbent assay （ELISA） and can be multiplexed. A panel of six cytokines （Vascular endothelial growth factor （VEGF）, Interleukin 1β （IL-1β）, Interleukin 4 （IL-4）, Interleukin 6 （IL-6）, Interferon γ （IFN-γ）, and Tumor necrosis factor （TNF）） were detected in the culture media of cancer cells. This work establishes a new method of high throughput multiplexed cytokine detection with higher sensitivity and dynamic range than ELISA.

Enhanced refolding of lysozyme with imidazolium-based room temperature ionic liquids: Effect of hydrophobicity and sulfur residue

The expression of recombinant proteins in microorganism frequently leads to the formation of insoluble aggregates, inclusion bodies （IBs）. Thus, the additional in vitro protein refolding process is required to convert inactive IBs into water-soluble active proteins. This study investigated the effect of sulfur residue and hydrophobicity of imidazolium-based room temperature ionic liquids （RTILs） on the refolding of lysozyme as a model protein in the batch dilution method which is the most commonly used refolding method. When lysozyme was refolded in the refolding buffer containing [BF4]-based RTILs with a systematic variety of alkyl chain on cations varying from two to eight, less hydrophobic imidazolium cations having shorter alkyl chains were effective to facilitate lysozyme refolding. Compared to the conventional refolding buffer, 2 times higher lysozyme re- folding yield was obtained in l-ethyl-3-methylimidazolium tetrafluoroborate （[EMIM][BF4]） containing refolding buffer. The refolding yield of lysozyme was even more increased by 2.5 times when 1-butyl-3-methylimidazolium methylsulfate （[BMIM][MS]） containing sulfur residue on anion was used. The sulfur residue in [BMIM][MS] is supposed to improve the refolding yield of lysozyme which has 4 intrarnolecular disulfide bonds. For dilution-based refolding of lysozyme, the opti- mum concentrations of RTILs in refolding buffer were found to be 1.0 M [EMIM][BF4] and 0.5 M [PMIM][MS], respectively. The optimum temperate for dilution-based refolding of lysozyme with RTILs was 4 ℃.

Light-triggered plasmonic vesicles with enhanced catalytic activity of glucose oxidase for programmable photothermal/starvation therapy

Glucose oxidase(GOx)-based nanotheranostic agents hold great promise in tumor starvation and its synergistic therapy. Self-assembled plasmonic gold vesicles(GVs) with unique optical properties, large hollow cavity, and strong localized surface plasmon resonance, can be used as multi-functional nanocarriers for synergistic therapy. Herein,GOx-loaded GVs(GV-GOx) were developed for light-triggered GOx release as well as enhanced catalytic activity of GOx, achieving programmable photothermal/starvation therapy. Under near-infrared laser irradiation, the GV-GOx generated strong localized hyperthermia due to plasmon coupling effect of GVs, promoting the release of encapsulated GOx and increasing its catalytic activity, resulting in enhanced tumor starvation effect. In addition, the high photothermal effect improved the cellular uptake of GV-GOx and allowed an efficient monitoring of synergistic tumor treatment via photoacoustic/photothermal duplex imaging in vivo. Impressively, the synergistic photothermal/starvation therapy demonstrated complete tumor eradication in 4 T1 tumorbearing mice, verifying superior synergistic anti-tumor therapeutic effects than monotherapy with no apparent systemic side effects. Our work demonstrated the development of a light-triggered nanoplatform for cancer synergistic therapy.