磷酸缓冲盐和硫酸钠对洋葱假单胞菌脂肪酶的非水相激活(英文)

研究了kosmotropic型磷酸缓冲盐和硫酸钠对洋葱假单胞菌脂肪酶(Pseudomonas cepacia lipase,PCL)非水相催化性能的影响.以往磷酸缓冲盐被用来调控体系的pH值,其掺杂量对酶的催化活性无明显影响,而适量硫酸钠的掺杂则可有效提高酶在非水相的催化活性.本文研究发现,通过精确调控冻干过程,磷酸缓冲盐掺杂能够将PCL在有机相中的转酯化活性提高近10倍,达到其水相本征活性的50%,这一激活效果甚至高于硫酸钠掺杂.利用热重方法分析了盐掺杂PCL的含水量和蛋白结构,并将失重结果同其在有机相中的催化活性相关联,发现PCL在磷酸缓冲盐和硫酸钠掺杂下的催化构型与蛋白含水量及其周围盐环境具有不同的依赖关系.利用2-(4’-氨基-2’-羟基苯基)苯并恶唑作为荧光探针,研究了磷酸缓冲盐和硫酸钠掺杂的PCL悬浮于有机相时对荧光探针发射光谱的影响,发现盐掺杂酶制剂的存在能够大大增加荧光探针稳定于极性溶剂的构型含量,这可能与蛋白周围掺杂盐键和的水分子有关.如果用探针分子稳定于极性溶剂和非极性溶剂的构型比值间接表示悬浮酶制剂的极性结构,在正己烷体系中硫酸钠掺杂的PCL具有比磷酸缓冲盐掺杂的PCL大得多的极性,且酶制剂的极性大小与其非水相转酯化活性之间具有相似的变化趋势.上述研究结果表明,掺杂盐对粗PCL酶制剂的激活可能部分归因于掺杂盐键和的水分子在蛋白周围构筑的极性环境.

磷酸缓冲盐(PBS)片剂的研制

目的:为使缓冲盐这类常用试剂的配制过程简便。方法:制成缓冲盐片剂,分析比较片剂配制成的缓冲液与散剂配制的缓冲液的特性和作用。结果:两者具有同样的特性和作用。结论:此项研究为把PBS这类试剂制成片剂使用提供了依据。

Biological,antibacterial activities and electrochemical behavior of borided commercially pure titanium in BSA-containing PBS

The effects of boride coating on the bioactivity, antibacterial activity, and electrochemical behavior of commercially pure titanium(CP-Ti) in phosphate buffer solution(PBS) with bovine serum albumin(BSA) were studied. The grazing incidence X-ray diffraction(GIXRD) pattern confirmed the formation of a Ti B/Ti B2 coating via boriding process. Scanning electron microscopy(SEM) observation indicated that the Ti B2 cross-linked particles covered the Ti B whiskers. Water contact angle measurements revealed that boriding led to the formation of a surface with intermediate water affinity. Potentiodynamic polarization(PDP) assays demonstrated that the Ti B/Ti B2 coating had acceptable passivation behavior in BSA-containing PBS. Electrochemical impedance spectroscopy(EIS) measurements revealed that the passivation behavior of the CP-Ti and the borided samples was improved by increasing exposure time. Based on the Mott-Schottky(M-S) tests, it was realized that the charge carriers of passive films of both samples decreased with increasing exposure time in BSA-containing PBS. The bioactivity test results in a simulated body fluid showed that the Ti B/Ti B2 coating switched the CP-Ti from bioinert to bioactive material. Finally, the antibacterial activity test of the Ti B/Ti B2 coating against Escherichia coli and Staphylococcus aureus indicated 99% antibacterial activity.

Induction of Recombinant Uridine Phosphorylase and Its Application in Biosynthesis of Pyrimidine Nucleosides

Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase（UPase）,was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth.The lactose-induced UPase was stable at 50°C.Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol·L 1uridine and 5-fluorouracil in phosphate buffer（pH 7.0）catalyzed at 50°C for 1.5 h and the yield of 5-fluorouridine was higher than 68%.Under the optimum reaction conditions for production of 5-fluorouridine,5-methyluridine and azauridine were synthesized from uridine by pUDP,the yield was 61.7%and 47.2%respectively.Deoxynucleosides were also synthesized by pUDP,but the yield was only about 20%.

Comparison of 3 M^TM Petrifilm^TM Aerobic Count Plates with Pour Plates for Determination of Aerobic Plate Counts in Fermented Chile Mash

Fermented chile pepper mash is a major food product in New Mexico. There are few reports on the fermentation process or on methods to monitor it, In the current study we examined a pour plate procedure with an overlay using plate count agar and 3 MTM PetrifilmTM Aerobic Count （AC） plates for determination of total aerobic bacterial counts during the fermentation of chile mash. Fifty chile mash samples were obtained directly from commercial fermentation vats and examined within 2 h of collection. Serial dilutions of the chile mash were prepared in Butterfield＇s Phosphate Buffer. 1 mL portions of the diluted samples were aliquoted in duplicate onto the AC plates and into empty Petri dishes. Plate count agar was poured and once the plates had solidified, they were overlaid with about 10 mL of PCA to minimize spreaders. Plates were incubated at 30 ℃ for 48 h and enumerated. Paired difference tests were conducted on log transformed data to compare the results of the two plating procedures. For commercial chile mash samples, we did not show any significant differences between the AC plate counts and the pour plate counts （α = 0.05）, 3 MTM PetrifilmTM AC plates are a good alternative to pour plates for the determination of the total aerobic counts in fermented chile mashes.

The Effect of Calcium Chloride on the Microbiological Characteristics of Fermented Chile Pepper （Capsicum annuum cv. Mesilla Cayenne） Mash

Fermented chile pepper mash （Capsicum annuum cv. Mesilla Cayenne） is a major industrial food product in New Mexico. The fermentation of chile pepper mash depends on temperature, acidity, salt concentration, dissolved air, available carbohydrates and enzymes. The objective of this study was to evaluate the effects of the addition of calcium chloride （CaCI2） on the microbial characteristics of the pepper mash fermentation. Nine five gallon buckets were prepared with pepper that had been washed and ground by the manufacturer, each with 15% sodium chloride added. The buckets were allotted randomly to 1 of 3 treatments. The treatments included no CaCl2, 0.2% CaCl2 and 0.4% CaCl2 added to the pepper mash. Samples were stored at room temperature and sampled over a 3 months period to examine changes in the microflora. Chile mash samples were serially diluted in butterfield＇s phosphate buffer for microbial enumeration. Aerobic plate counts were conducted by pour plating with an overlay. Petrifilms were used for E. coil/coliform counts and Enterobacteriaceae. Yeasts and molds were surface plated on Rose Bengal Chloramphenicol agar while streptococci and lactobacilli were plated onto M17 and acidified MRS respectively. Aerobic plate counts, coliforms, E. coli, Enterobacteriaceae, yeast, mold, lactobacilli, and streptococci were not affected by CaCl2 level. The addition of 0.4% CaCl2 was found to have no effect on the chile pepper mash fermentation.

Impact of the diameter of SonoVue microbubbles on binding characteristics of a new contrast agent targeted to choriocarcinoma cells in vitro

Objective:To evaluate the impact of the diameter of SonoVue microbubbles on binding characteristics, including the adhesion rate and stability, of a new contrast agent targeted to choriocarcinoma cells(JARs) in vitro, in order to establish a foundation to explore targeted ultrasound imaging for localization of tumor cell antigens and increase the early diagnostic rate for tumors.Methods:The objects were divided into three groups:the large microbubble group(n = 15), the middling microbubble group(n = 15) and the tiny microbubble group(n = 15).The rosette formation rate was counted.JARs were calculated by flow cytometry(FCM).The targeted contrast agent was prepared by mixing SonoVue microbubbles of different diam-eter with rabbit anti-human chorionic gonadotrophin(HCG) antibody.The binding rates of the targeted contrast agent to JARs before and after PBS rinse were analyzed.Results:The binding rate was significantly lower in the large microbubble group(61.7 ± 1.8)% than in the middling microbubble group(82.6 ± 4.5)% and the tiny microbubble group(91.3 ± 5.8)%(P < 0.05).The binding rates of different diameter microbubbles to JARs before and after PBS rinse were different.The middling microbubbles were the most stable ones, with the binding rate of(82.3 ± 4.5)% and(80.4 ± 3.9)% before and after PBS rinse(P > 0.05).The binding rates of the targeted microbubbles labeled with fluorescence to JARs were 68.6%, 81.3% and 89.3% in the large microbubble group, the middling microbubble group and the tiny microbubble group, respectively(P < 0.05).Conclu-sion:The binding capacity of the targeted SonoVue microbubbles to JARs is related to the diameter of the microbubble, which is determined by the shaking method before preparation.Modulating the diameter of SonoVue microbubbles may increase the binding rate and stability of targeted microbubbles to JARs, thus to improve the image of JARs.

Square Wave Voltammetric Behavior of N,N-DiethyI-P-Nitroso Aniline an Indirect Method for Determination of Chlorpheniramin Maleate, Application of Pharmaceutical Formulations

The electrochemical behavior of N,N-diethyl-p-nitroso aniline was carried out using SWV （square wave voltammetric） at HMDE. A well defined reduction peak was observed at （-0.214） volt versus the reference electrode （Ag/AgC1/sat. KCI）, calibration curve was constructed in phosphate buffer （pH = 7.0）, the relationship is linear within the concentration range 1.283 × 10.5 M - 3.66 × 10.5 M with the correlation （R = 0.9923）. The serial addition ofCPM （chlorpheniramine maleate） leads to the decrease in the reduction current peak （Ip）, quantitatively, the plot of Alp （Ip - Ip） where, Ip： Peak current of N, N-diethyl-p-nitroso aniline alone, lp： peak current of N,N-diethyl-p-nitroso aniline in the presence of CPM, versus concentration is linear within the concentration range 0.984 × 10-6 M - 9.756 × 10-6 M, the correlation coefficient was 0.9954. The method was successfully applied to determine CPM in different types of pharmaceutical formulations, and compared with standard method from British Pharmacopeia [1].

Electrochemical Determination of Reduced Glutathione at Multiwalled Carbon Nanotubes/Poly(bromocresol green) Modified Glassy Carbon Electrode

A multiwalled carbon nanotubes/poly(bromocresol green) modfied glassy carbon electrode (MWNTs-PBG/GCE) was used to investigate the electrochemical behavior of reduced glutathione(GSH). GSH showed an irreversible oxidation process on MWNTs-PBG/GCE with an oxidation peak at 0.77 V(vs. SCE) in a phosphate buffer solution(pH=4.0). The kinetic parameters of the electrochemical behavior of GSH on MWNTs-PBG/GCE were calculated. Under the optimal conditions and with the help of amperometric method, a linear relationship was obtained between the oxidation peak current and GSH concentration in the range from 2×10-7 mol/L to 5×10-6 mol/L with the detection limit as 1×10-8 mol/L(signal-to-noise ratio of 3). The current reached the steady-state current within about 5 s. The modified electrode surface had very good reproducibility and stability.

Effect of tumor suppressor in lung cancer-1 on growth inhibition of MG63 cell line

Objective： The aim of this study was to establish the osteosarcoma cell sublines which stably expressing tumor suppressor in lung cancer-1 （TSLC1） gene and evaluate its effect on growth inhibition of human osteosarcoma cell line MG63. Methods： The recombinant plasmid pCI-TSLC1 was stably transfected into MG63 cells with Lipofectamine 2000. The posi- tive clones were developed by selection by G418. Biological characteristics of one of the 6 cell lines which highly expressing TSLC1, namely, the M8T were studied. Cell growth was analyzed with MTT assay. 2 x 10^7cells suspended in 0.2 mL phosphate buffered saline （PBS） were injected into the two flanks of 5-6-week-old female BALB/C nu/nu athymic nude mice. The volumes of subcutaneous of tumor growth were evaluated and calculated by the formula V= Length x Width x Height x 0.5 once a week. Results： The MST cell subline which stably expressing TSLC1 was characterized by Western blot. The genetic stability and purity of M8T cells were stable. TSLC1 significantly suppressed the growth of M8T cells in vitro. Moreover, the tumorigenicity of MST cells was suppressed in vivo. Conclusion： The osteosarcoma cell sublines MST which stably expressing TSLC1 had been successfully established. The ability of growth and metastasis of MST was significantly suppressed both in vitro and in vivo.

Kinetics of Desolvation of the Glycation of Hemoglobin Catalyzed by Buffer Phosphate and Arsenate

The glycation of hemoglobin is catalyzed by buffer phosphate and arsenate. The catalytic constant （kB） for aqueous arsenate is two-fold larger than for aqueous phosphate. The catalytic constant （ks） of phosphate in sorbitol mixtures increase from （1.67 ± 0.11） × 10-10 s-1·M-1 to （5.78 ± 0.39） × 10-10 s-1·M-1 and the catalytic constant is enhanced 3.5 times, relative to that in water; the catalytic constant （kB） of arsenate in sorbitol mixtures increase from （2.98±0.07）× 10-10 s-1·M-1 to （6.62 ± 0.53） × 10-10 s-1·M-1 and the catalytic constant is enhanced 2 times, relative to that in water. The spontaneous rate constants are independent of sorbitol concentration for phosphate and arsenate. The catalytic power of phosphate and arsenate in sorbitol are the same. Desolvation of strongly hydrated species such as HPO42 and HAsO42 should make a contribution to the energy cost of the formation of anion-hemoglobin complexes and can be a possible explanation for higher catalytic potential of HAsO42 in water. The same catalytic constant （ksB） for phosphate and arsenate in sorbitol indicates that the same catalyst base group on the hemoglobin molecule may be involved in the abstraction of proton in the Amadori rearrangement.

Development of Co-agglutination Method for Viral Nervous Necrosis from Grouper （Epinephelus sp,） in Batam

Viral nervous necrosis （VNN） has been reported to infect larvae and juvenile of humpback grouper, and until now, this virus is one of main problem for humpback grouper. Co-agglutination test proved to be a simple, rapid and reliable diagnostic test suitable for use in the field or laboratory without any special apparatus. The study aimed to study application of a co-agglutination test using Staphylococci sensitized with specific antibody for the diagnostic of VNN in grouper. First, brain and eye organ samples from diseased fish are homogenized with phosphate buffer saline （PBS） of pH 7.2. Then, the supernatant is collected after centrifugation at 8,000 rpm for 20 min, and finally one drop of the supematant and one drop of anti VNN antibody sensitized Staphylococci suspension are mixed on a glass slid and observation of the agglutination is performed after 5-10 min. The result shows that co-agglutination technique detects positive VNN in the brain and eye samples. The co-agglutination technique may provide valid result in a very short time as compared with complex method, such as enzyme-linked immunosorbant essay （ELISA） and fluorescient antibody technique （FAT） that require a high cost. Thus, this test can detect VNN faster, simple and more economic.

Purification and Antioxidant Activities of Phycocyanin from Spirulina Platensis

This study aimed to purify and determine antioxidant activities of different fractions obtained during the purification process of phycocyanin from Spirulina platensis. The dried powder of Spirulina platensis, after ground with sands, was extracted with 50 mM sodium phosphate buffer at pH 6.8 before centrifuged to precipitate unwanted proteins. Then the supernatant was separated by celit column to obtain semi-pure phycocyanin and further purified by treated with ammonium sulfate. The purity of phycocyanin was monitored by measuring the absorbance spectrum from 200 to 700 nm. Its purity ratio A620A280 was determined. The antioxidant activities of the obtained phycocyanin were determined by 2,2＇-azino-bis（3-ethylbenzthiazoline-6-sulphonic acid） （ABTS） assay and lipid peroxidation （linoleie acid） assay. The purity ratio of phycocyanin in the Spirulina crude extract was 0.36 and increased to 2.68 after purification. The fraction with the highest purity ratio of phycocyanin demonstrated the hightest antioxidant activities. For ABTS assay, it presented the Vitamin C Equivalent Antioxidant Capacity （VCEAC） value of 0.0405 ±0.0002 mg of ascorbic acid/mg of sample and the Trolox Equivalent Antioxidant Capacity （TEAC） value of 0.0485 ±0.0002 mg oftrolox/mg of sample respectively. The result from lipid peroxidation assay exhibited IC50 value of 5.9336 ±0.2565 mg/mL. The purification of Spirulinaplatensis crude extract obtained from this study increased the purity ratio of phycocyanin and its antioxidant activities. This will be further investigated for the development into anti-aging cosmetic products.

Study on in Vitro Degradation of Sutures for Decomposable Esophageal Stent

PLLA and PGLA sutures for decomposable esophageal stent were investigated in phosphate buffer solution （PBS） （pH=7.4） at 37 ℃ for a period of 8 weeks. In vitro degradation was studied by determining the change of weight loss, pH value, intrinsic viscosity, tensile strength, orientation degree, degree of crystallinity, melting point and surface morphology of the suture samples. The results showed that all properties of PLLA sutures had no obvious changes, however, the properties of PGLA sutures all changed significantly. The pH value, intrinsic viscosity, tensile strength, orientation degree and degree of crystallinity decreased gradually, and the weight loss of PGLA sutures increased with the degradation time. At 6th week, tensile strength of PGLA sutures nearly reached O, and weight loss approached to 70% at 8th week. The results of DSC showed that melting point of crystalline region of PGLA sutures substantially remained unchanged and melting heat enthalpy increased gradually during in vitro degradation, and the new ordered regions appeared in the amorphous area. The results of SEM showed that surface coating of PGLA sutures spalled initially, and then the sutures occured transverse rupture. Therefore, PGLA suture is suitable to prepare decomposable esophageal stent to expand benign esophageal stenosis or stricture, but stent prepared by PLLA suture is not appropriate for the treatment of benign esophageal stenosis because it is decomposed for more than 2 months.

Combination of ribavirin and reduning protects mice against severe pneumonia induced by H1N1 influenza a virus

OBJECTIVE:To investigate the effects of ribavirin administration combined with Reduning in a mouse model of influenza A(H1N1)-induced severe pneumonia.METHODS:Influenza A/Beijing/501/2009(H1N1)-infected C57BL/6 mice were randomly divided into four experimental groups treated with either a mock injection of phosphate-buffered saline(PBS),ribavirin(66.6 mg/kg daily)or Reduning(86.6 mg/kg daily),or a combination of both,for 7 days.Mice were monitored for clinical signs and survival,and body weight was measured daily for 14 days.Virus titer,lung wet-to-dry ratios,pathology and cytokines including interleukin(IL)-6,IL-10,and interferon(IFN)-γwere assayed on different days.RESULTS:In the untreated group injected with phosphate buffer saline,all the mice died of the infection.The survival rate of mice treated with Reduning was only 10%,whereas 100%of the ribavirin-and the combination-treated mice survived.Low lung viral loads indicated that ribavirin significantly inhibited virus replication,whereas Reduning did not.Lung wet-to-dry ratios demonstrated that both ribavirin and Reduning,administered together or separately,reduced acute lung edema compared with results in the untreated group.Pathology analyses also showed that treatment with a combination of both drugs relieved pathological lesions,whereas the single drug treatment did not.Levels of IL-6,IL-10 and IFN-γin mice treated with ribavirin or the combination of both ribavirin and Reduning were all significantly lower than in the untreated group,especially in the combination-treated group.In addition,Reduning administration significantly decreased both IL-6 and IL-10production but had no effect on IFN-γ.CONCLUSION:Due to the synergistic effect of antiviral and antiinflammation,the combination of ribavirin and Reduning could be an effective treatment for severe H1N1 which was considered to be significant to delayed antiviral and drug resistant.

Investigation of flexible electrodes modified by TiN,Pt black and IrO_x

TiN, platinum （Pt） black and iridium oxide are introduced to the stimulating sites to improve the performance of the flexible electrode. Low temperature process is used to fabricate the modifying films. TiN is coated on the gold sites by magnetron sputtering while platinum black and iridium oxide are coated by electroplating and electrodeposifion, respectively. The impedance of the electrode decreases dramatically after modification. The combined analysis of surface morphology and cyclic voltammograms （CV） in phosphate buffer saline （PBS） solution indicates that the modified electrode sites have larger electrode-electrolyte capacitance and smaller faradic resistance than unmodified sites, thus they have smaller electrochemical impedances.

Experimental study on rehydration conditions of freeze-dried platelets

A rehydration process for freeze-dried human platelets was studied on 1 ml of samples. The effects of prehydration duration, prehydration temperature, an rehydration solution on the recovery rate, mean platelet volume （MPV）, and platelet distribution width （PDW） were investigated. The mass changes during the prehydration process were also studied. Three prehydration durations： 0, 1.5, and 3.5 h, and two rehydration solutions： platelet-poor plasma and phosphate-buffered saline （PBS）, were tested. It was found that： （1） the prehydration was of significance; （2） 1.5 h of prehydration had better effects than 3.5 h of prehydration; （3） as a rehydration solution, the platelet-poor plasma behaved better than the PBS. The impacts of prehydration duration and temperature on the results were studied. There was almost no difference between 35 and 37 ℃. Among all the prehydration durations tested, 15, 30, 60, 90, and 120 min, the best result was achieved with the time duration of 15 min. The weights of prehydrated platelets at the end of each test were measured and the water contents were calculated. After 15 min ofprehydration, the water contents in the samples were about （4.8±0.01）% and （5.27±0.29）% （w/w） corresponding to the conditions of 35 and 37 ℃, respectively. These results will be helpful for further studies on the freeze-drying of mammalian cells.

Local effect of celecoxib on peripheral nerve repair com- bined with silicone tubulization in rat

Objective： To assess local effect of celecoxib on nerve regeneration in a rat sciatic nerve transec- tion model. Methods： Forty-five male healthy white Wistar rats were randomly divided into three experimental groups （n= 15 for each）： sham-operation （SHAM）, control （SIL） and celecoxib treated （SIL/CLX） groups. In SHAM group after anesthesia left sciatic nerve was exposed and after homeo- stasis muscle was sutured. In SIL group the left sciatic nerve was exposed in the same way and transected proximal to tibioperoneal bifurcation leaving a 10 mm gap. Proximal and distal stumps were each inserted into a silicone tube and filled with 10 gl phosphate buffered solution. In SIL/CLX group defect was bridged using a silicone tube filled with 10 μl celecoxib （0.1 g/L）. Results： Functional study and gastrocnemius muscle mass confirmed faster and better recovery of regenerated axons in SIL/CLX than in SIL group （P〈0.05）. Morphometric indices of regenerated fibers showed number and diameter of the myelinated fibers in SIL/CLX were significantly greater than those in control group. In immunohistochemistry, lo- cation of reactions to S-100 in SIL/CLX was clearly more positive than that in SIL group. Conclusion： Response to local treatment ofcelecoxib demonstrates that it influences and improves functional re- covery of peripheral nerve regeneration.

Biocompatible Zr-Al-Fe bulk metallic glasses with large plasticity

In the present study,high-zirconium ternary Zr-Al-Fe bulk metallic glasses(BMGs) with low Young's modulus and good plasticity were developed.Zr75 Al7.5 Fe17.5 BMG exhibits a low Young's modulus of 70 GPa and high Poisson's ratio of 0.403.Pronounced plasticity was demonstrated under both compression and bending conditions for the BMGs.Furthermore,the alloys show high corrosion resistance in phosphate buffered solution.The combination of desirable mechanical and chemical properties implies potential for biomedical applications.