Comparative studies on soluble and immobilized rabbit muscle pyruvate kinase

Rabbit muscle pyruvate kinase was immobilized by covalent attachment to a polyacrylamide support (Akrilex C) containing carboxylic functional groups. As a result of immobilization, the pH optimum for catalytic activity shifted into a more alkaline direction. The apparentK _m value with phosphoenolpyruvate increased, and that with ADP slightly decreased. With respect to the stability against urea and thermal inactivation, the immobilized pyruvate kinase seemed to be the more stable at lower urea concentrations and between 45 and 55°C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.     

用反相高效液相色谱法测定小鼠心肌、骨骼肌中AT P、AD P和AM P的含量

采用反相高效液相色谱法同时测定小鼠心肌、骨骼肌组织中ATP(三磷酸腺苷 )、ADP(二磷酸腺苷 )、AMP(一磷酸腺苷 )的含量。以150mmol/L磷酸二氢钾缓冲液 (pH=6.25)为流动相 ,采用BDShypersilC18 不锈钢色谱柱 ,紫外检测波长为254nm ,线性范围为1～200mg/L。ATP、ADP和AMP的平均加标回收率均在90 %以上 ,检出限分别为10ng、10ng、15ng。该法灵敏、准确 ,试剂费用低。     

Thermal stability of rat uterus during development

Summary The age dependence of thermal denaturation was monitored in rats anaesthetized after they're born at 7^th, 14^th, 21^st, 28^th, 35^thand 42^nddays. The samples were stored in rigor or physiological saline solution. The DSC scans in the early age groups show a low temperature exotherm (connective tissues: from gel to liquid crystal transition) and one endotherm (it is very probably the myosin). During further development the endotherms became more and more complex (due to the development of contractile system). At 42 days the scans seem to be similar to the adult ones. In the two buffers the endotherms markedly differ showing that the ATPase activity is present. In adult uterus, treated with nucleotides or estrogen, this activity significantly differs from the skeletal muscle. On the basis of our results we suppose, that the age dependent changes are decisive processes in the development of rat uterus.     

丝素蛋白修饰改性的聚羟基脂肪酸酯支架上人体平滑肌细胞的生长

聚三羟基丁酸脂和聚三羟基己酸脂的共聚物(PHBHH_x)是一种具有良好强度和韧性的生物可降解高分子材料, 可作为组织工程心脏瓣膜支架的选择材料之一. 但其生物相容性尚不甚理想. 为此, 本工作利用丝素蛋白修饰改性高分子多孔支架, 以提高支架的生物相容性. 并将人体平滑肌细胞接种在该复合支架上进行体外培养, 以证实改性效果. 其中, 用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)方法测试细胞生长, 评估复合支架的细胞相容性. 并用扫描电子显微镜观察细胞在支架上的生长形态. 结果显示, 丝素蛋白修饰改性后的复合支架更有利于细胞的粘附与生长, 平滑肌细胞在支架上表现出良好的生长形态. 这表明, 丝素能够改善多孔支架的生物相容性, 使PHBHH_x/丝素蛋白复合物能更适宜作为组织工程心脏瓣膜的支架材料. 结果对于进一步研究细胞外间质在复合支架上的生长以及体外培养的组织重建有重要的参考意义.     

Simple extraction method for quantification of phenothiazine residues in pork muscle using liquid chromatography–triple quadrupole tandem mass spectrometry

In this study, an analytical method was developed for quantification of residues of the anthelmintic drug phenothiazine (PTZ) in pork muscle using liquid chromatography–tandem mass spectrometry. Muscles were extracted using 0.2% formic acid and 10 mm ammonium formate in acetonitrile, defatted and purified using n ‐hexane. The drug was well separated on a Waters XBridge™ C₁₈ analytical column using a binary solvent system consisting of 0.2% formic acid and 10 mm ammonium formate in ultrapure water (A) and acetonitrile (B). Good linearity was achieved over a six‐point concentration range in matrix‐matched calibration with determination coefficient =0.9846. Fortified pork muscle having concentrations equivalent to and double the limit of quantification (1 ng/g) yielded recovery ranges between 100.82 and 104.03% and relative standard deviations <12%. Samples (n = 5) collected from large markets located in Seoul City tested negative for PTZ residue. In conclusion, 0.2% formic acid and ammonium formate in acetonitrile can effectively extract PTZ from pork muscle without solid‐phase extraction, a step normally required for cleanup before analysis and the validated method can be used for routine analysis to ensure the quality of animal products.     

Formation of Highly Aligned Collagen Nanofibers by Continuous Cyclic Stretch of a Collagen Hydrogel Sheet

A collagen sheet with highly aligned collagen fibers is fabricated by continuous cyclic stretch. The rearrangement of the collagen fibers depends on the different process parameters of the cyclic stretch, including magnitude, frequency, and period of stretch. The collagen fibers are aligned perpendicularly to the direction of the stretch. Corneal stromal cells and smooth muscle cells cultivated on the highly aligned collagen sheet show alignment along the collagen fibers without the stretch during culture. Thus, the sheet can be a suitable scaffold for use in regenerative medicine.

     

Validation of a method for the analysis of quinolones residues in bovine muscle by liquid chromatography with electrospray ionisation tandem mass spectrometry detection

A liquid chromatography-tandem mass spectrometry method for the determination and confirmation of nine quinolones was optimised and validated according to Commission Decision 2002/657/EC. Analytes were extracted from veal muscle with water and extracts purified with 96-well plates Oasis HLB cartridges. Separation was carried out in a silica-based C₁₈ column (50 mm × 2.1 mm) with mobile phases consisting of water/acetonitrile mixtures containing acetic acid. Linear calibration curves in the ranges 4-400 and 50-800 ng g⁻¹, with correlation coefficients at least 0.995, were obtained for all the analytes. At concentration levels above 10 ng g⁻¹, quantification errors were lower than 10% and repeatability and within-laboratory reproducibility standard deviations below 6% and 10%, respectively. Decision limits and detection capabilities are reported.     

高效液相色谱检测动物肌肉组织中7种喹诺酮类药物的残留

本研究建立了同时检测动物肌肉组织中环丙沙星、单诺沙星、恩诺沙星、沙拉沙星、二氟沙星、恶喹酸、氟甲喹7种喹诺酮类药物的HPLC-FLD检测方法。动物组织样品用磷酸盐水溶液提取,HLB固相萃取柱净化,采用甲酸水溶液-乙腈体系作为流动相,梯度洗脱,程序波长荧光检测器检测。方法的线性范围为0.3~100μg/L,相关系数(r)大于0.9989;检出限为0.1~0.3μg/kg,定量限为0.3~1.0μg/kg;7种喹诺酮类药物在鸡肉和猪肉的平均回收率分别为70.4%~102.1%和79.6%~105.8%;相对标准偏差分别为1.1%~8.9%和1.3%~9.3%。     

Development and validation of an HPLC confirmatory method for the determination of tetracycline antibiotics residues in bovine muscle according to the European Union regulation 2002/657/EC

A high-performance liquid chromatographic method with diode-array detection, at 351 nm, was developed and validated for the determination of five tetracyclines (TCs): minocycline, tetracycline, oxytetracycline, chlortetracycline, and doxycycline in bovine muscle. Samples were macerated with a buffer solution, centrifuged, and purified using Abselut Nexus SPE cartridges. The separation of the examined TCs was achieved on an Inertsil ODS-3 5 microm, 250 x 4 mm analytical column, at ambient temperature. A multistep gradient elution was followed using 0.05 M oxalic acid and CH3CN, at a flow rate of 1.65 mL/min. The procedure was validated according to the European Union regulation 2002/657/EC determining selectivity, stability, decision limit, detection capability, accuracy, and precision. The results of the validation process demonstrate that the method can be readily applied to European Union statutory veterinary drug residue surveillance programmes. Mean recoveries of TCs from bovine muscle samples spiked at three concentrations (100, 250, and 400 ng/g) were in the range of 98.7-103.3%. Method's LOQ values achieved were 40 microg/kg for MNC, CTC, and DC and 25 microg/kg for OTC and TC. The decision limits (CCalpha) were in the range of 104.7-109.8 microg/kg, while the detection capability (CCbeta) was in the range of 108.4-116.7 microg/kg for all compounds.     

Nucleotide Analogue Induces Global and Local Changes in Muscle Fibres

The effect of AMP.PNP on the thermal stability and dynamics of myosin head were investigated by using DSC and different spin label technique for chemically skinned muscle fibres prepared from rabbit. The thermal unfolding of the fibres in rigor, strong as well as weak-binding state showed a complex process characterizing at least three discrete domain regions with different stability (T _m =54, 58.4 and 62.3°C). The unfolding at 54°C refers to the catalytic domain of myosin, whereas transition at T _m =58.4°C represents the rod-like region. In the presence of AMP.PNP only the parameters of the last transition changed significantly (T _m =70.4°C) showing an increased interaction between actin and myosin heads being attached to actin. Measurements on MSL-fibres (labelled at Cys-707 of myosin) in the presence of AMP.PNP showed that about half of the cross-bridges dissociated from actin. This fraction had a dynamic disorder, the other population had the same spectral feature as in rigor. In contrast, on TCSL-fibres AMP.PNP increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum showing conformational and motional changes in the internal structure of myosin heads. ST EPR measurements reported increased rotational mobility of spin labels in the presence of AMP.PNP. The DSC and EPR results suggest that in the presence of AMP.PNP the attached heads have the same global orientation as in rigor, but the internal structure undergoes a local conformational change. This revised version was published online in July 2006 with corrections to the Cover Date.