Preparation of Tyr-C-peptide from genetically altered human insulin precursor

C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity.¹²⁵I labeled TyrC-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed inEscherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.     

Determination of sulfates of androsterone and epiandrosterone in human serum using isotope diluted liquid chromatography-electrospray ionization-mass spectrometry

A promising liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) for analysis of the sulfates of 5alpha-androgen, androsterone and epiandrosterone (A-S and EpiA-S) in human serum was developed. The method was used to assess one of the markers of 5alpha-reductase activity of males including patients with prostate cancer (PC). After deproteinization with acetonitrile, the androgen sulfates and the internal standard, [7,7,16,16-2H4]dehydroepiandrosterone-S, were extracted from human serum using a solid-phase extraction cartridge and washed with hexane. The extract was reconstituted and applied to the LC/ESI-MS system operated in the selected ion monitoring mode. The method was validated over the range 0.02-5 microg/mL (A-S) and 0.005-1.5 microg/mL (EpiA-S) using 10 microL of human serum. The method was a concise procedure without chemical or enzymatic hydrolysis of the conjugates, purification by high-performance liquid chromatography and/or derivatization, and proved to be satisfactory in its reproducibility and accuracy. The levels of these androgen sulfates tended to decrease during aging, and the A-S levels in the sera obtained from both healthy males and patients with PC were correlated with their EpiA-S levels.     

龋病人牙釉质表面硬度及抗磨损性能测试

采用人工方法制成人牙釉质浅龋及使用抗龋措施的龋损釉质样本，分别测量各组釉质表面显微硬度，并以微动摩擦磨损试验机测试其抗磨性能，试验结果表明：各组实验组釉质较对照组表面硬度都有不同程度下降，氟化钠前处理会使釉质表达硬度显著下降，人工浅龋牙釉质的抗磨损性能不如正常釉质，使用两种抗龋措施对其抗磨性能没有明显改善。     

Potassium-selective optrode based on an immobilized fluorogenic crown ether

A new optical sensor phase for potassium ions has been developed based on the immobilization of the pH-dependent fluorogenic crown ether 4-acryloylamidobenzo-18-crown-6 on the non-ionic polymeric resin Amberlite XAD-2.Two different optical designs, a flow-through sensor and a fibre optic probetype sensor (optrode), have been constructed and their analytical performance characteristics have been evaluated. The resulting fluorimetric sensors for K⁺ ions exhibited detection limits of 0.4 or 0.8 M of K⁺ (16 g/l or 31 g/l), depending on the design, while the linear response occurred from 1 to 25 M of the metal concentrations. The precision, evaluated as the relative standard deviation of measurements of K⁺ levels at around ten times the detection limit (e.g. 5 M), turned out to be around ±2%.Advantageous features of this fluorimetric sensing phase and optrode include ease of construction, simplicity of use, reversibility, short response times (ca. 1 min full scale deflection) selectivity and operational stability, suitable for sensing potassium at low levels in complex matrices such as biological fluids.The fluorimetric optical sensor has been successfully applied to the direct determination of potassium in clinically important samples (serum and urine) and in natural waters. Very good accuracy has been obtained just using adequate synthetic aqueous potassium standards for calibration.     

Independent exponential feeding of glycerol and methanol for fed-batch culture of recombinant Hansenula polymorpha DL-1

As a novel feeding strategy for aptomizing human epidermal growth factor (hEGF) production with a recombinant Hansenula polymorpha DL-1 using the methanol oxidase (MOX) promoter in H. polymorpha DL-1, independent exponential feeding of two substrates was used. A simple kinetic model considering the cell growth on two substrates was established and used to calculate the respective feeding rates of glycerol and methanol. In the fedbatch culture with methanol-only feeding, the optimal set point of specific growth rate on methanol was found to be 0.10 h⁻¹. When the fed-batch cultures were conducted by the independent feeding of glycerol and methanol, the actual specific growth rate on glycerol and methanol was slightly lower than the set point of specific growth rate. By the uncoupled feeding of glycerol and methanol the volumetric productivity of hEGF increased from 6.4 to 8.0 mg/(L·h), compared with methanol-only feeding.     

Probing in vitro digestion at oil–water interfaces

Interfacial layers have been widely applied to study the formation and stability of emulsion-based systems. However, the application of isolated interfaces to address digestibility of emulsions is often limited because of the complexity of experimental methods and results. This review summarizes the latest developments in analytical methods and literature data on effects of digestion on interfacial layers. Particular emphasis is given to understand the changes on interfacial magnitudes during oral, gastric, and duodenal digestion, either applied separately or sequentially. Limitations of interfacial aspects and key factors that influence emulsion microstructure in bulk and lipid digestion are identified. Understanding the behavior of interfacial layers upon gastrointestinal digestion promotes an accurate tracking of the physiological fate of emulsions.     

LC Determination of Propylene Glycol in Human Plasma After Pre-Column Derivatization with Benzoyl Chloride

A simple high-performance liquid chromatographic method, using photodiode array detection was developed for the determination of propylene glycol in human plasma and in the fluid retreived after continuous veno-venous hemofiltration. The method entailed alkaline derivatization with benzoyl chloride and ethylene glycol as internal standard. The separation of the compounds, after extraction with pentane, was carried out on a Pursuit C8 column with UV-detection at 230 nm. Validation samples were analyzed with an accuracy between 95 and 105%, and intra- and inter-day coefficients of variation of less than 8%. The calibration curve was linear over a concentration range of 5–100 mg L⁻¹ with a detection limit of 1 mg L⁻¹. Blood plasma samples of several patients were analysed by using the prescribed method with propylene glycol concentrations varying from 5 to 98 mg L⁻¹. Compared to previously described LC methods, this method is ten times more sensitive and thus suitable for use in pharmacokinetic studies of propylene glycol.     

MTT法测定稀土离子对BEL-7402及K562细胞的毒性及增殖毒性

用MTT法测定稀土离子在不同浓度、不同培养液中,与BEL 7402和K562细胞作用不同时间,对细胞的毒性和增殖毒性。结果表明,在含10%小牛血清培养液中,仅个别稀土离子在较高浓度时对BEL 7402细胞增殖有较弱的抑制作用;对于K562细胞,稀土离子在低浓度时对细胞增殖即表现出较强的抑制作用(P<0.05)。当培养液不含小牛血清时,较低浓度的稀土离子即可抑制BEL 7402细胞的增殖(P<0 05)。     

Spencer cohomologies and symmetry groups

We propose the construction of a spectral sequence converging to Spencer cohomologies. By using symmetry groups of differential equations systems, we manage to unify computations by reduction to the invariant systems over a homogeneous space. The conditions of coincidence of Spencer cohomologies with the cohomologies of an invariant Spencer complex we obtain from the arithmetic of a -characteristic manifold with respect to fundamental weights of the homogeneous space.     

Monotone path systems in simple regions

A monotone path system (MPS) is a finite set of pairwise disjoint paths (polygonal areas) in thexy-plane such that every horizontal line intersects each of the paths in at most one point. A MPS naturally determines a pairing of its top points with its bottom points. We consider a simple polygon in thexy-plane wich bounds the simple polygonal (closed) regionD. LetT andB be two finite, disjoint, equicardinal sets of points ofD. We give a good characterization for the existence of a MPS inD which pairsT withB, and a good algorithm for finding such a MPS, and we solve the problem of finding all MPSs inD which pairT withB. We also give sufficient conditions for any such pairing to be the same.The first author's research is supported by the Natural Sciences and Engineering Research Council of Canada