Purification of chicken brain choline acetyltransferase

Chicken brain choline acetyltransferase was purified to homogeneity using ammonium sulfate fractionation, followed by chromatography on DEAE-Sephadex (A-25), hydroxyapatite, Sephadex G-150, immunoabsorption and Sepharose-CoA columns. A purification of 3500-fold was achieved and the final preparation had a specific activity of 2:32 μmol acetylcholine formed per minute per milligram protein. The purified chicken choline acetyltransferase migrated as a single band on polyacrylamide gel electrophoresis in the presence and absence of sodium deodecyl sulfate. The native enzyme, with a molecular weight of 67,000 daltons, consists of two subunits of identical molecular weight. Chicken choline acetyltransferase has a sharp pH optimum of 7.4. It is activated by sodium chloride and potassium chloride but inhibited by cupric ion and N-ethylmaleimide.     

Laboratory Diagnosis of Canine Pyometra

The principal laboratory test used to confirm the pyometra diagnosis in the bitch has been the determination of the total white blood cell count in venous blood. A marked elevation is known to be a characteristic of the disease. In the present study, the white blood cell count was determined as well as the γ-globulin level. An elevation of the γ-globulin level and the total white blood cell count was very characteristic to the pyometra patients. The increase in the number of white blood cells nor the high γ-globulin level cannot be regarded specific for pyometra, therefore it was regarded important to find out a more specific test for pyometra. When sonicated E. coli bacteria were tested against sera from pyometra patients in electroimmunodiffusion, the precipitation was almost always detected when E. coli had been isolated from the uterus. This technique provides a quick method in detecting the causative E. coli infection. The present study suggests that whenever laboratory tests are used to confirm the pyometra diagnosis by the total white blood cell count, it is advantageous to analyze the total γ-globulin level in the serum as well as specific antibodies against a common E. coli antigen. Because of the reliability of the glutaraldehyde coagulation test and the simple technique, this can be suggested as the method of choice for an average small animal practice.     

Ultraviolet light induced preferential cross-linking of histone H3 to deoxyribonucleic acid in chromatin and nuclei of chicken erythrocytes

Histones have been cross-linked to DNA in chicken erythrocyte nuclei and chromatin by using ultraviolet light irradiation at 254 nm. Following irradiation, cross-linked histone-DNA adducts were isolated and purified by hydroxylapatite chromatography, and the DNA component was subjected to acid hydrolysis. Of several hydrolysis techniques investigated, trichloroacetic hydrolysis of the DNA component of the adducts was found to be most effective. Histones isolated from hydrolyzed histone-DNA adducts were characterized by gel electrophoresis and fingerprint analysis. No histone-histone protein adducts were observed. All histone fractions have been shown to cross-link DNA in nuclei or chromatin by utilizing the technique employed, but with different propensities. The order of observed cross-linking, deduced from kinetic experiments, is H1 + H5, H3 greater than H4 greater than H2A much greater than H2B. The preferential binding of the core histone H3, as compared to the other core histones, is discussed in light of recent data concerning histone-DNA interactions and nucleosome structure. The use of the ultraviolet light technique as a conformational probe to study chromatin is also discussed.     

氦氖激光对绵羊精清中酶活性效应的研究

本文研究结果表明低剂量的氦氖激光可以提高绵羊精清中GOT和LDH酶的活性,并对其机制作了初步的探讨。     

A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor

A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.     

Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.     

Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.