Selectable plasmid vectors with alternative and ultrasensitive histochemical marker genes

Three different histochemical marker genes--E. coli beta-galactosidase gene (lacZ), Drosophila alcohol dehydrogenase gene (ADH) and human placenta alkaline phosphatase gene (ALP)--were cloned into a eukaryotic expression vector also containing the neomycin resistance gene. After calcium phosphate transfection and G418 sulfate selection of recipient BALB/c 3T3 cells, stable transfectants were pooled for histochemical staining. The lacZ-bearing cells produce aqua blue staining for beta-galactosidase; ADH-bearing cells, blue-black staining for alcohol dehydrogenase; and ALP-bearing cells, red staining for alkaline phosphatase. Cells carrying different marker genes can be easily differentiated by double-staining protocols. In addition, various photographic films can be used to enhance the colors of specific histochemically tagged cell classes. These plasmid vectors, providing selectability with the neomycin resistance gene and ultrasensitivity of alternative histochemical marker genes, will be very effective in virtually any biological system requiring analyses of multiple cell clones or classes in culture model systems or in situ.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Preparation of enteric-coated microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: The effect of swine serum on micromeritic properties

Summary Cellulose acetate phthalate was used to prepare the Mycoplasma hyopneumoniae vaccine (MHV) microspheres using a solvent evaporation method. Swine serum was used as an additive in the antigen to form the core materials. The addition of serum had a significant effect on surface topography of the MHV microspheres. By using this modified solvent evaporation method, the recoveries of antigens in the MHV microspheres were generally over 90% of the weight and antigenicity of antigens originally added in the formulation.     

Differential scanning calorimetry study of mixed-chain phosphatidylcholines with a common molecular weight identical with diheptadecanoylphosphatidylcholine

To examine the thermotropic phase behavior of various mixed-chain phosphatidylcholines in excess water and to compare it with the known behavior of identical-chain phosphatidylcholines, we have carried out high-resolution differential scanning calorimetric (DSC) studies on aqueous dispersions of 10 different mixed-chain phosphatidylcholines. These lipids, C(16):C(18)PC, C(18):C(16)PC, C(15):C(19)PC, C(19):C(15)PC, C(14):C(20)PC, C(20):C(14)PC, C(13):C(21)PC, C(21):C(13)PC, C(12):C(22)PC, and C(22):C(12)PC, have a common molecular weight which is the same as that of C(17):C(17)PC, an identical-chain phosphatidylcholine with a molecular weight of 762.2. When the values of any of the thermodynamic parameters (Tm, delta H, and delta S) of the mixed-chain phosphatidylcholines and C(17):C(17)PC are plotted against the normalized chain-length difference (delta C/CL), a linear function with negative slope is obtained provided that the value of delta C/CL is within the range of 0.09-0.4. The linear relationship suggests that these mixed-chain phospholipids are packed in the gel-state bilayer similar to the bilayer structure of C(17):C(17)PC at T less than Tm; however, the negative slope suggests that the conformational statistics of the hydrocarbon chain and the lateral lipid-lipid interactions of these phosphatidylcholines in the gel-state bilayer are perturbed proportionally by a progressive increase in the chain-length inequivalence between the two acyl chains within each lipid molecule. When the value of delta C/CL for mixed-chain phosphatidylcholines reaches the range of 0.44-0.55, the thermotropic phase behavior deviates markedly from that of less asymmetric phosphatidylcholines, suggesting that these highly asymmetric lipids are packed into mixed interdigitated bilayers at T less than Tm. The heating and cooling pathways of aqueous dispersions prepared from the 10 mixed-chain phospholipids are also discussed.     

Expression and characterization of human factor IX and factor IX-factor X chimeras in mouse C127 cells

Human blood clotting factor IX, and two chimeric molecules of factor IX, in which the first epidermal growth factor-like domain or both epidermal growth factor-like domains have been replaced by that of human factor X, have been expressed in mouse C127 cells. The recombinants have been purified using a metal ion-dependent monoclonal antibody specific for residues 1-42 of human factor IX. All recombinant molecules are activated normally by human factor XIa in the presence of calcium ion. Activation of the factor IX recombinants by factor VIIa-tissue factor appears to be normal for the epidermal growth factor-1 exchange but considerably reduced for the construction containing both epidermal growth factor-like domains of factor X. The analysis of gamma-carboxyglutamic acid residues reveals that all of the purified recombinants are almost fully carboxylated. The extent of aspartic acid hydroxylation at residue 64 is 60% for all recombinants. The chimeric molecule with both epidermal growth factor-like domains from factor X has about 4% normal activity in the activated partial thromboplastin time assay. In contrast, the construct containing the first epidermal growth factor-like domain of factor X shows essentially normal clotting activity. Thus, it is unlikely that this domain is involved in a unique interaction with factor VIII.     

Simplified procedure for the analysis of 3- and 4-hydroxyproline

A column chromatographic analysis of 3-hydroxyproline (3-Hyp), 4-hydroxyproline (4-Hyp), and γ-carboxyglutamic acid (Gla) is described. The analyses of urine and plasma were performed with a JLC-6AH amino acid analyzer. A 0.15 M sodium citrate buffer, pH 2.1, was used for elution. Urinary Gla, 3-Hyp, and 4-Hyp were among the seventeen peaks eluted before asparti acid. Hyp, Gla, glutamine, and asparagine in plasma were separated by elution with 0.2 M sodium citrate buffer, pH 3.25, containing 10% methanol. This single-column procedure achieves the sequential separation and quantitation of Gla, 3-Hyp, and 4-Hyp in urine as well as plasma, and is applicable to the diagnosis of collager, metabolism disorders.     

The binding of androgen receptor to DNA and RNA

The androgen receptor from mouse kidney cytosol has been studied for its nucleic acid binding properties by DNA-cellulose centrifugation assay. The receptor appears to bind to RNA (mRNA, tRNA, rRNA) as well as to DNA. Salt and heat activation of the androgen receptor enhances both DNA and RNA binding. The receptor binds slightly better to denatured DNA than to native DNA. The androgen receptor binds about 2-fold tighter to poly(dG-dC) than to poly (dA-dT). The interaction of the receptor with DNA is not greatly affected by the BrdUrd substitution. The observation that androgen receptor shows a significant affinity to RNA may imply that androgen receptor-RNA interaction could play a role in gene regulation.     

Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads

A simple photolithographic technique has been developed which can be used to produce microscopic grid patterns on glass coverslips. The grid pattern is first photo-reduced onto film, and the resulting photographic negative is then used as a mask. A glass slide or coverslip, coated with a layer of photoresist, is then exposed to tungsten light through the mask. After developing and etching, the grid pattern is transferred permanently onto glass. This simple and rapid procedure allows one to mass-produce very small, high resolution grids which are useful for monitoring individual microinjected cells or chromosomal spreads under the microscope.