Topochemistry of blood cells of the Gallus domesticus (Aves, Galliforme).

Blood smears of both male and female chicken Gallus domesticus were analysed by using the following topochemical methods: a) Periodic acid-Schiff (PAS) for glycogen. b) Mercury-bromophenol blue for protein. c) O-Toluidine for myeloperoxidase. d) Sudan black B for lipid. The PAS reaction revealed glycogen in the cytoplasm of all thrombocytes and in a few heterophils. The presence of proteins was evidenced in all types of cells. However variation in the intensity of staining of protein granules was observed in the fusiform structures of the heterophils. A negative reaction for myeloperoxidase was found in all cells. Although some evidence of myeloperoxidase activity was show in the polymorphonuclears it was not enough to ascertain a positive reaction. Lipids were detected in the cytoplasm of few heterophils, eosinophils and monocytes.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

Dominant lethal mutations induced by MMS and mitomycin C in the fish Oryzias latipes

Males of the fish Oryzias latipes were treated with various chemicals and then mated with normal females. The fertility and hatchability of the eggs laid by the parents were examined, and the dominant lethal effects were estimated. Mitomycin C induced dominant lethals in the fish spermatids and spermatocytes after the males had been treated with concentrations of 2.5 and 25 micrograms/ml. Methyl methanesulfonate (MMS) induced dominant lethals in spermatozoa and spermatozoa and spermatids after the injection of 200 and 400 mg/kg. These results are in good agreement with the results obtained with mice. However, the effects of ethyl methanesulfonate (EMS) were not clear on spermatogenic cells at any stage. We could not recognize any significant induction of dominant lethals by urethanes, bleomycin, caffeine, and two kinds of food-color additives, at least under the present experimental conditions.     

Studies on extracellular ribonucleases of Ustilago sphaerogena. Purification and properties

1. Four ribonucleases were isolated from culture media of Ustilago sphaerogena. They were designated ribonucleases U(1), U(2), U(3) and U(4). 2. They were purified about 1600-, 3700-, 1100- and 16-fold respectively. 3. It was shown by gel filtration that ribonucleases U(1), U(2) and U(3) have molecular weights about 10000 like ribonuclease T(1), and that ribonuclease U(4) is much larger. 4. Ribonucleases U(1), U(2) and U(3) are thermostable, but ribonuclease U(4) is not. 5. The pH optimum of ribonucleases U(1) and U(4) is pH8.0-8.5, and that of ribonucleases U(2) and U(3) is pH4.5.     

Interaction of α2-macroglobulin with alkaline proteinase from an alkalophilicBacillus sp. grown in an extraordinarily alkaline environment

The interaction of ₂-macroglobulin (₂M) with an alkaline serine proteinase (ALPase I) from alkalophilicBacillus sp. grown in an extraordinarily alkaline environment was investigated. Stoichiometry of the reaction showed that ALPase I bound to ₂M in a molar ratio of about 21. The ₂M-ALPase I complex showed about 80% of the proteinase activity shown by ALPase I in the hydrolysis of succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and casein. The conformational changes in the ₂M molecule caused by the complex formation at pH 7.5 were determined from electron micrographs and difference spectra. The antigenic activity of the ₂M-ALPase I complex with the anti-ALPase I antiserum was found to be completely abolished. Immunoelectrophoresis of the complex incubated at pH 7.5 after 48 h showed no appreciable change, and the complex was recognized as exhibiting enhanced stability at pH 7.5.     

A new alkaline serine protease from alkalophilic Bacillus sp.: Cloning,sequencing, and characterization of an intracellular protease

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D37921.