Cryopreservation of wild mouse spermatozoa

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.     

Association of pp60src with Triton X-100-insoluble residue in human blood platelets requires platelet aggregation and actin polymerization.

Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena.     

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.     

Developmental analysis of the eye lens obsolescence (Elo) gene in the mouse: cell proliferation and Elo gene expression in the aggregation chimera.

This study investigates the primary effect of the eye lens obsolescence (Elo) gene of the mouse. Morphological features of the Elo lens were defined as follows: (1) deficient elongation of lens fiber cells, (2) morphological abnormality of nuclei of lens fiber cells, (3) lack of eosinophilic granules in the central fiber cells and (4) rupture of lens capsule in the posterior region. We have immunohistologically examined, by means of an in vivo BrdU incorporation system, whether or not the Elo gene regulates cell proliferation during lens development. The lens fiber cells were morphologically abnormal in day 13 embryonic Elo lens. However, there were no significant differences in morphology or cell proliferation between normal and Elo lens epithelium until day 14 of gestation. After day 15, the total cell number in the Elo lens epithelium was significantly less than that in the normal, but the total numbers of S-phase cells in the two genotypes were not significantly different. The ratio of the total S-phase cell number to the total number of lens epithelial cells may be affected by the developmental stage, but not directly by the genotype. The genotype, however, may be having a direct influence at later ages because malformation of Elo lens fiber cells must cause reduction of the total number of lens epithelial cells in older embryos. Although, at 30 days old, Elo lens cells were externally extruded through the ruptured capsule into the vitreous cavity, BrdU-labelled lens epithelial cells were detectable. To investigate whether the Elo lens phenotype is determined by its own genotype or by its cellular environment, we produced aggregation chimeras between C3H-Elo/+(C/C) and BALB/c(c/c). Most lenses of BALB/c dominant chimeras were oval in shape without the ruptured lens capsule. However, they were opaque in the center and slightly smaller in size than normal. The lenses of C3H-Elo/+ dominant chimeras were morphologically similar to the Elo lens. Although normal nuclei were regularly arranged in the anterior region, Elo-type nuclei were located in the posterior region. Immunohistological staining by using anti-C3H strain-specific antibody demonstrated that the lens fiber cells with abnormal nuclei were derived only from C3H-Elo/+, not from BALB/c. These observations suggest that the primary effect of the Elo gene in the developing lens may be specific to the fiber cell differentiation rather than to the cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells.

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.