Introduction of mouse C epsilon genes into Cos-7 cells and fertilized mouse eggs

A fragment of the cloned gene for the mouse C epsilon chain, coding for the first, second, third, and fourth domains, has been coupled to the SV40 promotor region (pSV2-mC epsilon). About 50 copies of pSV2-mC epsilon or its PvuII-EcoRI fragments were introduced into Cos-7 cells. Expression of PvuII-EcoRI fragments of pSV2-mC epsilon was observed in about 50% of the Cos-7 cells by indirect fluorescence staining. However, no expression of circular pSV2-mC epsilon was observed. About 200 copies of linearized pSV2-mC epsilon with EcoRI were introduced into fertilized mouse eggs. Two of 78 mice born from these eggs had integrated mouse C epsilon genes. Mouse C epsilon gene was shown to be integrated in a tandem array and as intact structures without undergoing gross deletions or rearrangements, judged from the Southern blotting patterns from several restriction enzymes. The first transgenic mouse was mated to a normal male to examine whether mouse C epsilon gene were stably transmitted to progeny. Among 5 mice to which the C epsilon gene had been transmitted, one deleted 5 copies of this gene and another deleted one junction fragment, thus demonstrating relatively unstable transmission. No C epsilon mRNA was detected in the liver, kidney, brain, lung, skeletal muscle, heart, testis, or spleen of a transgenic mouse.     

Effect of various catecholamine antagonists on prolactin secretion in conscious male rabbits

To clarify physiological roles of catecholaminergic systems in the control of rabbit prolactin (PRL) release, the effect of various catecholamine receptor antagonists on plasma PRL levels was examined in conscious, freely moving male rabbits. An intravenous (iv) injection of yohimbin (2.5 mg/kg body wt), an alpha 2-adrenoreceptor antagonist, but not prazosin (2 mg/kg body wt), an alpha 1-adrenergic receptor antagonist, resulted in a significant elevation of plasma PRL. Conversely, propranolol (2.5 mg/kg body wt, iv), a nonselective beta-adrenoreceptor antagonist, and metoprolol (2.6 mg/kg body wt, iv), a beta 1-adrenergic antagonist, slightly but significantly suppressed basal levels of plasma PRL. On the other hand, haloperidol (0.5 mg/kg body wt, iv), pimozide (0.3 mg/kg body wt, iv), sulpiride (5 mg/kg body wt, iv), chlorpromazine (3 mg/kg body wt, iv), and YM-09151-2 (0.2 mg/kg body wt, iv), all dopamine receptor antagonists caused a significant increase in plasma PRL. These results suggest that dopaminergic and alpha 2-adrenergic mechanisms exert a tonic inhibitory role and beta-adrenergic mechanisms, probably beta 1, a tonic stimulatory role in the regulation of PRL release in the rabbit.     

Selective enhancement in striatal [H]nitrendipine binding following chronic treatment with morphine

Two parameters of Ca²⁺ dynamics in brain preparations (⁴⁵Ca-uptake to slices and [³H]nitrendipine binding to membrane fractions) were measured in naive and chronic morphine-administered rats. While morphine did not have any effect on ⁴⁵Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K⁺-stimulated ⁴⁵Ca-uptake to slices. Furthermore, the effect of morphine was antagonized by naloxone. Inhibition of K⁺-stimulated ⁴⁵Ca-uptake to striatal slices by morphine was not observed in preparations obtained from chronic morphine-administered rats (6 mg/kg/b.i.d./7 days). In membrane fractions, [³H]nitrendipine binding increased by 34% in striatum following chronic morphine treatment, whereas no change was observed in the cortex and hippocampus. The results will be discussed in relation to the phenomena underlying chronic morphine administration.     

Accumulation of ornithine decarboxylase-antizyme complex in HMOA cells.

A new method was developed for the assay of ornithine decarboxylase (ODC)-antizyme complex, in which alpha-difluoromethylornithine (DFMO)-inactivated ODC was used to release active ODC competitively from the complex. ODC-antizyme complex was present in the extracts of hepatoma tissue-culture (HTC) cells and of ODC-stabilized variant HMOA cells, in much larger amounts in the latter. Cellular amounts of the complex fluctuated after a change of medium in a similar manner in HTC and HMOA cells, increasing during the period of ODC decay. After treatment with cycloheximide, the decay of ODC-antizyme complex in HMOA cells was more rapid than the decay of free ODC, but it was much slower than the decay of free ODC or complexed ODC in HTC cells. Administration of putrescine caused a rapid increase in the amount of ODC-antizyme complex in both HTC and HMOA cells, but nevertheless the decay of total ODC (free ODC plus ODC-antizyme complex) was more rapid with putrescine than with cycloheximide. These results suggested the possibility that ODC is degraded through complex-formation with antizyme. In contrast with complexed antizyme, free antizyme was not stabilized in HMOA cells.     

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.     

New genus and species of the family stichaeidae from Hokkaido,Japan

Specimens of a new genus and species of the stichaeid fish,Leptostichaeus pumilus, were collected from the Okhotsk Sea off Hokkaido in Japan. The present new genus and species clearly differs from all the other genera and species of the stichaeid fishes in the following characters: 3 or 4 pectoral fin rays; 10 or fewer caudal principal rays; 79–82 dorsal spines; no pelvic fin; last interneural spine supporting a single dorsal spine; infraorbital, occipital and lateral line canals absent; moderate size of dorsal spine shorter than eye diameter; membranes of dorsal and anal fins widely connected with caudal fin; a large black spot divided by a yellow band present just above gill cover.     

Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

Summary The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4°C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5 60 min at 20° and 37°C in Con-A-free serum resulted in a temperature-dependent internalization of membranebound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37°C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constitutents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Electrophoretic variants of blood proteins in Japanese. IV. Prevalence and enzymologic characteristics of glucose-6-phosphate dehydrogenase variants in Hiroshima and Nagasaki

Summary Electrophoretic screening of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6PD) was conducted one sample of 9,260 children born to the atomic bomb survivors in Hiroshima (Honshu) and Nagasaki (Kyushu). The prevalence of electrophoretic variants was 0.11% in males and 0.42% in females in Hiroshima, and 0.16% in males and 0.31% in females in Nagasaki. Enzymologic characteristics of 10 variants obtained from three males and seven hemizygous fathers of heterozygous females were examined. As a result, three new types of G6PD variants were identified among five variants detected in Hiroshima, and three new types among five variants in Nagasaki. All the variants except one belonged to Class 3, as defined by Yoshida et al. (1971).     

Morphological aspects on pancreatic islets of non-obese diabetic (NOD) mice

The pancreatic islets of female non-obese diabetic (NOD) mice (a model of insulin-dependent diabetes mellitus), have been examined by both light and electron microscopy. At about the age of 2 weeks, mononuclear cells began to infiltrate in or near the islets and some of these cells were in contact with the islet cells. Following this degeneration of islet B-cells took place, the process occurring in two ways. In many cells numerous secretory granules with extremely dense cores occupied the cytoplasm. Other cells, however, were filled with low-density secretory granules and the nuclei of these cells became pycnotic. After degeneration of B-cells, the islets were effaced by numerous mononuclear cells. With the onset of the diabetic state these mononuclear cells gradually disappeared, and thereafter small islets remained. By electron microscopy, retrovirus-like particles were observed in cisternae of the rough endoplasmic reticulum in islet B-cells at all stages. With an anti-retrovirus serum (goat anti-KiMSV-NIHxeno serum), positive immunofluorescence was observed in some pancreatic islet cells of NOD mice aged 1 day and 4, 6, 8, 9, 10 and 14 weeks. It is suggested that these virus particles may be intimately related to the inflammatory reaction occurring in the islets and to the development of diabetes mellitus.     

Gene structures of low-neurovirulent vaccinia virus LC16m0, LC16m8, and their Lister original (LO) strains

Vaccinia viruses LC16m0 and LC16m8 are temperature-sensitive and low-neurovirulent variants derived from the Lister (Elstree) (LO) strain. Analyses of genome DNAs by digestion with restriction endonucleases and cross-hybridization of the digested fragments revealed that LC16m0 and LC16m8 possess a new XhoI site in addition to the 14 XhoI sites of LO. This new site is located at about 12 X 10(6) daltons from the right terminal end. There was no significant difference in the genome structures between the LC16 variants and LO except the new XhoI site and their terminal fragments which were not identified in LO owing to their heterogeneity. With HindIII digested fragments, there was no difference among the three viruses. This complete mapping raised the possibility that the putative gene responsible for temperature sensitivity and neurovirulence is located at the region of the XhoI site found in LC16m0 and LC16m8.