Genetic diversity among sublines originating from a single somatic hybrid cell of Duboisia hopwoodii + Nicotiana tabacum

Summary The genetic instability of an intertribal hybrid cell line, Duboisia hopwoodii + Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied. Ten subclones of calli derived from this hybrid cell line were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to which tobacco chromosomes were eliminated varied among the subclones — evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybrid subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.     

Effects of cold exposure on inhibition by isoproterenol of release of alanine in the rat

The effects of isoproterenol on the release of alanine during perfusion with pyruvate and valine were studied in perfused hindlimbs from rats that had been kept for 5 or 20 days at 4 degrees C. In hindlimbs perfused with Krebs bicarbonate buffer in a flow-through mode, the rate of release of alanine during perfusion with 2 mM pyruvate plus 5 mM valine was 250 nmol.min-1.leg-1, a rate that is comparable with that reported in hindlimbs perfused with complex medium. Neither the pyruvate-stimulated nor valine plus pyruvate-stimulated rates of release of alanine changed after 20 days of exposure to cold. Isoproterenol inhibited the release of alanine during perfusion with pyruvate, with valine, and with valine plus pyruvate in hindlimbs from a control group of rats. However, in hindlimbs from cold-exposed groups, isoproterenol failed to inhibit the release of alanine during perfusion with valine plus pyruvate and stimulated the release of alanine during perfusion with valine. Aminooxyacetate inhibited the effects of valine, pyruvate, and isoproterenol. The results obtained suggested that cold exposure decreases the responses to isoproterenol of the mechanism of alanine release and causes an increased supply of alanine to the liver.     

Recent problems in cell culture systems using normal human cells

Some remarkable studies of human cell culture have contributed to many basic and applied sciences on "normal human cells"; developments of biological products or physiologically activated substances. In this reviews, some problems concerning in vitro culture systems were discussed and recent advances on the researches of normal human cells were described.     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.     

Histochemical demonstration of zinc in rat epididymis using a sulphide-silver method

Summary We studied the histochemical distribution of zinc in rat epididymis using a sulphide-silver method. In the supranuclear cytoplasm of the principal cells that line the epididymis of rats, varying amounts of sulphide-silver-reactive zinc were visualized. In adult mating rats, significant amounts of zinc were found in the proximal portion of the epididymis, whereas in non-mating, mature and immature young rats, this heavy metal was most prominent in the distal portion of this organ. In all of the rats studied, zinc was sparsely distributed in the intermediate portion of the epididymis. From these results, it can be assumed that the zinc present in the epithelial lining of rat epididymis plays an important role in the maturation of spermatozoa. The present results represent a useful contribution to our understanding of the functional morphology of rat epididymis.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

Summary To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

Human hybrid immunoglobulin M containing immunoglobulin A

Summary Some hybridoma clones made by fusion of a human lymphoblastoid cell line, HO323 with human B lymphocytes, secreted not only IgA but also IgM-like immunoglobulin molecules. The IgM-like immunoglobulin had a molecular size of 900 K which corresponded to that of IgM. Immunochemical analyses revealed that the IgM-like immunoglobulin contained two monomeric IgA and three monomeric IgM molecules. In the IgA moieties, half of original light chains were replaced withx chains derived from the IgM, and vice versa.     

Studies on the association of NIDDM in Japanese patients with hyperthyroid Graves' disease.

We attempted to analyze the association of hyperthyroid Graves' disease with non-insulin-dependent diabetes mellitus (NIDDM). Forty-nine patients (23 males and 26 females; 7.6%) of a total of 647 patients with hyperthyroid Graves' disease had NIDDM, several years before or after Graves' disease was diagnosed. Only 1 patient had insulin-dependent diabetes mellitus. Compared with the general Japanese population (n = 9,133), the incidence of NIDDM (n = 348; 3.9%) in patients with Graves' disease was higher in all age groups. Only 4 patients (8.2%) of the 49 hyperthyroid patients with NIDDM had a history of being overweight (body mass index > 25). In contrast, 276 (79.9%) of the 348 diabetic patients were currently or previously overweight. Moreover, the incidence of a family history of diabetes (13 of the 49 hyperthyroid Graves' patients with NIDDM; 26.5%) was also lower in the patients with NIDDM in the general Japanese population (50% incidence). The male:female ration in patients with Graves' disease and NIDDM was 1:1.1; much different from that in the total Graves' disease population (1:4.1). Analysis of the HLA loci A, B, C, DR and DQ (35 determinations) in 35 hyperthyroid patients with NIDDM and in 386 subjects from the general population revealed a highly significant difference between them in the incidence of HLA-Cw4, -DR2, -DQw1, -DQw3 and -DQw4. This study suggests that there was an association of Graves' disease with NIDDM. A significant association of HLA-DR and -DQ loci was observed in hyperthyroid Graves' patients with NIDDM.