Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Growth Regulation in Dwarf Barley Coleoptiles by the Minor Cell Wall Components, Galactose and Mannose

Sugar compositions of cell walls of dark-grown coleoptiles from12 barley strains, 11 of which were coleoptilar semi-dwarf strains,were analyzed on days 2 and 3 after germination. Major wallcomponents of the 12 strains were arabinose, xylose, and glucosein hemicellulose and cellulose; minor components were galactoseand mannose. The sugar content of each wall component per unit length wasnot correlated to any growth parameters calculated from a logisticequation simulating coleoptile growth, but the relative contentsof galactose and mannose in relation to the total wall sugarcontent was correlated to the growth rate on day 3 and the growthcontinuing period. These facts suggest that growth of these12 barley strains in the late growth stage is regulated by theminor wall components, galactose and mannose. ¹ Dedicated to the late Professor Joji Ashida. (Received October 12, 1982; Accepted January 12, 1983)     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

Elektronenmikroskopische Untersuchungen am Nucleus praeopticus der Kröte (Bufo vulgaris formosus)

Zusammenfassung Die Feinstruktur der neurosekretorischen Nervenzellen des Nucleus praeopticus magnocellularis der Kröte (Bufo vulgaris formosus) und ihre Umgebung wurde untersucht.Die neurosekretorischen Zellen enthalten drei Arten von osmiophilen Gebilden: die neurosekretorischen Elementargranula, die neurosekretorischen Kügelchen und die Einschlußkörper.Die neurosekretorischen Elementargranula besitzen einen Durchmesser von 1000–3000 Å, durchschnittlich von 1300–1500 Å. Sie entstehen im Golgi-Apparat (Perikaryon) der betreffenden Zellen wie bei den schon beschriebenen anderen Tierarten.Die neurosekretorischen Kügelchen haben einen Durchmesser von 4000 Å bis zu mehreren . Sie kommen zuerst in den Ergastoplasmacisternen des Perikaryons vor und wandern dann innerhalb des Axons caudalwärts ab, ebenso wie die Elementargraunla, verlieren sich aber vor dem Erreichen der Neurohypophyse. Nach Lage und Gestalt entsprechen sie den Kolloidtropfen, die von vielen Lichtmikroskopikern für die neurosekretorischen Zellen niederer Vertebraten beschrieben wurden.Die Einschlußkörper treten vornehmlich im zentralen Bezirk des Perikaryons in Erscheinung. Sie sind so groß wie die Mitochondrien und besitzen verschiedene Innenstrukturen. Auf Grund der Struktur und der histochemischen Reaktion möchten wir diese Einschlußkörper den Lipofuscingranula mit saurer Phosphatase zuordnen.Die neurosekretorischen Nervenzellen schmiegen sich an den die Kapillare umgebenden Perivaskularraum unmittelbar an, innerhalb dessen die Basalmembran unvollkommen ausgebildet ist oder ganz fehlt.Stellenweise dehnt sich ein Abschnitt des Endothels durch den Perivaskularraum hindurch entlang der Außenfläche des Perivaskularraums aus, wobei sich die Endothelzellen der Kapillare und die neurosekretorischen Nervenzellen direkt berühren können. Eine poröse Bauweise des Endothels wurde nicht nachgewiesen. Zwischen den Ependymzellen des III. Ventrikels und den darunterliegenden neurosekretorischen Nervenzellen sind oftmals auffallend große Extrazellularräume zu beobachten, die durch den Spaltraum der benachbarten Ependymzellen mit dem Ventrikellumen kommunizieren. Sie enthalten mikrovilliartige Ausläufer der Ependymzellen und die geschilderten, neurosekretorische Bildungen führenden Axone. Eine Ausstoßung dieser Axone in den Ventrikel wurde nicht festgestellt.Diese Untersuchung wurde zum Teil mit finanzieller Unterstützung durch das Japanische Unterrichtsministerium im Jahre 1963 durchgeführt.Der kurze Inhalt dieser Arbeit wurde unter dem Thema 'Electron microscopic studies on the praeoptic nucleus in the toad am 5. und 6. September 1963 auf dem Kongreß für Endokrinologie in Gunma, Japan, vorgetragen.     

The Effect of Electrotonus on the Olfactory Epithelium

The effect of electrotonus on the slow potential of the olfactory epithelium of the frog was studied. The "on"-slow potential induced by a general odor like amyl acetate increased its magnitude in accordance with increase of anodal current, while it decreased its magnitude with increase of cathodal current. Similar relations were also found in the case of the vapors of organic solvents like ethyl ether of low concentrations. Conversely, the on-slow potential induced by the vapors of organic solvents of high concentration decreased its magnitude in accordance with the increase of anodal current, while it increased its magnitude with the increase of cathodal current. The "off"-slow potential induced by the vapors of organic solvents of high concentration showed a potential change under the action of electrotonic currents which is similar to the change of the on-slow potential induced by general odors. It was concluded that there are two receptive processes in the olfactory cell. One is an ordinary excitatory process which produces an electronegative slow potential in response to general odors. The other is a process of a different kind which is activated only by the vapor of an organic solvent of high concentration and which shows an entirely opposite reaction from that generally found in excitable tissues when an electrotonic current is applied.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.