Quantification of cell nuclei isolated from hepatocytes by cell lysis with nonionic detergent in citric acid

A method was developed for determining the number of nuclei of hepatocytes cultured on collagen gel using a nonionic detergent, Nikkol BO-10TX. The cells were recovered in a test tube after solubilizing the gel by incubating it with the detergent in 0.1 M citric acid and then centrifuging the mixture. Nuclei were isolated from the cells with the same detergent solution and collected by centrifugation. The numbers of nuclei in cultures, scored with a hemocytometer or an electronic particle counter, were proportional to the lactate dehydrogenase activities of the cells. This method was also applicable for scoring the number of nuclei of hepatocytes cultured on collagen-coated plastic.     

Localization of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae

The subcellular distribution of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae cells was determined by subcellular fractionation and indirect immunofluorescence microscopy using the bcy1 mutant deficient in the regulatory subunit as control. The regulatory subunit of cAMP-dependent protein kinase showing cAMP-binding activity was identified as a single protein of 50 kDa by photoaffinity labeling and immunoblotting. The regulatory subunit was concentrated in a nuclear fraction in addition to a cytoplasmic fraction. By comparison of the regulatory subunit distribution with the DNA localization, the area detected by the indirect immunofluorescence was identified as the nucleus.     

Membrane-stabilizing effect of vitamin E: effect of alpha-tocopherol and its model compounds on fluidity of lecithin liposomes

The effects of vitamin E (alpha-tocopherol) and its model compounds on the fluidity of liposomes composed of dipalmitoylphosphatidylcholin (DPPC) and fatty acids were investigated by the measurement of the fluorescent polarization (P) using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plobe. Although all tocopherols decreased the fluidity of liposomes which was perturbed by the inclusion of an unsaturated fatty acid having more than one double bond, alpha-tocopherol was more effective than the others. The fluidity in arachidonic acid-containing liposomes was decreased most in the presence of alpha-tocopherol and was decreased considerably by the inclusion of model compounds having a side chain at least one isoprene unit or a long straight chain instead of isoprenoid side chain. However, the chromanol with methyl group instead of the above side chain, and phytol, having no chromanol moiety, had no effect. These results show that a structural requirement for a membrane stabilization is to be either the chromanol moiety with methyl groups born on its aromatic ring or a side chain of appropriate length; an isoprenoid side chain of full length or one containing 4'a- and 8'a-methyl groups is not necessarily needed.     

A Possible Regulation of Ethylene Production by the Epidermis in Mung Bean Hypocotyl Sections

IAA-induced and l-aminocyclopropane-l-carboxylic acid (ACC)-dependentethylene production in etiolated mung bean (Vigna radiata [L]Wilczek) hypocotyl sections does not occur in epidermal cells(Todaka and Imaseki 1985). Mung bean hypocotyls contain a proteinwhich inhibits auxin-induced ethylene biosynthesis in hypocotylsections (Sakai and Imaseki 1975a, b). This inhibitory proteinwas also found to inhibit ACC-dependent ethylene productionin hypocotyl sections, but not in hypocotyl sections from whichthe epidermis had been removed. Uptake of ACC by both unpeeledand peeled sections was not inhibited by the protein. Similarly,IAA-induced ethylene production was inhibited by the proteinin unpeeled hypocotyl sections, but not in peeled sections.The protein was not inactivated in peeled sections, as proteinsynthesis by peeled sections was inhibited to the same extentas in unpeeled sections. The protein inhibited incorporationof 3,4-[¹⁴C]-methionine into ACC and ethylene in unpeeled sections,but not in peeled sections, whereas oxidation of the labeledmethionine into CO₂ was inhibited by the protein to a similarextent in both types of hypocotyl sections. KCN, a potent inhibitorof ethylene production, inhibited both IAA-induced and ACC-dependentethylene production in both peeled and unpeeled hypocotyl sections.It is likely that the epidermis plays some role in controllingethylene production which occurs in stem cells other than epidermalcells. (Received July 16, 1985; Accepted October 21, 1985)     

Cloning in Saccharomyces cerevisiae of a cycloheximide resistance gene from the Candida maltosa genome which modifies ribosomes.

We have previously shown that cycloheximide resistance can be induced in a strain of Candida maltosa by modifying ribosomes (M. Takagi, S. Kawai, Y. Takata, N. Tanaka, M. Sunairi, M. Miyazaki, and K. Yano, J. Gen. Appl. Microbiol. 31:267-275, 1985). The present paper describes the cloning of the gene involved in this resistance (designated RIM-C for ribosome modification by cycloheximide) by using a host-vector system of Saccharomyces cerevisiae.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Bradykinin-induced transient accumulation of inositol trisphosphate in neuron-like cell line NG108-15 cells

Studies were undertaken to further elucidate the mechanism(s) by which bradykinin-dependent phosphoinositide metabolism takes place in neuroblastoma X glioma hybrid NG108-15 cells [(1984) J. Biol. Chem. 259, 10201-10207] using [3H]inositol-labelled cells. Bradykinin produced net increases in the level of [3H]inositol phosphates, especially of [3H]inositol trisphosphate which is formed transiently and most rapidly. The results indicate that bradykinin activates a phosphodiesterase to break down phosphatidylinositol 4,5-bisphosphate, generating two recently recognized intracellular messengers, 1,2-diacylglycerol and inositol trisphosphate.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Increase of translatable mRNA for major microsomal proteins in n-alkane-grown Candida maltosa.

In an n-alkane-assimilating Candida sp., transfer from glucose- to n-alkane-containing medium induced changes in the microsomal proteins, and several distinctive polypeptides were demonstrated in the solubilized microsomal fraction derived from n-alkane-grown cells. Long-term-labeling and pulse-labeling experiments in vivo demonstrated the synthesis of the specific microsomal polypeptides. The polypeptides were synthesized as in vitro translation products directed by polyadenylated RNA extracted from n-alkane-grown cells. Two major polypeptides were partially purified from the microsomal fraction from n-alkane-grown cells, and antiserum was prepared in a rabbit. Immunoprecipitation of these two polypeptides was accompanied by an increase in the amount of translatable mRNA. The molecular weights of the polypeptides derived from long-term-labeling, pulse-labeling and in vitro translation experiments appeared to be identical.