Hiromi Mitsui

Preadipocytes possess cellular retinoid binding proteins and their differentiation is inhibited by retinoids

Summary ST 13 preadipocytes can be induced to differentiate into mature adipocytes by maintaining them as a confluent monolayer in a medium containing insulin. The differentiated cells synthesize and accumulate a large amount of triglyceride in their cytoplasm as multiple lipid droplets, and at the same time, their insulin binding activity increased 50 fold over that of the undifferentiated cells. Vitamin A, an essential nutrient for animals, inhibited the preadipocyte differentiation reversibly at a non-toxic dose. Retinol- and retinoic acid binding proteins were detected in the cytoplasm of preadipocytes suggesting the inhibition of differentiation might be mediated by these proteins.

Establishment of a clonal cell line that differentiates into adipose cells in vitro

SummaryFrom the fibroblastic cells of murine mammary tumor tissue we isolated a clonal cell line that could be induced to differentiate into fat-accumulating cells in vitro. Differentiation began after the cultures had reached confluence and was accompanied by (a) an increase in the incorporation of sodium acetate into the triglyceride (TG) fraction of cellular lipids, (b) a more than 50-fold increase in cellular TG content per milligram cell-layer protein basis and (c) the cessation of cellular DNA synthesis. The addition of insulin to the culture medium enhanced lipid accumulation and increased the fraction of cells that differentiated into adipocytes. When insulin (5 to 10 μg/ml) was added exogenously, 80% or more of the cells were induced to differentiate into mature adipocytes within 2 weeks. This cell line can be used as a model for mammalian cell differentiation and also as a convenient material for the study of lipid metabolism in adipocytes.

Reversible G1 arrest of a human Burkitt lymphoma cell line(Raji) induced by tunicamycin

Summary The effects of tunicamycin on the growth of a human Burkitt lymphoma cell line(Raji) were examined. The growth of cells was reversibly inhibited in the presence of tunicamycin (

cDNA cloning and nucleotide sequence of rat muscle-specific enolase (ββ enolase)

The nucleotide sequence of rat muscle‐specific enolase cDNA was determined by sequencing three cDNA clones encoding this enolase isozyme. The nearly full‐length cDNA consists of 13‐bp 5′‐ and 84‐bp 3′‐noncoding regions and a poly(A) tail in addition to a 1302‐bp coding region encoding a polypeptide composed of 434 amino acid residues. The deduced primary structure of this enolase isozyme is about 80% similar to those determined previously for rat neuron‐specific and non‐neuronal enolase isozymes. Southern blot analysis suggested strongly the existence of a single copy of the muscle‐specific enolase gene per haploid genome. The mRNA for this enolase isozyme was detected in rat skeletal muscle on day 1 after birth and its level increased rapidly during 10–30 days after birth without any change in its size (1500 bases).

Altered molecular structure of HLA-DR antigens synthesized in the presence of tunicamycin

Summary Effects of tunicamycin on the synthesis of proteins and glycoproteins, especially the surface antigens of a human lymphoblastoid cell line (Raji), were investigated. Electrophoretic profiles of [3H]leucine labeled membrane components were similar in the tunicamycin treated cells and untreated cells. However, the respective electrophoretic profiles of [3H]glucosamine labeled membrane components were remarkably different. In the presence of tunicamycin, the relative amount of HLA-DR antigens in the newly synthesized membrane was remarkably reduced, and both subunits of the antigens showed reduced apparent molecular weights. Moreover, one of the subunits completely lost its [3H]glucosamine labeled portion(s), and the other subunit still contained [3H]glucosamine labeled portion(s). These results imply that HLA-DR antigens possess either lipid carrier dependent oligosaccharides or other type(s) of oligosaccharide(s).

Tunicamycin inhibits the differentiation of ST 13 fibroblasts to adipocytes with suppression of the insulin binding activity

Abstract ST 13 cells are a clonal line of murine fibroblasts that are capable of differentiating into adipocyte-like cells in vitro . When the cells were maintained as a confluent monolayer, they began to accumulate lipid droplets and to exhibit a rapid increase of insulin binding activity. Tunicamycin, a specific inhibitor of dolichol-mediated protein glycosylation, blocked this adipose conversion without affecting cell growth and total protein synthesis. The inhibitory effect of tunicamycin was dose-dependent and reversible. Enhancement of the incorporation of [ 14 C]acetate into triglyceride fraction accompanying the adipose conversion was completely inhibited by tunicamycin, whereas the incorporation into phospholipid fraction was only partially affected. The insulin binding activity increased about 10-fold during differentiation, but was completely suppressed in tunicamycin-treated cells.

MESENCHYME CELLS IN STARFISH DEVELOPMENT: EFFECT OF TUNICAMYCIN ON THEIR DIFFERENTIATION, MIGRATION AND FUNCTION

The effect of tunicamycin, an inhibitor of protein glycosylation, on starfish development was investigated. Specific developmental events such as 1) bulging of the archenteron tip, 2) migration of mesenchyme cells, 3) formation of coelomic pouches and 4) mouth formation, are inhibited in the presence of this drug. These events are discussed in connection with differentiation, migration and function of mesenchyme cells. The possibility is discussed that tunicamycin exerts its effect by interfering with de novo synthesis of a cell surface factor(s) supporting dynamic cell surface activities.

Stimulation of cell proliferation by vitamin A derivatives on murine sarcoma virus-transformed mouse cells in serum-free culture

The effect of retinoids (Rds) on cell proliferation was studied in serum-free culture condition, using non-transformed and transformed derivatives of BALB 3T3. Cell proliferation of an SV40-transformed line was inhibited significantly by Rd treatment. However, proliferation of two cell lines that were transformed by a Kirsten and Moloney strain of murine sarcoma virus (MSV) and produced growth factor into culture medium, was remarkably stimulated by Rds. Addition of serum masked both the inhibitory and stimulatory effects of Rds.

The high affinity receptor for ω-conotoxin represents calcium channels different from those sensitive to dihydropyridines in mammalian brain

A monoclonal antibody recognizing the alpha 2 delta complex of the dihydropyridine (DHP)-sensitive calcium channel of skeletal muscle immunoprecipitated most of the DHP receptor solubilized from bovine and rabbit brains, and bovine cardiac muscle. However, it did not significantly immunoprecipitate the high affinity omega-conotoxin receptor solubilized from these brains. These results indicate that the DHP receptor and the high affinity omega-conotoxin receptor are different molecules in mammalian brain.

Specific changes in the surface glycoprotein pattern of a human leukemic null cell line NALL-1 associated with morphologic and biological alterations induced by phorbol-ester

Summary 12-0-tetradecanoylphorbol-13-acetate, a highly active tumor-promoting agent, inhibits cell proliferation of human leukemic null cell line NALL-1 at a dose range of 10 −9 – 10 −7 M. Soon after the addition of 12-0-tetradecanoylphorbol-13-acetate to suspension culture, cells began to adhere to the substratum. Associated with the change in cell behavior, rate of DNA synthesis decreased rapidly but rate of RNA and protein synthesis remained essentially unchanged. After 48hr treatment with 12-0-tetradecanoylphorbol-13-acetate, adherent and growth arrested cells were all alive. Changes in surface glycoproteins of these cells were analyzed by the neuraminidase/galactose oxidase or periodate/NaB[ 3 H 4 ] surface-labeling technique followed by polyacrylamide gel electrophoresis and fluorography. In 12-0-tetradecanoylphorbol-13-acetate treated cells, a glycoprotein with an apparent molecular weight of 145,000 was strongly labeled. The amount of HLA-DR antigen was also increased. These and other observations suggest that NALL-1 cells are induced by 12-0-tetradecanoylphorbol-13-acetate to differentiate into mature cells having some properties of B-cell blasts.