A simple method forin situ freezing of anchorage-dependent cells including rat liver parenchymal cells

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at –80°C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.     

A novel Vero cell line for use as a mammalian host-vector system in serum-free medium

We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots S3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33°C to 39.5°C.     

A new DNA profiling system for cell line identification for use in cell banks in Japan

Summary Using the polymorphic DNA probes, ChdTC-15, ChdTC-114, pYNH24, and λTM-18, a DNA profiling system was developed that verified identities of individual cultured cell lines collected in the Japanese cell banks, JCRB, RCB, and IFO. These highly polymorphic DNA probes include both VNTR (Variable Number of Tandem Repeats) sequences and substantial lengths of unique regions. In the mixed probe system, several distinct bands from four to eight can be used for cell line identification. These bands were widely spread in a range of molecular sizes, and were stable and reproducible under stringent conditions of Southern blot hybridization. Because the DNA profile was specific for each individual human cell line, it is useful not only to authenticate many existing cultured cell lines but also to monitor their identity during propagation in a laboratory, and to confirm newly established lines as unique.     

Preliminary crystallographic study of phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake)

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P21, with a = 44.1, b = 55.7, c = 48.8 A, and beta = 92.4 degrees. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.     

A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation

A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.     

Two distinct cDNAs for human IMP dehydrogenase

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.     

Reactivity of Limulus amoebocyte lysate towards (1----3)-beta-D-glucans

The structure activity relationship for beta-D-glucans for the gelation of the amoebocyte lysates of the horseshoe crab (Limulus) has been investigated. beta-D-Glucans that had no (1----3) linkages induced little or no gelation. The (1----3)-beta-D-glucans curdlan (unbranched), grifolan (approximately 33% branched), schizophyllan (approximately 33% branched), lentinan (approximately 40% branched). SSG (approximately 50% branched), and OL-2 (approximately 66% branched) induced significant gelation. The optimum concentration for gelation was correlated with the content of branching. Single chain (rather than a triple helix) conformation and higher molecular weight were associated with higher reactivity.     

A murine thymocyte clone expressing gamma delta T cell receptor mediates natural killer-like cytolytic function and TH1-like lymphokine production

The murine CD4- CD8- (double negative, DN) thymocyte cell line and clones expressing T cell receptor gamma delta chains in association with CD3 complex have been established and characterized. This line and a representative clone (DN7.12.11) which appear to derive from the minor population of CD3+ DN thymocytes can be stimulated to proliferate and to produce lymphokines by anti-CD3 or anti-Thy-1 antibodies or calcium ionophore plus phorbol ester. Autocrine proliferation is dependent on binding of interleukin (IL)2 to functional IL2 receptor. Upon stimulation, these cells produce IL2 and IFN-gamma but not IL4, resembling conventional CD4+ TH1 cells in this regard. The cloned line also mediates spontaneous cytolysis against a variety of tumor targets without regard for the presence of conventional major histocompatibility complex molecules on the target cell surface. Blocking and modulation experiments suggest that target recognition by the gamma delta/CD3 complex is not involved in the spontaneous lysis, resembling natural killer (NK) cells. The results suggest that gamma delta +DN T cells are able to have mature functions such as NK-like cytotoxicity and lymphokine secretion as peripheral gamma delta +T cells. They also provide a possible role of gamma delta + DN thymocytes in establishing a intrathymic environment for differentiation and selection of alpha beta-expressing T cells.     

A protein essential for recovering oxygen evolution in cholate-treated chloroplasts

A novel method for the reconstitution of oxygen evolution in cholate-extracted spinach thylakoid membranes was established and a protein essential for the reconstitution was purified from cholate extracts. Purification of the protein was accomplished by chromatography on a DEAE-Sephacel column. This protein (M_r 17 000) was reinserted into vesicular membranes reconstituted from cholate-extracted thylakoids in the presence of 25% glycerol to reactivate oxygen evolution.     

Sesquiterpenoids of Riccardia and Pallavicinia species

Riccardia species (Metzgeriales) contain various types of sesquiterpenes. R. jackii produces ent-selinane-, ent-aromadendrane-and ent-bicyclogermacrane-type sesquiterpenes together with (R)-cuparene and α-barbatene. Aneura pinguis (= Riccardia pinguis) is chemically quite different from R. multifida and R. jackii. The former produces a large amount of pinguisone. R. multifida contains 6-(3-methyl-2-butenyl)-indole and (+)-β-elemene as the major components. Pallavicinia longispina (Dilaenaceae; Metzgeriales) produces mainly spathulenol. The chiral properties of the sesquiterpenes isolated from R. jackii are quite similar to those of red algae, Laurencia species.