Phospholipid modulates in vitro replication of autonomous replicating sequence from human cells

A cloned plasmid, pmyc(H-K), containing sequences derived from human c-myc gene replicated in vitro in Raji nuclear extract in a semiconservative manner. Using this system, it was found that phosphatidylinositol and cardiolipin strongly inhibited the replication of pmyc(H-K) in vitro, whereas other phospholipids, i.e., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin, had no appreciable effect. The concentrations of phosphatidylinositol and cardiolipin producing 50% inhibition of the replication were 4.6 and 5.4 microM, respectively. Phosphatidylinositol and cardiolipin inhibited the relaxation of pmyc(H-K) supercoiled DNA, but showed little or weaker effects on DNA polymerase alpha and topoisomerase II in Raji nuclear extract. These results suggest that phosphatidylinositol and cardiolipin antagonize the replication of pmyc(H-K) in vitro, through, at least in part, the interaction with topoisomerase I.     

Protozoan hemoglobin from Tetrahymena pyriformis. Isolation, characterization, and amino acid sequence

A hemoprotein that can be defined as hemoglobin based on oxygen binding was isolated from Tetrahymena pyriformis. The protein exists in monomeric form and is separated into four fractions (Ia, Ib, IIa, and IIb) on a CM-cellulose column. From examinations of the absorption spectra and the N-terminal sequence, fractions Ia and Ib were assigned to the oxy-form and its met-form, respectively, of the one protein, while IIa and IIb corresponded to those of the other one. The complete amino acid sequence was therefore determined of fractions I and II. The I was composed of 121 amino acid residues, with the N-terminal serine being blocked. The II, on the other hand, consisted of 119 amino acid residues, its sequence being exactly identical to that of the third residue, lysine, to the C-terminal lysine of the fraction I. Although the genomic multiplicity cannot be ruled out completely, we have concluded that fraction II is a degradation product of the fraction I by endogeneous proteases. The amino acid sequence of T. pyriformis hemoglobin is very unique and showed no notable degree of similarity with the other hemoglobins sequenced so far, but it was found to be 33.9% identical with Paramecium caudatum hemoglobin by a maximal alignment.     

Synergistic action of phorbol ester and IL-3 in the induction of "connective tissue-type" mast cell proliferation

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induced the proliferation of connective tissue-type mast cells (CTMC) synergistically with IL-3 in a methylcellulose culture, as well as with IL-4. The culture of single CTMC and the serum-free culture of CTMC fractionated by Percoll density gradient centrifugation showed that this synergistic action of IL-3 and TPA required no effects of accessory cells or other humoral factors. Although the populations of CTMC acted on by TPA and IL-4 seemed to be close to each other, the velocity of colony growth induced by the simultaneous stimulation of the combination of TPA and IL-4 was faster than that induced by either TPA or IL-4 in the presence of IL-3. In addition, the addition of anti-IL-4 antibody did not neutralize the effect of TPA on the proliferation of CTMC. These results suggest that TPA and IL-4 act on the proliferation of CTMC synergistically with IL-3 via a different pathway. Beside TPA, other phorbol derivatives capable of activating protein kinase C (PKC) induced the proliferation of CTMC synergistically with IL-3, but phorbol derivatives which were unable to activate PKC did not. These results indicate that the activation of PKC is involved in the process of TPA action on the proliferation of CTMC. Furthermore, the facts that 1-oleoyl-2-acetylglycerol, which activated membrane PKC transiently, and staurosporine, which has been reported to inhibit PKC, did not induce the proliferation of CTMC in the presence of IL-3 and that the effect of TPA was exhibited by the sustained stimulation suggest that the action of TPA on the proliferation of CTMC requires at least two steps. The first one is the primary activation of membrane PKC and the second one is the disappearance of PKC from the cells, "down-regulation."     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta VIII. Effect of Mevinolin on Photoinduced Carotenogenesis

Mevinolin, which is a highly specific competitive inhibitorof 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase,was used in a search for photoinducible enzyme(s) other thanHMG-CoA reductase in the pathway of carotenoid biosynthesisin Rhodotorula minuta. The photoinduced production of carotenoids was competitivelyinhibited by mevinolin. The concentration of mevinolin thatis required to inhibit completely the production of carotenoidsdepends on the light dose given to the cells. However, the relationshipbetween the inhibition ratio and the concentration of mevinolinwas almost identical regardless of the light dose. These resultssuggest that the activity of enzymes involved in the formationof HMG-CoA may not be affected by light. When an adequate amount of mevalonate was added to the growthmedium that contained sufficient mevinolin for the completeinhibition of the photoinduction of the production of carotenoids,the same quantity of carotenoids was produced as in the absenceof mevinolin. Moreover, the production of carotenoids in thepresence of both mevinolin and mevalonate was inhibited by cycloheximide. It appears from these results that one or more photoinducibleenzymes, such as HMG-CoA reductase, may be present in the carotenogenicpathway beyond mevalonate. (Received April 12, 1989; Accepted January 16, 1990)     

Quantitative changes of free-base,riboside, ribotide and glucoside cytokinins in developing rice grains

Rice grains at various growth stages were analysed for endogenous free-base, riboside, ribotide and glucoside cytokinins on the basis of GC/MS and GC/SIM using deuterium-labeled internal standards. Cytokinins identified were trans- and cis-zeatins, trans- and cis-ribosylzeatins, isopentenyladenosine, isopentenyladenosine monophosphate, trans- and cis-ribosylzeatin monophosphates, trans- and cis-zeatin-O-glucosides, trans- and cis-ribosylzeatin-O-glucosides and zeatin-9-glucoside (trans/cis geometry was not determined). The highest amounts of cytokinins were recorded at the early growth stage, namely either heading, anthesis or milk stage, suggesting that cytokinins may play important roles in the development of the grain. Cis isomers of zeatin derivatives were always present and more abundant than trans isomers. It seemed unlikely that cis isomers were released from t-RNAs during the extraction procedure.     

An extra maternally derived X chromosome is deleterious to early mouse development

An extra copy of the X chromosome, unlike autosomes, exerts only minor effects on development in mammals including man and mice, because all X chromosomes except one are genetically inactivated. Contrary to this contention, we found that an additional maternally derived X (XM) chromosome, but probably not a paternally derived one (XP), consistently contributes to early death of 41,XXY and 41,XXX embryos in mice. Because of imprinted resistance to inactivation, two doses of XM remain active in the trophectoderm, and seem to be responsible for the failure in the development of the ectoplacental cone and extraembryonic ectoderm, and hence, from early embryonic death. Discordant observations in man indicating viability of XMXMXP and XMXMY individuals suggest that imprinting on the human X chromosome is either weak, unstable or erased before the initiation of X-inactivation in progenitors of extraembryonic membranes.     

Galactosylation at side chains of branched cyclodextrins by various beta-galactosidases.

The galactosyl transfer reaction to branched cyclodextrins (CDs) was investigated using lactose as a donor substrate and branched CDs as acceptors by various beta-galactosidases. Bacillus circulans beta-galactosidase synthesized galactosyl transfer products to branched CDs, of which the galactose residues were linked at side chains of branched CDs, not directly at CD rings. Aspergillus oryzae and Penicillium multicolor beta-galactosidases also produced derivatives galactosylated at side chains of branched CDs. The structures of main transgalactosylation products of branched CDs by these beta-galactosidases seem to be different from those by B. circulans beta-galactosidase, judging from the retention times on high performance liquid chromatography.     

Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells

Summary Bovine granulosa cell — conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5μg/ml insulin, and 0.5μg/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P<0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P>0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0.7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56° C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80° C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.     

Coexpression of the receptor-associated protein gephyrin changes the ligand binding affinities of alpha 2 glycine receptors.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein, whose ligand binding alpha subunit occurs in several isoforms in the mammalian central nervous system. Here we show that coexpression of the GlyR-associated protein gephyrin changes the agonist and antagonist binding affinities of GlyRs generated by alpha 2 subunit expression in 293 kidney cells. Thus, a receptor-associated protein modifies the functional properties of a neurotransmitter receptor. This may contribute to an optimization of the postsynaptic neurotransmitter response.