Low Mr GTP-binding proteins in human platelets: cyclic AMP-dependent protein kinase phosphorylates m22KG(I) in membrane but not c21KG in cytosol

We have purified and characterized two kinds of GTP-binding proteins with Mr of 22,000 in human platelet membrane (main; m22KG(I), minor; m22KG(II)) (Nagata, K. and Nozawa, Y. (1988) FEBS Lett. 238, 90-94). In this study, the main GTP-binding protein (m22KG(I)) was found to be phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), but not by protein kinase C. About 0.5 mol of phosphate was maximally incorporated into one mol of the protein and this phosphorylation was inhibited in the presence of A-kinase inhibitor. Phosphorylation of m22KG(I) did not alter either its GTP-binding or GTPase activity. When m22KG(I) was incubated alone or in the presence of 100 microM guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and then exposed to A-kinase, no significant changes in the level of phosphorylation were observed. On the other hand, the most abundant GTP-binding protein with Mr of 21,000 (c21KG) in human platelet cytosol, which was identified as a transformation suppressor gene product (rap 1 protein, smg p21 and Krev-1 protein), was not phosphorylated by A-kinase under the same condition. However, c21KG was phosphorylated by A-kinase after pretreatment with alkaline phosphatase.     

Characteristics of major mutagenicity of instant coffee

Detailed quantitative studies on the mutagenicity of methylglyoxal showed that its contribution to the total mutagenicity of instant coffee on S. typhimurium TA100 was minor although we reported previously (Kasai et al., 1982) that its contribution to the mutagenicity of freshly brewed coffee was about 50%. Cysteine suppressed the mutagenicity of methylglyoxal and of methylglyoxal when added to instant coffee, but did not affect the mutagenicity of coffee itself. Catalase suppressed most of the mutagenicity of coffee, but not that of methylglyoxal or of methylglyoxal added to coffee.     

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

Summary To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.     

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.     

Augmentation of retinoic acid-induced granulocytic differentiation in HL-60 leukemia cells by serine/threonine protein phosphatase inhibitors.

To evaluate the involvement of protein phosphatases (PP) in differentiation of human myelogenous leukemia HL-60 cells, we made use of potent inhibitors of PP1 and PP2A, calyculin-A (CAL-A) and okadaic acid (OKA). CAL-A and OKA could augment all-trans retinoic acid (ATRA)-induced granulocytic differentiation, whereas the differentiation toward macrophage lineage by 12-o-tetradecanoylphorbol acetate (TPA) was unchanged in the presence of CAL-A. CAL-A augmented the phosphorylation of 18K, 23K and 30K proteins induced by ATRA. The PP1 and PP2A were identified and were present mainly in the cytosol of HL-60 cells. These results suggest that either PP1 or PP2A or both may be involved in regulating granulocytic differentiation of HL-60 cells.     

Site-directed mutagenesis of glutamine residue of calmodulin. Activation of guanylate cyclase of Tetrahymena plasma membrane

Tetrahymena calmodulin (CaM) differs from mammalian CaM in its ability to activate Tetrahymena guanylate cyclase. Of 12 differences in amino acid sequence, two occur near the carboxyl terminus (Gln-143----Arg and Thr-146----deletion). To investigate the functional significance of the carboxyl-terminal region in activation of the guanylate cyclase, three mutated CaMs were engineered by using cassette mutagenesis of rat CaM cDNA: Gln-143----Arg (CaM.A), Thr-146----deletion (CaM.D), and Gln-143----Arg/Thr-146 deletion (CaM.AD). Recombinant wild type CaM (wCaM), CaM.A, CaM.D, and CaM.AD were indistinguishable in their ability to activate cyclic AMP phosphodiesterase. The two mutated CaMs (CaM.A and CaM.AD) with the Gln-143 replacement activated guanylate cyclase of Tetrahymena plasma membrane in the presence of Ca2+, with the maximal activation being half of that produced by Tetrahymena CaM. In contrast, neither CaM.D nor wCaM could stimulate the cyclase activity. A CaM antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), prevented the cyclase activation by either Tetrahymena CaM, CaM.A, or CaM.AD. Thus, we conclude that Arg-143 is in a region of the molecule involved in activation of Tetrahymena guanylate cyclase. The data also suggest that the cyclase activation by Tetrahymena CaM requires complex macromolecular interactions between the entire CaM molecule and the enzyme.     

Fibroblast colonies in monolayer cultures of human bone marrow

In a liquid culture of human bone marrow, the development of fibroblast colonies takes place on days 6 to 9. Twenty percent fetal calf serum is used as the stimulus for fibroblast colony growth. Human bone marrow cells are plated as 2 × 10₅ cells in the culture. Normal human bone marrow yields 47 ± 4 fibroblasts colonies per 2 × 10₅ cells plated. Bone marrow fibroblast cultures using agar or methylcellulose restrict colony formation. Marked colony suppression was observed in acute leukemia, and a discrete colony number was observed in hypoplastic anemia. This fibroblast culture method should be applied to a larger number of patients to determine whether it has a pathognomonic value and clinical significance.     

Elongation of oligonucleotides in the 3'-direction with activated mononucleotides and their analogs using RNA ligase.

P1-Adenosine 5'-P2-2',3'-ethoxymethylidene nucleosides [A(5')ppN(Em)] from four common nucleosides have been prepared and used for single addition of nucleotides to elongate oligonucleotide chains in the 3'-direction in RNA ligase reaction. U-U-C, T-U-C and A-C-C were used as acceptors. Structural dependence in these acceptors was found to be smaller compared to joining reactions between oligonucleotides. Adenosine analogs including 8-bromo-, 2'-fluoro-, 2'-azido-, 8,2'-O-cyclo-, 8,2'-S-cyclo-adenosine, arabinosyladenine and 2'-deoxyadenosine were added to the 3'-end of A-C-C by adenylation chemically followed by joining with RNA ligase. Symmetrical 5'-pyrophosphates of 8-bromo-, 2'-fluoro- and 2'-azido-adenosine were not recognized as donor substrates.