Suppression and stimulation of DNA synthesis by ADP-ribosylation of nuclear proteins from adult hen and chick embryo liver.

The stimulation of DNA synthesis by ADP-ribosylation of nuclear proteins was observed in chick embryo liver nuclei. In contrast, a significant decrease in template activity was detected in hen liver nuclei treated with NAD. When a 0.35 M NaCl extract from embryo, but not adult, liver nuclei was treated with NAD and then combined with either adult or embryo liver nuclear residue, the ability to activate the template was greatly enhanced. These results indicate that in the chick embryo liver, the ADP-ribosylation of the nuclei plays an important role as a regulator of DNA synthesis.     

Effect of poly(ADP-ribose) formation on DNA synthesis in chick-embryo-liver nuclei. Possible mechanism of template activation.

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.     

Membrane fluidity change in erythrocytes induced by complement system.

The structural change in erythrocyte membranes induced by antibody and complement was studied using phospholipid spin-labels. Sheep erythrocytes were labeled with phosphatidylcholine spin-label and various intermediate cells (erythrocyte-antibody complex (EA), EA bound with complement components from C1 to C7 (EAC1-7), EAC1-8, and EAC1-9) were prepared. Electron spin resonance spectra of EA, EAC1-7, and EAC1-8 were very similar to that of the erythrocytes, while that of EAC1-9 was markedly different. The overall splitting value for the lysed EAC1-9 (53 G) was much smaller than that for the erythrocytes (57 G), indicating a marked fluidization around the phosphatidylcholine label. The unlysed EAC1-9 membranes contained a limited fraction of the fluidized area. When EA was reacted with complement in the presence of 36% bovine serum albumin, the membranes were fluidized similarly to the lysed EAC1-9, although the hemolysis was largely blocked. The membranes of unlysed EAC1-9 prepared in isotonic (ethylenedinitrilo)tetraacetic acid were also fluidized, but to somewhat smaller extent. The role of C9 in the modification of erythrocyte membranes was also demonstrated using Mg2+ ghosts, which were prepared by hypotonic hemolysis in the presence of Mg2+. The membranes of Mg2+ ghost of EAC1-7 were markedly fluidized when bound with C8 and C9, but not affected by binding of C8 only. The component C8 was found to give a latent effect on the membranes that caused irreversible fluidization upon osmotic shock. The terminal component thus creates a fluidized area in the erythrocyte membranes through which small ions and molecules may diffuse more easily and the resulting osmotic unbalance may finally cause hemolysis.     

Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH₂-terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.     

Effect of polyanions and polycations on adenosine 3',5'-monophosphate-binding and protein kinase activity.

Histone, protamine, poly-L-arginine, and poly-L-lysine enhance the binding of adenosine 3′,5′-monophosphate (cyclic AMP) to rat liver cyclic AMP-dependent protein kinase as determined by Millipore filtration assay. Poly-L-glutamic acid and poly-L-aspartic acid suppress cyclic AMP-binding stimulated by histone. Poly-L-glutamic acid and poly-L-aspartic acid are effective against protein kinase and result in decrease in initial reaction velocity when histone is used as a protein substrate. Incubation of cyclic AMP-dependent protein kinase with 6 μg poly-L-glutamic acid produces half-maximal inhibition of cyclic AMP-dependent protein kinase when 30 μg histone is used as substrate.     

COLONY-FORMING ABILITY OF BONE MARROW CELLS OF W ANAEMIC MICE ON MACROPHAGE LAYER FORMED IN PERITONEAL CAVITY OF MICE

The colony-forming ability of haematopoietic cells of W anaemic mice was examined on the macrophage layer formed in the peritoneal cavity of mice. Bone marrow cells of W anaemic mice formed a considerable number of colonies on the macrophage layer, notwithstanding they did not form any colonies in the spleen of the same recipients. As the colony-forming ability of the bone marrow cells was not reduced by the incubation with ³H-thymidine, most of the cells which formed colonies on the macrophage layer seemed to stay in G₀ state. The interrelationship between the spleen colony-forming cells, the macrophage-layer colony-forming cells, and in vitro colony-forming cells was discussed.     

Image-based parameter inference for epithelial mechanics

Measuring mechanical parameters in tissues, such as the elastic modulus of cell-cell junctions, is essential to decipher the mechanical control of morphogenesis. However, their in vivo measurement is technically challenging. Here, we formulated an image-based statistical approach to estimate the mechanical parameters of epithelial cells. Candidate mechanical models are constructed based on force-cell shape correlations obtained from image data. Substitution of the model functions into force-balance equations at the cell vertex leads to an equation with respect to the parameters of the model, by which one can estimate the parameter values using a least-squares method. A test using synthetic data confirmed the accuracy of parameter estimation and model selection. By applying this method to Drosophila epithelial tissues, we found that the magnitude and orientation of feedback between the junction tension and shrinkage, which are determined by the spring constant of the junction, were correlated with the elevation of tension and myosin-II on shrinking junctions during cell rearrangement. Further, this method clarified how alterations in tissue polarity and stretching affect the anisotropy in tension parameters. Thus, our method provides a novel approach to uncovering the mechanisms governing epithelial morphogenesis.     

Genetic variations in humans associated with differences in the course of hepatitis C

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.     

Characterization of soluble and putative membrane-bound UDP-glucuronic acid decarboxylase (OsUXS) isoforms in rice

Arabinoxylans in crop plants are the major sugar components of the cell walls, and UDP-xylose is a key substrate in the biosynthesis of xylans. In this study, the six putative UDP-D-glucuronic acid decarboxylase genes from rice (Oryza sativa UDP-xylose synthase; OsUXS) were cloned. Except for the soluble form of OsUXS3 (GenBank Accession No. \AB079064), the remaining five OsUXS enzymes contain a putative membrane-bound region. The six OsUXS genes were classified into three types by phylogenetic analysis and were expressed during the development of rice seeds. The HPLC retention times of the enzyme products and NMR data, indicate that the recombinant OsUXS2 enzyme catalyzes the conversion of UDP-D-glucuronic acid to UDP-D-xylose. Interestingly, the reactions catalyzed by the recombinant OsUXS2 and OsUXS3 enzymes were inhibited by NADP+, and accelerated by NADPH. The catalytic activities of the recombinant OsUXS2 and OsUXS3 enzymes were strongly inhibited by UDP, UTP, TDP, and TTP. The expression levels of OsUXS genes changed in different manners during the development of rice seeds, suggesting that each corresponding OsUXS enzyme plays a significant role in rice seed development at a certain stage. In the present study, we report that the UXS2-type enzyme of rice is not only characterized for the first time but also show significant findings involved in the gene expression of OsUXSs.     

Cooperative conformational change and aggregation of concanavalin A in alkaline solutions

Three properties, the binding activity to Sephadex G-75, conformation, and the extent of aggregation, of concanvalin A. (con A) in alkaline pH solutions were examined with special attention to the time course and their time-independent final values. Highly cooperative conformational changes among four subunits were suggested which were coupled either with protonation in the case of demetallized con A or with metal binding in the case of metal-liganded con A. Midpoints of the conversions of the metal-liganded con A were about pH 8.8, 9.1 and 9.1 with respect to the activity, the conformational change and the aggregation, respectively. These values were about 1 pH higher than the corresponding values of demetallized con A: 7.9, 8.05 and 8.2. Each conversion took place in narrow pH ranges. The pH range for the loss of activity was found to be significantly lower than those of the other two. The aggregation was suggested not to be coupled with the conformational change. Dissociation into subunits did not take place indicating strong interactions among four subunits in the tetramer.