Lysis of growing fissin-yeast cells induced by aculeacin A,a new antifungal antibiotic

Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.     

Theophylline reduces poly (ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [3H]adenosine-labelled poly(ADP-ribose)molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose)molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecule. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.     

Role of proton dissociation in the transport of cystine and glutamate in human diploid fibroblasts in culture

The effect of extracellular pH on the transport interaction of cystine and glutamate in cultured human diploid cells was examined over the pH range of 5.8-8.0. The initial rates of uptake of cystine increased with an increase in pH and glutamate potently inhibited the cystine uptake independently of pH. The uptake of glutamate was almost invariable within the pH range, but it was inhibited by cystine in a pH-dependent manner; the inhibition increased with an increase in pH. Regardless of pH, the uptake of cystine and glutamate was strongly inhibited by alpha-aminoadipate, alpha-aminopimelate, and homocysteate. From the pK values of cystine and other amino acids, it is suggested that cystine is transported in the same ionic form as is glutamate.     

Reconstitution of mating active membrane vesicles in Paramecium

Mating-active membrane vesicles isolated from cilia of Paramecium caudatum by the urea-EDTA method were dissolved in 9 mM lithium diiodosalicylate (LIS). Membrane vesicles were reconstituted from the LIS-soluble fraction by dialysis. They showed an ability to induce conjugating pairs without prior occurrence of mating agglutination. The same kind of membrane vesicles was obtained when cilia were treated with 4 mM LIS and stored after removing LIS for two weeks.     

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.