Preferential ADP-ribosylation of arginine-3 in synthetic heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly.

Hen liver nuclear ADP-ribosyltransferase modified the synthetic heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) at arginine-2 and/or arginine-3. Trypsin treatment of ADP-ribosyl-Kemptide revealed that the ADP-ribosylation of arginine-3 was constantly more abundant than that of arginine-2. ADP-ribosylation of Kemptide suppressed the subsequent phosphorylation by cyclic AMP-dependent protein kinase.     

A molecular basis for familial hypertrophic cardiomyopathy: an alpha/beta cardiac myosin heavy chain hybrid gene

An alpha/beta cardiac myosin heavy chain (MHC) hybrid gene is coinherited with familial hypertrophic cardiomyopathy (FHC) in one kindred. FHC is a disease of the heart muscle characterized by a thickening of the left ventricular wall with myocyte and myofibrillar disarray that is inherited as an autosomal dominant trait. We demonstrate here and in the accompanying article that the cardiac MHC genes, which encode integral myofibrillar components, are mutated in all affected individuals from two unrelated families with FHC. In one kindred, an unequal crossover event during meiosis may have produced the alpha/beta cardiac MHC hybrid gene that is present in affected individuals. We conclude that mutations in the cardiac MHC genes can cause FHC.     

Mutual Contact of Adherent Polymorphonuclear Leukocytes Inhibits Their Generation of Superoxide

Superoxide generation by polymorphonuclear leukocytes (PMNs) in suspension, or adherent to glass or plastic, after stimulation with /V-formylmethionyl-leucyl-phenylalanine or phorbol myristate acetate was measured by cytochromec reduction and spin trapping. Amounts of superoxide generated by adherent PM Ns were inversely related to cell density. The generation of hydrogen peroxide was also inhibited at higher cell densities. In contrast to adherent cells, superoxide released by PMNs in suspension linearly increased with respect to cell number over a wider range. Microscopic observation indicated that the number of cells in mutual contact increased rapidly at cell densities higher than 4 × 10⁴ cells/cm², and inhibition of superoxide became apparent at higher cell densities. Mediators which could be released by PMNs, such as NO and adenosine, were not the cause of inhibition. Thesedatu suggest that mutual contact of PMNs suppresses their generation of superoxide. Survival rates of PMNs after stimulation increased at higher densities, indicating that the mutual contact-induced inhibition of superoxide generation by PMNs may be physiologically relevant at sites of inflammation.     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.     

Possible participation of Ca2+, Mg2+-dependent endonuclease in liver DNA fragmentation after N-methyl-N-nitrosourea treatment

We examined the fragmentation of DNA treated with N-methyl-N-nitrosourea under conditions in which Ca2+, Mg2+-dependent endonuclease is active. The molecular mass of DNA found in mouse liver slices treated with methylnitrosurea in the presence of Ca2+ plus Mg2+ was 4 X 10(5) Da. Similar results were obtained with a reconstituted system containing partially purified Ca2+, Mg2+-dependent endonuclease and methylnitrosurea-treated DNA. The enzyme extensively cleaved methylnitrosurea-treated DNA, compared with non-treated DNA. The methylnitrosurea-treated nuclear proteins obtained from mouse liver nuclei had no effect on the DNA fragmentation by the enzyme. Using closed-circular DNA treated with methylnitrosurea, the enzyme produced single-strand cuts in the DNA, as was seen in non-treated, closed-circular DNA, however, the rate of hydrolysis was increased. Ca2+, Mg2+-dependent endonuclease thus warrants further investigation, with regard to the precise mechanism of extensive degradation of DNA in cells treated with carcinogenic alkylating agents.     

Activation of ATPase Activity in the Chromatin Fraction of Pea Nuclei by Calcium and Calmodulin

ATPase, especially the Ca^2$-dependent enzyme, in the chromatinfraction isolated from nuclei of pea seedlings was activatedby bovine brain calmodulin and by protein that was judged tobe calmodulin from its Ca^2$-dependent activation of NAD kinase.This calmodulin-dependent ATPase activity was blocked by calmodulin-specificinhibitors. (Received July 11, 1983; Accepted October 24, 1983)     

Possible participation of glucocorticoid in elevation of 3',5'-cyclic AMP levels through inhibition of cyclic AMP phosphodiesterase.

Administration of prednisolone and cholate to rats elevated levels of cAMP (adenosine 3',5'-cyclic monophosphate) by 1.5- to 2.0-fold. Compounds such as prednisolone, hydrocortisone, cholate, and deoxycholate were found to be potent inhibitors of partially purified cAMP phosphodiesterase prepared from rat liver. Kinetic analysis showed that the prednisolone inhibition was noncompetitive with a Ki of 8.9 x 10(-4) M. These results suggest that in addition to increasing DNA-dependent RNA polymerase activity in vivo, a large application of glucocorticoid may incur elevation of intracellular cAMP levels.     

Differential sensitivity of pancreatic beta-cells to phorbol ester TPA and C-kinase inhibitor H-7 in nonpregnant and pregnant rats

In order to elucidate the possible role of C-kinase in exaggerated insulin release in pregnancy, the effects of phorbol ester TPA and a C-kinase inhibitor H-7 were investigated using the isolated perfused pancreas from nonpregnant and pregnant rats. At the termination of perfusion, the insulin content of the perfused pancreas was determined to estimate insulin biosynthesis. Insulin release from the perfused pancreas was markedly augmented by 20 nM TPA in the presence of 4.4 mM glucose in pregnant rats, but not in nonpregnant rats. When glucose concentrations in the perfusate were raised to 16.7 mM, insulin release from the perfused pancreas was profoundly enhanced in pregnant rats. TPA further augmented insulin release, but the insulin content was not affected by TPA. In contrast to the considerable effect of TPA in the presence of 4.4 mM glucose, the potentiating effect of TPA on insulin release was rather weaker in pregnant than in non-pregnant rats in the presence of 16.7 mM glucose. The release of insulin induced by 16.7 mM glucose was inhibited by the addition of 100 microM H-7 in nonpregnant rats, whereas insulin release from pregnant rat pancreases was not altered. Thus, the effect of TPA and H-7 on insulin release can be more clearly observed in the beta-cells of nonpregnant rats than those of pregnant ones when maximal concentrations of glucose are used as a stimulant. Exaggerated insulin release caused by glucose in pregnancy may be due to already fully activated C-kinase in the beta-cells.     

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.     

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.