Differential oligomerization of membrane-bound CD38/ADP-ribosyl cyclase in porcine heart microsomes

A protein fraction displaying ADP-ribosyl cyclase activity was purified from porcine heart microsomes which appeared as a major band of 45 kDa on Coomassie blue stained SDS-PAGE gel under reducing condition. Protein immunoblot analysis with antiserum to recombinant rat CD38 showed a series of bands (45-285 kDa) under nonreducing condition, while only the 45 kDa monomer under reducing condition. The high molecular weight oligomers of CD38 were found to be stable even upon treatment with various concentrations of SDS in the sample buffer and also upon incubation at lower temperature. These oligomers of CD38 also displayed higher ADP-ribosyl cyclase activity than that of the monomer.     

Retinoic acid and triiodothyronine stimulate ADP-ribosyl cyclase activity in rat vascular smooth muscle cells

Cyclic ADP-ribose (cADPR) is a nucleotide synthesized from beta-NAD- that can trigger or facilitate Ca2+-release through ryanodine-channels. We investigated the synthesis of cADPR (ADPR-cyclase activity) in cultured vascular smooth muscle cells (VSMC) from rat aorta in response to incubation with all-trans-retinoic acid (RA), 3,3',5'-triiodothyronine (T3), cortisol, beta-estradiol and 1-dehydrotestosterone. Only RA and T3 caused concentration-dependent (10(-9)-10(-6) M) stimulation of ADPR-cyclase activity in VSMC. Maximum stimulatory responses to RA (+100%) and T3 (+40%) were additive and the stimulatory effects of both hormones on ADPR-cyclase were due to an increase in Vmax without changes in the apparent Km. These observations indicate that in VSMC synthesis of cADPR can be upregulated by RA and T3. We propose that some of the actions of RA on VSMC such as enhancement of contractile competence, differentiation, and anti-proliferative effects might be elicited, at least in part, via upregulation of the cADPR/Ca2+-release signaling system.     

Muscarinic receptor-mediated dual regulation of ADP-ribosyl cyclase in NG108-15 neuronal cell membranes

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma x glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30-52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.     

Purification and characterization of a soluble form of mammalian adenylyl cyclase

An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 micromol/min/mg, corresponding to a turnover number of 720 min-1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by Gsalpha and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein betagamma subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.     

CD38 and ADP-ribosyl cyclase catalyze the synthesis of a dimeric ADP-ribose that potentiates the calcium-mobilizing activity of cyclic ADP-ribose

CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californica is the exchange of the base group of NAD+ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD+ leading to the formation of a dimeric ADPR ((ADPR)2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38(+) Namalwa cells from as low as 10 microM NAD+, was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5'-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR)2 was identified as that between the N1 of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me2SO:tert-butoxide. Although (ADPR)2 itself did not release Ca2+ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca2+-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR)2 is a new product of CD38 that amplifies the Ca2+-mobilizing activity of cADPR.     

A subfamily 2 homo-dimeric glutathione S-transferase mYrs-mYrs of class theta in mouse liver cytosol

A homo-dimeric subfamily 2 glutathione (GSH) S-transferase (GST) mYrs-mYrs of the class theta was isolated from mouse liver cytosol and purified to homogeneity. The first 28 N-terminal amino acid sequence of the GST was completely identical to that of rat subfamily 2 GST Yrs-Yrs of the class theta. GST mYrs-mYrs cross-reacted with anti-rat GST Yrs-anti-sera but not with anti-sera raised against rat GSTs Ya-Ya (alpha), Yb1-Yb1 (mu), and Yp-Yp (pi) and represented more than 95% of the mouse liver cytosolic GST activity to scavenge the reactive sulfate ester 5-sulfoxymethylchrysene of the potent carcinogen 5-hydroxymethylchrysene. The mouse class theta GST had little activity toward 1-chloro-2,4-dinitrobenzene and was unretainable on GSH and an S-hexyl-GSH affinity columns. GST mYrs-mYrs had a much higher GSH peroxidase activity toward fatty acid hydroperoxides than did the other classes of mouse GSTs.     

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P 相似文献   

Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase

CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.     

关于高维波动方程的Cauchy问题的解

Ｐｏｉｓｏｎ公式给出了高维波动方程的Ｃａｕｃｈｙ问题的解,在三维情况下,解的形式为一曲面积分;在二维情况下,解的形式为二重积分．本文证明,当初值条件φ,ψ及自由项ｆ满足一定条件时,Ｃａｕｃｈｙ问题的解可以以偏导数的形式给出．     

The TATA-binding protein from Saccharomyces cerevisiae oligomerizes in solution at micromolar concentrations to form tetramers and octamers

Equilibrium analytical ultracentrifugation has been used to determine the stoichiometry and energetics of the self-assembly of the TATA-binding protein of Saccharomyces cerevisiae at 30 degreesC, in buffers ranging in salt concentration from 60 mM KCl to 1 M KCl. The data are consistent with a sequential association model in which monomers are in equilibrium with tetramers and octamers at protein concentrations above 2.6 microM. Association is highly cooperative, with octamer formation favored by approximately 7 kcal/mol over tetramers. At high [KCl], the concentration of tetramers becomes negligible and the data are best described by a monomer-octamer reaction mechanism. The equilibrium association constants for both monomer <--> tetramer and tetramer <--> octamer reactions change with [KCl] in a biphasic manner, decreasing with increasing [KCl] from 60 mM to 300 mM, and increasing with increasing [KCl] from 300 mM to 1 M. At low [KCl], approximately 3 mole equivalents of ions are released at each association step, while at high [KCl], approximately 3 mole equivalents of ions are taken up at each association step. These results suggest that there is a salt concentration-dependent change in the assembly mechanism, and that the mechanistic switch takes place near 300 mM KCl. The possibility that this self-association reaction may play a role in the activity of the TATA-binding protein in vivo is discussed.     

一类散度型椭圆方程弱解的最大模估计

研究了散度型椭圆方程的弱解的性质。利用De Giorgi迭代技术做出了比Poisson方程稍一般的方程的弱解的最大模估计。     

The compact form of DNA in solution. V. The heat effect preceding compactization of double-chained DNA in PEG containing water-salt solutions]

The half-life of antipyrine has been estimated from saliva samples in ten subjects by a gas chromatographic method. Half-life, apparent volume of distribution and total body clearance estimated from saliva and plasma concentrations of antipyrine are not significantly different. The concentration of antipyrine in saliva is independent of flow rate within the range expected in healthy subjects in response to mechanical and sapid stimuli. Antipyrine estimation in saliva could facilitate many areas of phaamacokinetic research limited by the difficulty of obtaining serial plasma samples.     

The adenylyl and guanylyl cyclase superfamily

Antibodies and their recombinant fragments have enormous potential for therapy of malignant and other diseases, but there can be problems associated with their production and purification in the quantities required for therapeutic use. We investigated the use of gene therapy for the production of such recombinant antibody fragments in vivo. We generated two recombinant adenoviruses expressing the single chain Fvs (scFvs) fused to murine GM-CSF (mGM-CSF). The scFvs used are MFE-23 which binds to a human tumour-associated marker carcino-embryonic antigen (CEA) and B1.8 which binds the hapten 4-hydroxy-3-nitro-5-iodo-phenylacetyl (NIP). Using scFvs to target GM-CSF to tumour cells should reduce the systemic toxicity of GM-CSF but retain its ability as a cytokine to induce systemic immune responses to tumour targets. Cell lines infected with the recombinant adenoviruses in vitro express and secrete high levels of the scFv.mGM-CSF fusion proteins. The scFv retains specificity while the mGM-CSF portion is fully bioactive and there is no detectable degradation of the fusion product. We also demonstrated effective in vivo expression of the scFv.mGM-CSF proteins. C57BI/6 mice injected intravenously with the adenovirus encoding the MFE-23.mGM-CSF fusion produce high levels of the fusion protein by 2 days after infection. The scFv.mGM-CSF protein can be detected in the serum, at biologically active levels, for at least 20 days and the level of protein produced is related to the amount of adenovirus injected. This approach has the potential to streamline the testing of the many therapeutic strategies based on recombinant scFvs and we are currently testing these constructs in an animal model for antitumour activity.     

Evolution of the epidemic process as a form of solution to its internal contradictions

The author formulated the main contradition of the epidemic process consisting of contradiction between the interaction of the motive forces of the epidemic process, since this contradiction at the same time served as the imperative condition of the origination and as the cause of the subsequent arrest of the spread of infection. The main contradiction of the epidemic process is expressed in reduction of the number and limited activity of the sources of infection, attenuation or arrest of the mechanism of transmission of the causative agents, formation of immunity in the population, the appearance of hereditary resistance in the hosts to the causative agent of the infection. The action of the principal internal contradiction of the epidemic process in the course of evolution conditions genetic variability of the causative agents of the infectious diseases, intensification of the mechanisms of excretion of the parasites from the host organism, and increase of their resistance in the external environment, formation of latent forms of infection.