Biomedical subjects

A De Flora

Publications and source records attributed to A De Flora.

At least 19 recordsLinked to original sources

PubMed2000

Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors.

Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD(+) (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 microM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3-deaza-cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.

Adenosine Diphosphate Ribose↗

PubMed1999

Structural and enzymatic characterization of human recombinant GDP-D-mannose-4,6-dehydratase.

GDP-D-mannose-4,6-dehydratase (GMD) is the key enzyme in the 'de novo' pathway of GDP-L-fucose biosynthesis. The reported cDNA sequences for human GMD predict three forms of different length, whose 'in vivo' occurrence and molecular properties are completely undefined. Here, we report the expression in Escherichia coli and the properties of each native recombinant GMD form. Only the 42 kDa long GMD (L-GMD) and the 40.2 kDa (M-GMD) forms were recovered as soluble functional proteins, while the 38.7 kDa form, short GMD (S-GMD), lacking an N-terminal domain critical for dinucleotide binding, was inactive and formed a precipitate. Both L-GMD and M-GMD are homodimers and contain 1 mol of tightly bound NADP+. Their kinetic properties (Km, Kcat) are apparently identical and both forms are non-competitively feedback-inhibited by GDP-L-fucose to a similar extent. M-GMD is the predominant enzyme form expressed in several human cell lines. These data seem to suggest that modulation of the 'de novo' pathway of GDP-L-fucose biosynthesis involves mechanisms other than differential 'in vivo' expression of GMD forms.

Amino Acid Sequence↗

PubMed1999

Role of macrophage protection in the development of murine AIDS.

Macrophages play a key role in AIDS pathogenesis and thus controlling infectivity and viral replication in these cells is a key issue in any antiretroviral therapy. In the present study, using a murine model of AIDS, we evaluated new therapeutic approaches specifically designed for the protection of macrophages. Based on previous observations, we took advantage of the unique ability of autologous erythrocytes to deliver drugs selectively to macrophages. The antiviral drugs selected were a new homodimer of AZT (AZTp2AZT) and reduced glutathione (GSH). The addition of an oral drug for the protection of lymphocytes (i.e., AZT) was also investigated. C57BL/6 mice infected with the retroviral complex LP-BM5 were treated with GSH-loaded erythrocytes, GSH-loaded erythrocytes plus oral AZT, or GSH/AZTp2AZT-loaded erythrocytes plus oral AZT. The treatments including AZT and erythrocytes loaded with GSH alone or with GSH plus AZTp2AZT provided similar results and were most effective in inhibiting the progression of MAIDS; they reduced splenomegaly, lymphadenopathy, and hypergammaglobulinemia by about 70%, 90% and 83%, respectively, when compared with infected animals at 10 weeks postinfection. Evaluation of BM5d proviral DNA content in infected organs revealed that both treatments were able to almost completely protect most infected animals. They were also able to normalize the blood lymphocyte phenotype and to restore the responses of T and B cells to mitogens significantly. Treatment with GSH-loaded erythrocytes alone did not provide significant results for most parameters investigated, but a marked reduction in proviral DNA content was obtained in infected organs, including the brain. The results reported in this paper confirm the important role of macrophages in retroviral infection and moreover prove that erythrocytes, by selectively protecting these cells, strongly affect MAIDS progression. Furthermore, the combination of GSH- or GSH/AZTp2AZT-loaded erythrocytes with an oral nucleoside analogue (AZT) for the protection of lymphocytes provides additive responses in all the parameters investigated.

Animals↗

PubMed1999

Heterodimer-loaded erythrocytes as bioreactors for slow delivery of the antiviral drug azidothymidine and the antimycobacterial drug ethambutol.

Disseminated infection with Mycobacterium avium complex (MAC) remains the most common serious bacterial infection in patients with advanced AIDS. The organisms that make up this complex are found ubiquitously in the environment, yet rarely cause disseminated disease in nonimmunocompromised human patients; on the contrary, up to 50% of patients with AIDS may ultimately develop the pathology. Hence, therapeutic strategies able to inhibit HIV and Mycobacterium replication are needed. Because of the rapid plasma elimination and toxicity of the most commonly used drugs, daily multiple-drug therapies must often be continued throughout life, frequently causing major side effects and, as a consequence, poor patient compliance. Therefore, alternative strategies that reduce the toxicity of the drugs and allow prolonged application intervals are sorely needed. Since erythrocytes (RBCs) can behave as bioreactors able to convert impermeant prodrugs to membrane-releasable active drugs, new compounds (AZTpEMB, AZTpEMBpAZT, and AZTp2EMB) consisting of both an antiretroviral and an antimicrobial drug were designed and synthesized. Among these, only AZTp2EMB was hydrolyzed by erythrocyte enzymes and could be encapsulated inside RBCs. AZTp2EMB-loaded RBCs slowly released AZT and EMB in culture medium, reducing its concentration by one-half about every 48 hr of incubation at 37 degrees C. Moreover, when AZTp2EMB-loaded erythrocytes were incubated for 6 days in the presence of human macrophages infected with Mycobacterium avium (M. avium) a marked bactericidal effect (>1 log) was observed. Thus, AZTp2EMB-loaded erythrocytes could be used as endogenous bioreactors for AZT and EMB delivery in the treatment of HIV and M. avium infection.

Anti-HIV Agents↗

PubMed1999

Ligand-induced internalization of CD38 results in intracellular Ca2+ mobilization: role of NAD+ transport across cell membranes.

CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38(+) cells with either NAD+ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that the cADPR-generating activity of the endocytotic vesicles results in remarkable and sustained increases of intracellular free calcium concentration in different cells exposed to either NAD+, or GSH, or N-acetylcysteine. This effect of CD38-internalizing ligands on intracellular calcium levels was found to involve a two-step mechanism: 1) influx of cytosolic NAD+ into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8-N3-NAD+, and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed conversion of NAD+ to cADPR, followed by outpumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigargin-sensitive stores. This unknown intracellular trafficking of NAD+ and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD38(+) cells.

3T3 Cells↗

PubMed1998

Dimeric and tetrameric forms of catalytically active transmembrane CD38 in transfected HeLa cells.

CD38, a type II transmembrane glycoprotein, behaves as a catalytically active transporter responsible for ectocellular generation of cyclic ADP-ribose (cADPR) from NAD+ and for subsequent influx of cADPR across membranes [Franco, L., Guida, L., Bruzzone, S., Zocchi, E., Usai, C. and De Flora, A. (1998) FASEB J. in press]. cADPR regulates intracellular calcium homeostasis by releasing calcium from responsive stores. The cADPR-transporting function of CD38 requires channel-generating oligomeric forms of the protein rather than the 46 kDa monomers that have been described so far in CD38+ cells. Here we demonstrate that CD38, both in reconstituted proteoliposomes and in CD38-transfected HeLa cells, is a mixture of catalytically active monomers, homodimers and homotetramers. A soluble recombinant form of CD38 corresponding to its ectocellular region proved to be monomeric. Thus, association of native CD38 with either artificial or natural membranes seems to result in a reversible juxtaposition of monomers suitable to cADPR-transporting activity.

ADP-ribosyl Cyclase↗

PubMed1998

Preliminary crystallographic investigations of recombinant GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from E. coli.

The GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GM_ER) isolated from E. coli has been overexpressed as a GST-fusion protein and purified to homogeneity. The enzyme, an NADP+(H)-binding homodimer of 70 kDa, is responsible for the production of GDP-L-fucose. GM_ER shows significant structural homology to the human erythrocyte protein FX, which is involved in blood-group glycoconjugate biosynthesis, displaying 3,5 epimerase/reductase activity on GDP-4-keto-6-deoxy-D-mannose. GM_ER has been crystallized in a trigonal crystalline form, containing one molecule per asymmetric unit, suitable for high-resolution crystallographic investigations.

Amino Acid Sequence↗

PubMed1998

Defective intracellular activity of GDP-D-mannose-4,6-dehydratase in leukocyte adhesion deficiency type II syndrome.

Leukocyte adhesion deficiency type II (LAD II) is a rare genetic disease characterized by severe immunodeficiency which is related to defective expression in leukocytes of sialyl-Lewis X (SLeX), a fucosylated ligand for endothelial selectins. The molecular basis of LAD II is still unknown, but has been tentatively localized in the de novo pathway of GDP-L-fucose biosynthesis from GDP-D-mannose. Here, we demonstrate that in cell lysates from a LAD II patient, GDP-D-mannose-4,6-dehydratase (GMD), the first of the two enzymes of the pathway has a defective activity compared to control subjects. GMD in cell lysates from both parents showed intermediate activity levels. Cloning of GMD from patient and control lymphocytes ruled out any mutation affecting the amino acid GMD sequence and the purified recombinant proteins from both controls and the patient showed identical specific activities. Since the levels of immunoreactive GMD in cell lysates were comparable in the patient and in controls, the biochemical deficiency of intracellular GMD activity in LAD II seems to be due to mutation(s) affecting some still unidentified GMD-regulating protein.

Cloning, Molecular↗

PubMed1998

Expression of CD38 increases intracellular calcium concentration and reduces doubling time in HeLa and 3T3 cells.

CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38(-) HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38(+) HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.

3T3 Cells↗

PubMed1998

Macrophage protection against human immunodeficiency virus or herpes simplex virus by red blood cell-mediated delivery of a heterodinucleotide of azidothymidine and acyclovir.

Human herpesvirus (HSVs) are distributed worldwide and are among the most frequent causes of viral infection in HIV-1-immunocompromised patients. Hence, therapeutic strategies able to inhibit HSV-1 and HIV-1 replication are sorely needed. Until now, the most common therapies against HSV-1 and HIV-1 infectivity have been based on the administration of nucleoside analogs; however, to be active, these antiviral drugs must be converted to their triphosphorylated derivatives by viral and/or cellular kinases. At the cellular level, the main problems involved in the use of such drugs are their limited phosphorylation in some cells (e.g., antiretroviral drugs in macrophages) and the cytotoxic side effects of nucleoside analog triphosphates. To overcome these limitations, a new heterodinucleotide (AZTp2ACV) consisting of both an antiretroviral and an antiherpetic drug, bound by a pyrophosphate bridge, was designed and synthesized. The impermeant AZTp2ACV was encapsulated into autologous erythrocytes modified to increase their recognition and phagocytosis by human macrophages. Once inside macrophages, metabolic activation of the drug occurred. The addition of AZTp2ACV-loaded erythrocytes to human macrophages provided effective and almost complete in vitro protection from HIV-1 and HSV-1 replications, respectively. Therefore, AZTp2ACV acts as an efficient antiviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

Acyclovir↗

PubMed1998

Inhibition of murine AIDS by a new azidothymidine homodinucleotide.

A new antiretroviral drug (azidothymidine homodinucleotide, AZTp2AZT), designed for the protection of macrophages against retroviral infection, was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS) alone and in combination with oral azidothymidine (AZT). C57BL/6 mice were infected with the retroviral complex LP-BM5 and treated for 3 months by weekly administrations of 15 nmol of AZTp2AZT encapsulated into autologous erythrocytes for macrophage protection. AZTp2AZT treatment was found to reduce lymphoadenopathy (48%), splenomegaly (26%), and BM5d proviral DNA content in lymph nodes, spleen, and brain of 37%, 40%, and 36%, respectively, compared with untreated animals. AZT administration in drinking water (0.25 mg/ml) was more effective than administration of AZTp2AZT encapsulated into erythrocytes in reducing lymphoadenopathy, splenomegaly, gammaglobulinemia, and proviral DNA content in lymph nodes, but it caused a reduction in erythrocyte count and hematocrit levels. Although combined treatments do not provide additive responses in the several parameters investigated, they were found to be much more effective in reducing the proviral DNA content in brain (67%) than were monotherapies. Furthermore, no apparent signs of hematotoxicity were observed. Thus, macrophage delivery of antiviral drugs may contribute to brain protection from retroviral infections by mechanisms other than those exerted by oral AZT administration.

Animals↗

PubMed1998

Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate. Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity. In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+ release assay, and a thin layer chromatographic assay. Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR. The soluble cyclase was half-maximally stimulated by 5.3 microM cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP. The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes. The stimulatory effect of cGMP required ATP or ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction. The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA). The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mM and 10 mM, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.

ADP-ribosyl Cyclase↗

PubMed1998

Ectocellular CD38-catalyzed synthesis and intracellular Ca(2+)-mobilizing activity of cyclic ADP-ribose.

CD38 is a type-II transmembrane glycoprotein occurring in several hematopoietic and mature blood cells as well as in other cell types, including neurons. Although classified as an orphan receptor, CD38 is also a bifunctional ectoenzyme that catalyzes both the conversion of NAD+ to nicotinamide and cyclic ADP-ribose (cADPR), via an ADP-ribosyl cyclase reaction, and also the hydrolysis of cADPR to ADP-ribose (hydrolase). Major unresolved questions concern the correlation between receptor and catalytic properties of CD38, and also the apparent contradiction between ectocellular generation and intracellular Ca(2+)-mobilizing activity of cADPR. Results are presented that provide some explanations to this topological paradox in two different cell types. In cultured rat cerebellar granule neurons, extracellular cADPR (either generated by CD38 or directly added) elicited an enhanced intracellular Ca(2+)-response to KCl-induced depolarization, a process that can be qualified as a Ca(2+)-induced Ca2+ release (CICR) mechanism. On the other hand, in the CD38+ human Namalwa B lymphoid cells, NAD+ (and thiol compounds as well) induced a two-step process of self-aggregation followed by endocytosis of CD38, which resulted in a shift of cADPR metabolism from the cell surface to the cytosol. Both distinctive types of cellular responses to extracellular NAD+ seem to be suitable to elicit changes in the intracellular Ca2+ homeostasis.

ADP-ribosyl Cyclase↗

PubMed1998

The metabolism of 6-deoxyhexoses in bacterial and animal cells.

L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates. In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens. L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features. The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases. This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose. These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates. The enzyme involved in the first step of GDP-L-fucose biosynthesis in E. coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory. We have also cloned and fully characterized a human protein, formerly named FX, and an E. coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production. Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis. The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose. While several sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.

Animals↗

PubMed1998

The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP-ribose across membranes.

CD38 is a type II transmembrane glycoprotein expressed in many vertebrate cells. It is a bifunctional ectoenzyme that catalyzes both the synthesis of Cyclic ADP-ribose (cADPR) from NAD+ and the degradation of cADPR to ADP-ribose by means of its ADP-ribosyl cyclase and cADPR-hydrolase activities, respectively. The cyclase also converts NGD+ to cyclic GDP-ribose (cGDPR), which is refractory to cADPR-hydrolase. cADPR, but not cGDPR, is a potent calcium mobilizer from intracellular stores. It has been demonstrated to be a new second messenger involved in the regulation of calcium homeostasis in many cell types, from plants to mammals. The number of physiological processes shown to be regulated by cADPR is steadily increasing. A topological paradox exists because ectocellularly generated cADPR acts intracellularly. Here we demonstrate that the catalytic functioning of CD38 is accompanied by a cADPR (cGDPR) -transporting activity across natural and artificial membranes. In resealed membranes from CD38(+) human erythrocytes, transport of catalytically generated cADPR or cGDPR was saturation dependent and occurred against a concentration gradient. Likewise, CD38-reconstituted proteoliposomes were active in concentrating NAD+ (NGD+) -derived cADPR (cGDPR) inside the vesicle compartment. Moreover, the cADPR-transporting activity in CD38 proteoliposomes prevented the hydrolase-catalyzed degradation to ADPR that occurs conversely with detergent-solubilized CD38, resulting in selective influx of cADPR. In the CD38 proteoliposomes, catalytically active CD38 exhibited monomeric, dimeric, and tetrameric structures. In CD38 sense- but not in antisense-transfected HeLa cells, externally added NAD+ resulted in significant, transient increases in cytosolic calcium. These data suggest that transmembrane juxtaposition of two or four CD38 monomers can generate a catalytically active channel for selective formation and influx of cADPR (cGDPR) to reach cADPR-responsive intracellular calcium stores.

ADP-ribosyl Cyclase↗

PubMed1997

Expression, purification and characterization of GDP-D-mannose 4,6-dehydratase from Escherichia coli.

GDP-D-mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP-D-mannose to GDP-L-fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST-GMD protein and the thrombin-cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST-GMD fusion protein has a Km of 0.22 +/- 0.04 mM and a specific activity of 2.3 +/- 0.2 micromol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP-L-beta-fucose, the end-product of the pathway, inhibits it specifically. The GST-GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer. Apparently, this NADP+ is involved in the catalytic mechanism of GMD.

Chromatography, Gel↗

PubMed1997

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.

ADP-ribosyl Cyclase↗

PubMed1997

Effects of the murine L929 and L1210 cell lines on nitric oxide and TNF-alpha production by RAW 264.7 murine macrophages.

Co-cultures of the murine macrophagic cell line RAW 264.7 with the L929 fibrosarcoma cell line, but not with the leukemia L1210 cell line, showed enhanced NO production over control RAW 264.7 cells. This potentiating effect, which was observed in detectably mycoplasma-free conditions and required low concentrations of recombinant murine IFN-gamma, was due to soluble factors released from L929 cells and not to physical contact between the two cell types. The soluble factors were able to induce TNF-alpha in the macrophages and to potentiate the TNF-alpha release induced by IFN-gamma. Increased generation of NO in RAW 264.7 cells co-cultured with L929 cells was prevented by a neutralizing anti-TNF-alpha antibody, suggesting that TNF-alpha is an autocrine factor for iNOS expression in these conditions. Also the L929 cell line showed a 4- to 5-fold enhanced NO production following co-culture with RAW 264.7 cells, thus indicating that exposure of tumor cells to macrophages can lead to an increased iNOS expression in tumor cells themselves.

Animals↗