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H Morimoto

Publications and source records attributed to H Morimoto.

At least 91 records · Page 5Linked to original sources
PubMed1993

Site-directed mutagenesis in hemoglobin: functional and structural study of the intersubunit hydrogen bond of threonine-38(C3)alpha at the alpha 1-beta 2 interface in human hemoglobin.

To clarify the functional and structural roles of Thr-38 alpha at the alpha 1-beta 2 interface, two artificial alpha-chain mutants, in which Thr-38 alpha is replaced by Ser (Hb T38 alpha S) or Val (Hb T38 alpha V), were prepared. Thr-38 alpha is one of the highly conserved amino acid residues in hemoglobins and forms a hydrogen bond to Asp-99 beta, which is a crucial residue to stabilize the T state, via a water molecule in the deoxygenated form. We investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Their oxygen equilibrium curves showed small changes in the oxygen binding properties. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering, and 1H-NMR spectra also indicated that the oxy and deoxy forms of these mutants show spectra characteristic of the R and T states, respectively, and the R-T transition is not very disturbed. The present structural and functional data of the mutants imply that the hydrogen bond between Thr-38 alpha and Asp-99 beta does not play a key role in stabilizing the deoxy T structure, which is in sharp contrast to the role of the hydrogen bond between Tyr-42 alpha and Asp-99 beta, and suggest that the interactions via the intersubunit hydrogen bonds are highly site-specific, depending on the amino acid residue which participates in them.

Globins↗
PubMed1993

Oxygen equilibrium properties of asymmetric nickel(II)-iron(II) hybrid hemoglobin.

Asymmetric Ni(II)-Fe(II) hybrid hemoglobin, XL[alpha(Fe)beta(Fe)][alpha(Ni)beta(Ni)], in which the alpha 1 beta 1 dimer containing ferrous protoporphyrin IX and the complementary alpha 2 beta 2 dimer containing Ni(II) protoporphyrin IX were cross-linked between Lys-82 beta 1 and Lys-82 beta 2 by reaction with bis(3,5-dibromosalicyl) fumarate, was synthesized and characterized. We have previously shown that (i) Ni(II) protoporphyrin IX, which binds neither oxygen nor carbon monoxide, mimics a fixed deoxyheme with respect to its effect on the oxygen equilibrium properties of the counterpart iron subunits in both symmetric Ni(II)-Fe(II) hybrid Hbs [Shibayama, N., Morimoto, H., & Miyazaki, G. (1986) J. Mol. Biol. 192, 323-329] and (ii) the cross-linking used in this study little affects the oxygen equilibrium properties of hemoglobin [Shibayama, N., Imai, K., Hirata, H., Hiraiwa, H., Morimoto, H., & Saigo, S. (1991) Biochemistry 30, 8158-8165]. These remarkable features of our model allowed us to measure the oxygen equilibrium curves for the first two steps of oxygen binding to the alpha 1 beta 1 dimer within the hemoglobin tetramer. At all pH values examined, the affinities of this asymmetric hybrid for the first oxygen molecule are as low as those of native hemoglobin. The hybrid did not show cooperative oxygen binding at pH 6.4, while significant cooperativity was observed with rising pH; i.e., the Hill coefficient was increased from 1.41 to 1.53 upon a pH change from 7.4 to 8.4. The electronic absorption spectrum of Ni(II) protoporphyrin IX in the alpha 2 subunit was changed upon carbon monoxide (or oxygen) binding to the alpha 1 beta 1 dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Linking Reagents↗
PubMed1993

Effects of adenosine A2 receptor agonists on the excitation of capsaicin-sensitive afferent sensory nerves in airway tissues.

We examined the effects of adenosine analogues on the asthmatic reactions induced by the stimulation of capsaicin-sensitive afferent sensory nerves. Intravenous (i.v.) injection of adenosine A2 receptor agonists, 5'-(N-ethylcarboxamido)-adenosine (NECA) and 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamido-adenos ine (CGS 21,680), dose dependently inhibited capsaicin-induced guinea-pig bronchoconstriction (1-1000 nmol kg-1), whereas i.v. administration of the adenosine A1 receptor agonist, N6-cyclo-hexyladenosine (CHA), did not affect it (1000 nmol kg-1). Intratracheal injection of NECA (0.05-5 nmol site-1) and CGS 21,680 (0.05-5 nmol site-1) also reduced capsaicin-induced constriction in a dose-dependent manner. However, NECA (1000 nmol kg-1) failed to inhibit substance P-induced guinea-pig bronchoconstriction. NECA (1-1000 nmol kg-1) dose-dependently inhibited cigarette smoke-induced rat tracheal plasma extravasation, but not substance P-induced reaction. NECA (0.1-10 microM) and CGS 21,680 (10 microM) significantly blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea-pig lung, whereas CHA (10 microM) had no effect. This evidence suggests that adenosine A2 receptors modulate negatively the excitation of capsaicin-sensitive afferent sensory nerves and substance P release from their endings in airway tissues.

Adenosine↗
PubMed1993

Studies on neurokinin antagonists. 3. Design and structure-activity relationships of new branched tripeptides N alpha-(substituted L-aspartyl, L-ornithyl, or L-lysyl)-N-methyl-N-(phenylmethyl)-L-phenylalaninamides as substance P antagonists.

As an extension of our study on discovering a novel substance P (SP) antagonist, we designed new branched tripeptides containing L-aspartic acid (2 and 5), L-ornithine (3 and 6), and L-lysine (4 and 7) by reconstructing the structure of the previously reported tripeptide SP antagonist [Ac-Thr-D-Trp(CHO)-Phe-NMeBzl (1), FR113680]. The strategy for this design was based on the postulate that the dipeptide half D-Trp(CHO)-Phe-NMeBzl in 1 is essential for receptor recognition. Molecular modeling studies implied that these newly designed tripeptides could mimic the spatial orientations of the essential dipeptide structure. As expected, all of these compounds potently inhibited 3H-SP (1 nM) binding to guinea pig lung membranes in the 10(-8) M range. The 1H-indol-3-ylcarbonyl derivatives (5-7) were slightly more potent than the corresponding 1H-indol-2-ylcarbonyl derivatives (2-4), as predicted by the molecular modeling studies. The structure-activity relationships studies on the selected 1H-indol-3-ylcarbonyl derivatives indicated that the threonine moiety at the side chain can be modified into a variety of structures without any significant loss of the activity. Furthermore in the L-lysine series, even dipeptide compounds having nothing or a simple acyl group at the epsilon-amino group, such as N alpha-[N alpha-(1H-indol-3-ylcarbonyl)-L-lysyl]-N-methyl-N-(phenylmethyl)- L-phenylalaninamide (18b), exhibited potent activity. These dipeptides belong to a new structural class of SP antagonist.

Animals↗
PubMed1993

The porphyrin-iron hybrid hemoglobins. Absence of the Fe-His bonds in one type of subunits favors a deoxy-like structure with low oxygen affinity.

Protoporphyrin-protoheme hybrid hemoglobins (Hb), in which the protohemes (Fe) in either the alpha- or beta-subunits were substituted with protoporphyrins IX (PP) alpha(PP)2 beta(Fe)2 and alpha(Fe)2 beta(PP)2 have been prepared. The structural and functional properties of these hybrid Hbs were investigated by measuring oxygen equilibrium curves and proton nuclear magnetic resonance spectra. The equilibrium constants of the first ligand, K1, observed for alpha(PP)2 beta(Fe)2 were much smaller than K1 values of HbA. The effects of pH and inositol hexaphosphate on K1 were substantially diminished. On the other hand, K1 values of alpha(Fe)2 beta(PP)2 were similar to those of HbA, including the pH and inositol hexaphosphate effects. The deoxy forms of alpha(PP)2 beta(Fe)2 and alpha(Fe)2 beta(PP)2 showed exchangeable proton resonances at 11 and 14 parts/million arising from the hydrogen bonds at the alpha 1 beta 2 contact in a deoxy-like structure. In the liganded form, these signals were dependent upon solution conditions. As K1 became larger, the reduction in the intensity of these signals was observed for both liganded forms. The resonance position of E11 Val originating from the beta subunits of alpha(PP)2 beta(Fe-CO)2 also varied in accordance with K1. We compare properties of PP-Fe hybrids with those of Co-Fe and Ni-Fe hybrids and conclude that the first oxygen binding to the beta heme may be linked to the metal-proximal His interaction in the alpha subunits. However, the first oxygen binding to the alpha heme is linked minimally to the metal-proximal His interaction in the beta subunits but may be correlated instead to the position of E11 Val relative to the porphyrin plane in the beta subunits.

Hemoglobins↗
PubMed1993

Kinetic study of free-radical-scavenging action of biological hydroquinones (reduced forms of ubiquinone, vitamin K and tocopherol quinone) in solution.

Kinetic study of free-radical-scavenging (FRS) action of eight kinds of biologically important hydroquinones (HQ's) (ubiquinol-10 (UQ10H2 1), ubiquinol-0 (UQ0H2 2), vitamin K1 HQ (VK1H2 3), vitamin K3 HQ (VK3H2 4), alpha-, beta-, gamma-tocopherol HQ's (alpha-, beta-, gamma-TQH2 5, 6, 7), and 2,3,5-trimethyl-1,4-HQ (TMQH2 8)) has been performed. The second-order rate constants, k3, for the reaction of HQ's 1-8 with substituted phenoxyl radical (PhO.) in ethanol, diethyl ether, benzene, and n-hexane have been measured with a stopped-flow spectrophotometer, as a model reaction of HQ's with unstable free radicals (LOO., LO., and HO.) in biological systems. The rate constant of UQ10H2 1 is similar to that of alpha-tocopherol in ethanol. The HQ's 3-8 showed higher reactivity than alpha-tocopherol in ethanol. Especially, the rate constants of VK1H2 3 and VK3H2 4 were found to be 31- and 21-fold larger than that of alpha-tocopherol, respectively, which has the highest reactivity among natural tocopherols. The rate constant of these HQ's increased by decreasing the polarity of solvents. The approximate order of magnitude of k3 value was (i) VK1H2 and VK3H2 > (ii) alpha-, beta-, and gamma-TQH2's and TMQH2 > (iii) alpha-tocopherol > (iv) UQ10H2 and UQ0H2 in solution. The result suggests that these biological HQ's also scavenge the active oxygen free radicals and prevent lipid peroxidation in various tissues and membranes. On the other hand, the reaction between substituted phenoxyl and biological quinones has not been observed.

Free Radical Scavengers↗
PubMed1993

Overcoming tumor necrosis factor and drug resistance of human tumor cell lines by combination treatment with anti-Fas antibody and drugs or toxins.

Monoclonal mouse anti-Fas antibody is directed against Fas antigen, a M(r) 36,000 encoded polypeptide that belongs to the family of cell surface proteins which includes nerve growth factor receptor, tumor necrosis factor (TNF) receptors, B-cell antigen CD40, and T-cell antigens OX40. Anti-Fas antibody mimics TNF-alpha in its cytolytic activity but not in other TNF-alpha-mediated activities. Thus, we examined if anti-Fas antibody synergizes in cytotoxicity with toxins and drugs. The present studies demonstrate that anti-Fas antibody in combination with diphtheria toxin (DTX), Adriamycin, or cis-platinum results in enhanced cytotoxicity and synergy and also overrides resistance to TNF, drugs, or toxins when tested against a battery of human tumor cell lines. Synergy with anti-Fas and DTX requires that DTX is enzymatically active, since inhibitors of DTX-mediated protein synthesis inhibition resulted in loss of synergy. When the plant toxin ricin was used, there was no synergy with anti-Fas antibody but rather an additive effect. The synergy was not obtained in a TNF receptor-negative line but was achieved with other anti-Fas-resistant lines. Cell lines resistant to either Adriamycin or cis-platinum were rendered sensitive by the combination of drug and anti-Fas antibody. Further, combination treatment of anti-Fas and Adriamycin overcame resistance of the gp 170-expressing, multidrug-resistant MDR ovarian line. In all cases, cytotoxicity was augmented by pretreatment of target cells with gamma-interferon which upregulates Fas antigen expression. These results show that anti-Fas antibody can synergize in cytotoxicity with toxins and chemotherapeutic drugs, and combination treatment can reverse resistance to TNF, toxins, and/or drugs.

Antibodies, Monoclonal↗
PubMed1993

Biosynthesis and characterization of phosphatidylglycerophosphoglycerol, a possible intermediate in lipoteichoic acid biosynthesis in Streptococcus sanguis.

A membrane enzyme preparation from Streptococcus sanguis was shown to convert sn-[14C]glycerol 3-phosphate and CDP-diacylglycerol (or deoxyCDP-diacylglycerol) into a series of progressively higher-molecular-weight [14C]oligophosphoglycerophospholipids in vitro. The first oligophosphoglycerophospholipid to accumulate (termed lipid-1) was purified to homogeneity; chemical analysis, gas-liquid chromatography and chemical degradation studies indicated the most likely structure to be phosphatidylglycerophosphoglycerol (PGpG). PGpG is formed directly from two molecules of phosphatidylglycerol (PG), one molecule of PG serving as a sn-glycerol 1-phosphate (pG) donor and the second serving as the pG acceptor, with co-production of diacylglycerol. These oligophosphoglycerophospholipids may be intermediates in the biosynthesis of lipoteichoic acids.

Carbon Radioisotopes↗
PubMed1993

Inhibitory effects of a cholecystokinin antagonist, loxiglumide (CR-1505), on the growth of freshly separated and xenografted human pancreatic cancer.

The effects of cholecystokinin (CCK) and a CCK antagonist, loxiglumide (CR-1505), on four freshly separated and six xenografted human pancreatic cancers, were investigated. The level of DNA synthesis in only one of five tested pancreatic cancers was enhanced by CCK at concentrations of 0.01-10 nM, while in the other four cancers DNA synthesis was not affected. The levels of DNA, RNA, and protein synthesis (by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation tests, respectively) in all the tested cancers were dose-dependently inhibited by loxiglumide at concentrations of 20-2000 microM, and the IC50 of loxiglumide for DNA synthesis in pancreatic cancers was 156 +/- 80 microM (means +/- SD). The in vivo effect of loxiglumide was assessed using a xenografted line (PC-HN) transplanted in nude mice. The in vivo 50% lethal dose of loxiglumide for nude mice was about 500 mg/kg. Death was caused by respiratory failure due to severe congestion of the lung after the administration of a large dose of loxiglumide. The growth of a PC-HN transplanted in the nude mice was significantly inhibited by subcutaneous loxiglumide at 250 mg/kg, twice a day for 28 days, which did not cause death. It is suggested that loxiglumide inhibits the in vivo and in vitro growth of human pancreatic cancer, perhaps independently of its action as a CCK antagonist, and this study also suggests that loxiglumide may be a new type of therapeutic agent to be used for the treatment of human pancreatic cancer.

Animals↗
PubMed1993

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1.

The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes. The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0 degrees C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 degrees C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-2H]ADP.Vi shifts both peaks slightly more upfield without changing their widths or relative areas.

Adenosine Diphosphate↗
PubMed1993

Cytotoxic properties of a new synthetic demethylpodophyllotoxin derivative, BN 58705, against human tumor cell lines.

The in vitro cytotoxic properties of a newly synthesized demethylpodophyllotoxin derivative, 4-o-butanoyl-4'-demethylpodophyllotoxin (BN 58705), were determined by using several human tumor cell lines of different histological origin and of different sensitivity to conventional chemotherapeutic drugs (Adriamycin and cis-diammine-dichloride platinum). BN 58705 is shown to be cytotoxic against various human tumor cell lines as assessed by the MTT assay. Furthermore, BN 58705 is shown to be cytotoxic against several drug-resistant tumor cell lines. BN 58705 is cytotoxic at concentrations 100- to 1000-fold lower than those of Adriamycin or cis-diammine-dichloride platinum required to achieve similar cytotoxicity. BN 58705 did not mediate DNA fragmentation of target cells, whereas the epipodophyllotoxin-like etoposide induced DNA cleavage by stabilizing the DNA-enzyme intermediate. Like vinca alkaloids, BN 58705 induced a block in the mitotic phase of the cell cycle. By comparison, BN 58705 exerted a stronger cytotoxic activity in vitro than did either etoposide, an epipodophyllotoxin, or vincristine, a vinca alkaloid. When BN 58705 was applied in vivo in mice, it resulted in low toxicity (50% lethal dose, 150 mg/kg). These results demonstrate than BN 58705 is cytotoxic to drug-resistant human tumor cell lines and is manyfold more potent than conventional drugs. The cytotoxic potency and low toxicity of BN 58705 are important criteria to establish its potential chemotherapeutic efficacy in vivo.

Animals↗
PubMed1993

[A simple method for flow cytometric measurement of Ki-67 reactive antigen on small solid tissues].

Monoclonal antibody Ki-67 is a useful and easy means for evaluating cell proliferative activity and malignancy of tumor cells. However, almost all studies for expression of Ki-67 reactive antigen on solid tumors have been performed only by microscopic observation of immunostained sections, and not by flow cytometry. The main reason for this is the difficulty associated with preparation of numerous suspended cells so as to be analyzable with FCM from small solid tissues. In this study, a simple and easy method for flow cytometric measurement of Ki-67 reactive antigen on small solid tissues was shown. The important steps in this method are cell dispersion with Triton X-100 and washless staining.

Antibodies, Monoclonal↗
PubMed1993

[TNF and TNF receptors: structure, mechanism of action, role in disease and therapy].

In this article, the topics on TNF and TNF receptors presented in the 4th International Conference on Tumor Necrosis Factor and Related Cytokines (Netherlands, May, 1992) are briefly summarized in Japanese, based on Dr. Benjamin Bonavida's report (Cancer Update, supplement 8, 1992) with permission. Following categorized themes were discussed in the meeting: 1) regulation of TNF synthesis, especially at the transcriptional level, 2) TNF receptors, including type I (55 kd), type II (75 kd) and soluble forms, 3) TNF cytotoxicity and immunomodulation with considerations for mechanisms, 4) TNF and infectious diseases such as Plasmodium falciparum infection, 5) TNF and inflammatory diseases such as rheumatoid arthritis and 6) TNF and cancer, in attempts to overcome drug resistance and TNF toxicity.

Arthritis, Rheumatoid↗
PubMed1992

Effects of FR 113680 and FK 224, novel tachykinin receptor antagonists, on cigarette smoke-induced rat tracheal plasma extravasation.

We examined the effects of novel tachykinin antagonists, FR 113680 (N alpha-[N alpha-(N alpha-acetyl-L-threonyl)-N1-formyl-D-tryptophyl]-N- methyl-N-phenylmethyl-L-phenylalaninamide) and FK 224 (N-[N2-[N-[N-[N-[2,3-didehydro-N-methyl-N-[N[3-(2-phenthylpheny l) - propionyl]-L-threonyl]-tyrosyl]-L-leucynyl]-D-phenylalanyl]-L-allo - threonyl]-L-asparaginyl]-L-serine nu-lactone) on rat tracheal plasma extravasation induced by cigarette smoke. Intravenous injection of FK 224 (0.032-3.2 mg kg-1) inhibited rat tracheal plasma extravasation induced by cigarette smoke and capsaicin. FR 113680 (32 mg kg-1 i.v.) also significantly inhibited cigarette smoke-induced plasma extravasation, whereas D-chlorpheniramine maleate, FPL 55712, atropine sulfate and indomethacin had no effect. Tracheal plasma extravasation induced by substance P (SP) and neurokinin A (NKA), but not histamine, was also reduced by intravenous administration of FR 113680 and FK 224. These findings suggest that cigarette smoke stimulates primary afferent sensory nerves, releases tachykinins and evokes plasma extravasation in rat trachea.

Amino Acid Sequence↗
PubMed1992

Stopped-flow kinetic study of vitamin E regeneration reaction with biological hydroquinones (reduced forms of ubiquinone, vitamin K, and tocopherolquinone) in solution.

A kinetic study of the regeneration reaction of vitamin E (tocopherol) with eight biological hydroquinones (HQs) (ubiquinol-10 (Q10H2 1); ubiquinol-0 (Q0H2 2); vitamin K1 HQ (VK1H2 3); vitamin K3 HQ (VK3H2 4); alpha-, beta-, and gamma-tocopherol-HQs (alpha-, beta-, and gamma-TQH2 5-7); and 2,3,5-trimethyl-1,4-HQ (TMQH2 8)) in solution was performed. The second-order rate constants (k4) for the reaction of HQs 1-8 with alpha-tocopheroxyl and 5,7-diisopropyltocopheroxyl radicals in ethanol, benzene, and isopropyl alcohol/water (5:1, v/v) solutions were measured with a stopped-flow spectrophotometer. The order of magnitude of k4 values obtained for HQs is VK1H2 > VK3H2 > alpha-TQH2 > beta-TQH2 approximately gamma-TQH2 approximately TMQH2 > Q10H2 > Q0H2, being independent of the kinds of tocopheroxyl radicals and the polarity of the solvents. The log of the k4 values obtained for HQs was found to correlate with their peak oxidation potentials. Comparing the k2 value (2.68 x 10(6) M-1 s-1 obtained for the reaction of alpha-tocopheroxyl with vitamin C (sodium ascorbate) with those (k4 = 2.54 x 10(5) and 8.15 x 10(5) M-1 s-1) obtained for the reaction of alpha-tocopheroxyl with Q10H2 and alpha-TQH2 in isopropyl alcohol/water mixtures, the former is approximately 11 and 3 times as reactive as the latter, respectively. On the other hand, the k2 value obtained for sodium ascorbate is smaller than the k4 values obtained for VK1H2 and VK3H2. These results suggest that mixtures of vitamin E and these HQs (as well as those of vitamins E and C) may function synergistically as antioxidants in various tissues and mitochondria.

Ascorbic Acid↗
PubMed1992

Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha.

We have reported that diphtheria toxin (DTX) mediates target cell lysis and intranucleosomal DNA fragmentation (apoptosis) and also synergizes with TNF-alpha. In this paper, we examined which step in the pathway of DTX-mediated inhibition of protein synthesis was important for induction of cytolytic activity and for synergy. Using a DTX-sensitive tumor cell line, we first examined the activity of the mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2). CRM 197 was not cytolytic for target cells and did not mediate intranucleosomal DNA fragmentation of viable cells. The failure of CRM 197 to mediate target cell lysis suggested that the catalytic activity of DTX is prerequisite for target cell lysis. This was corroborated by demonstrating that MeSAdo, which blocks the biosynthesis of diphthamide, inhibited DTX-mediated protein synthesis inhibition and also blocked target cell lysis. Furthermore, the addition of nicotinamide, which competes with NAD+ on the DTX action site of EF-2, also blocked DTX-mediated lysis. These findings suggest that ADP-ribosylation of EF-2 may be a necessary step in the pathway leading to target cell lysis. In contrast to the sensitive line, the SKOV-3 tumor cell line is sensitive to protein synthesis inhibition by DTX but is not susceptible to cytolysis and apoptosis by DTX. Thus, protein synthesis inhibition by DTX is not sufficient to mediate target cell lysis. The synergy in cytotoxicity obtained with the combination of DTX and TNF-alpha was examined in order to determine the pathway mediated by DTX in synergy. Like the direct lysis by DTX, synergy was significantly reduced by MeSAdo and by nicotinamide. Furthermore, synergy was not observed with combination of CRM 197 and TNF-alpha. These results demonstrate that, in synergy, DTX may utilize the same pathway required for its cytolytic activity. Pseudomonas aeruginosa exotoxin shared most the properties shown for DTX. Altogether, these findings demonstrate that DTX-mediated apoptosis is initiated at a step beyond the ADP ribosylation of EF-2.

ADP Ribose Transferases↗