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E Hamel

Publications and source records attributed to E Hamel.

At least 181 records · Page 10Linked to original sources
PubMed1990

Reexamination of the role of nonhydrolyzable guanosine 5'-triphosphate analogues in tubulin polymerization: reaction conditions are a critical factor for effective interactions at the exchangeable nucleotide site.

Recently it was proposed [O'Brien, E. T., & Erickson, H. P. (1989) Biochemistry 28, 1413-1422] that tubulin polymerization supported by guanosine 5'-(beta,gamma-imidotriphosphate) [p(NH)ppG], guanosine 5'-(beta,gamma-methylenetriphosphate) [p(CH2)ppG], and ATP might be due to residual GTP in reaction mixtures and that these nucleotides would probably support only one cycle of assembly. Since we had observed polymerization with these three compounds, we decided to study these reactions in greater detail in two systems. The first contained purified tubulin and a high concentration of glycerol, the second tubulin and microtubule-associated proteins (MAPs). In both systems, reactions supported by nucleotides other than GTP were most vigorous at lower pH values. In the glycerol system, repeated cycles of polymerization were observed with ATP and p(CH2)ppG, but not with p(NH)ppG. With p(NH)ppG, a single cycle of polymerization was observed, and this was caused by contaminating GTP. In the MAPs system, repeated cycles of polymerization were observed with both nonhydrolyzable GTP analogues, even without contaminating GTP, but ATP was not active at all in this system. Binding to tubulin of p(NH)ppG, p(CH2)ppG, and, to a lesser extent, ATP was demonstrated indirectly, since high concentrations of the three nucleotides displaced radiolabeled GDP originally bound in the exchangeable site, with p(NH)ppG the most active of the three compounds in this displacement assay. The failure of GTP-free p(NH)ppG to support tubulin polymerization in our glycerol system even though it displaced GDP from the exchangeable site was further investigated by examining the effects of p(NH)ppG on polymerization and polymer-bound nucleotide with low concentrations of GTP. The two nucleotides appeared to act synergistically in supporting polymerization, so that a reaction occurred with a subthreshold GTP concentration if p(NH)ppG was also in the reaction mixture. Analysis of radiolabeled exchangeable-site nucleotide in polymers formed in reaction mixtures containing both GTP and p(NH)ppG demonstrated that p(NH)ppG which entered polymer did so primarily at the expense of GDP originally bound in the exchangeable site rather than at the expense of GTP. It appears that in the glycerol reaction condition, tubulin-p(NH)ppG cannot initiate tubulin polymerization but that it can participate in polymer elongation. ATP and p(CH2)ppG also entered the exchangeable site during polymerization without GTP in glycerol, as demonstrated by displacement of radiolabeled GDP from polymer when these alternate nucleotides were used.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1990

Antitubulin effects of derivatives of 3-demethylthiocolchicine, methylthio ethers of natural colchicinoids, and thioketones derived from thiocolchicine. Comparison with colchicinoids.

Esterification of the phenolic group in 3-demethylthiocolchicine and exchange of the N-acetyl group with other N-acyl groups or a N-carbalkoxy group afforded many compounds which showed superior activity over the parent drug as inhibitors of tubulin polymerization and of the growth of L1210 murine leukemia cells in culture. A comparison of naturally occurring Colchicum alkaloids with thio isosters, obtained by replacing the OMe group at C(10) with a SCH3 group, showed the thio ethers to be invariably more potent in these assays. The comparison included 3-demethylthiodemecolcine prepared from 3-demethylthiocolchicine by partial synthesis. Thiation of thiocolchicine with Lawesson's reagent afforded novel thiotropolones which exhibited high antitubulin activity. Their structures are fully secured by spectral data. Colchicine and several of its analogues show good antitumor effect in mice infected with P388 lymphocytic leukemia, and all of them show high affinity for tubulin and inhibit tubulin polymerization at low concentration. Consequently, antitubulin assays with this class of compounds can serve as valuable prescreens for the initial evaluation of potential antitumor drugs.

Animals↗
PubMed1990

Synthesis and biological evaluation of 2-styrylquinazolin-4(3H)-ones, a new class of antimitotic anticancer agents which inhibit tubulin polymerization.

A novel series of 2-styrylquinazolin-4(3H-ones which inhibited tubulin polymerization and the growth of L1210 murine leukemia cells was discovered. Extensive structure-activity relationship studies suggest that the entire quinazolinone structure was required, but activity was further enhanced by halide or small hydrophobic substituents at position 6. These analogues did not substantially interfere with the binding of radiolabeled colchicine, vinblastine, or GTP to tubulin and weakly stimulated GTP hydrolysis uncoupled from polymerization. Several analogues have shown in vivo tumor growth inhibitory activity in the L1210 leukemia model, with the lead compound 5o exhibiting good antitumor activity against murine solid tumors as well as human tumor xenografts.

Animals↗
PubMed1990

Endothelial cells inhibit the vascular response to adrenergic nerve stimulation by a receptor-mediated mechanism.

Rabbit central ear arteries, with and without endothelium, were perfused at a constant flow rate and the perfusion pressure was measured as an index of the vessel resistance. Transmural nerve stimulation (TNS) induced a frequency-dependent increase in perfusion pressure in all vessels that was blocked by tetrodotoxin, phentolamine, and prazosin. Removal of endothelium significantly enhanced contractions induced by TNS. The inhibitory effect of endothelium was not modified by indomethacin but was abolished by hemoglobin, indicating that endothelium-derived relaxant factor (EDRF) was the vasodilator involved. The endothelium-dependent inhibitory effect (rubbed vessel minus control vessel contractions) increased with time during the first 10-20 s after the beginning of TNS, and was frequency dependent and inhibited by low doses of phentolamine, which suggest a receptor-mediated mechanism. To analyze whether amine neurotransmitters are able to permeate the artery wall and contact the endothelial cell membrane, the passage of [3H]acetylcholine from the abluminal side to the lumen was studied in intact vessels. [3H]acetylcholine readily permeated the vessel wall, as assessed by radioautography, and appeared in the perfusion fluid at a concentration that explains the relaxation induced by perivascular acetylcholine. These data suggest that endothelial cells modulate the effect of perivascular neurotransmitters by a receptor-mediated mechanism. In the case of the sympathetic innervation, such modulation would be more relevant at low levels of transmitter release and would be minimized during intense sympathetic stimulation.

Acetylcholine↗
PubMed1989

Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibition of the binding of colchicine to tubulin.

Combretastatin A-4 (CS-A4), 3,4,5-trimethoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, and combretastatin A-2 (CS-A2), 3,4-(methylenedioxy)-5-methoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, are structurally simple natural products isolated from the South African tree Combretum caffrum. They inhibit mitosis and microtubule assembly and are competitive inhibitors of the binding of colchicine to tubulin [Lin et al. (1988) Mol. Pharmacol. 34, 200-208]. In contrast to colchicine, drug effects on tubulin were not enhanced by preincubating CS-A4 or CS-A2 with the protein. The mechanism of their binding to tubulin was examined indirectly by evaluating their effects on the binding of radiolabeled colchicine to the protein. These studies demonstrated rapid binding of both compounds to tubulin even at 0 degrees C (binding was complete at the earliest times examined), in contrast to the relatively slow and temperature-dependent binding of colchicine. Although the binding of the C. caffrum compounds to tubulin was quite tight, permitting ready isolation of near-stoichiometric amounts of drug-tubulin complex even in the absence of free drug, both CS-A4 and CS-A2 dissociated rapidly from tubulin in the presence of high concentrations of radiolabeled colchicine. Apparent rate constants for drug dissociation from tubulin at 37 degrees C were 3.2 x 10(-3) s-1 for CS-A4, 4.8 x 10(-3) s-1 for CS-A2, and 2.9 x 10(-5) s-1 for colchicine (half-lives of 3.6, 2.4, and 405 min, respectively). Thus, the effectiveness of the C. caffrum compounds as antimitotic agents appears to derive primarily from the rapidity of their binding to tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Identification of the cysteine residue of beta-tubulin alkylated by the antimitotic agent 2,4-dichlorobenzyl thiocyanate, facilitated by separation of the protein subunits of tubulin by hydrophobic column chromatography.

The mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate (DCBT) has been examined in detail. Shown in previous studies to inhibit tubulin polymerization [Abraham, I., Dion, R. L., Duanmu, C., Gottesman, M. M., & Hamel, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6839-6843] and to form a covalent bond preferentially with beta-tubulin [Bai, R., Duanmu, C., & Hamel, E. (1989) Biochim. Biophys. Acta 994, 12-20], DCBT has now been documented to interact at low concentrations with a high degree of specificity at cysteine residue 239 of beta-tubulin. These low DCBT concentrations also result in the partial inhibition of tubulin polymerization. Such findings strongly indicate that cysteine-239 of beta-tubulin is essential for microtubule assembly. Although alpha-tubulin is alkylated almost as well as beta-tubulin when the drug:tubulin ratio = 5:1 (Bai et al., 1989), beta-tubulin is alkylated about 25 times as extensively as alpha-tubulin, almost exclusively at Cys-239, when the drug:tubulin ratio = 1:5. In addition, we find that low concentrations of DCBT do not affect the binding of colchicine to tubulin but that colchicine and related compounds do reduce the alkylation of tubulin by DCBT. This suggests that Cys-239 of beta-tubulin is not involved in the binding of colchicine to tubulin but that this amino acid residue is at least partially masked by the drug when it is bound to the protein. We also describe a column chromatography procedure (hydrophobic chromatography on decylagarose) useful for the preparative resolution of unalkylated, although denatured, alpha- and beta-tubulin.

Alkylation↗
PubMed1989

Tubulin-dependent hydrolysis of guanosine triphosphate as a screening test to identify new antitubulin compounds with potential as antimitotic agents: application to carbamates of aromatic amines.

Tubulin-dependent GTP hydrolysis was evaluated for its potential as a relatively simple screening assay for new antimitotic drugs. Carbamates of aromatic amines were chosen as the test system because of the relatively diverse structures of compounds in this class already known to have antimitotic properties and because of the large number of such compounds in the NSC collection of the National Cancer Institute. Of 162 compounds evaluated, significant alterations in the GTPase reaction were observed with 26 agents. Sixteen of these had substantial inhibitory effects on tubulin polymerization (true positives), while ten did not (false positives). There were no false negatives (i.e., no agent inactive in the GTPase assay inhibited tubulin polymerization). The true positives were examined for effects on cell growth and mitosis, and four compounds had 50% inhibitory concentration values of 2 microM or less with L1210 murine leukemia cells. All four caused the accumulation of cells in metaphase arrest. We conclude that tubulin-dependent GTP hydrolysis can be used effectively to select new antitubulin compounds with potential as antimitotic agents from a large group of compounds of unknown activity.

Antineoplastic Agents↗
PubMed1989

Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4.

The African tree Combretum caffrum (Combretacae) has been found to contain a powerful inhibitor of tubulin polymerization (IC50 2-3 microM), the growth of murine lymphocytic leukemia (L 1210 and P 388 with ED50 approximately 0.003 microM and human colon cancer cell lines [(e.g. LoVo (ED50 = 0.005 microgram/ml), HT 29 (ED50 0.02 microgram/ml, Colo 205 (ED50 = 0.07 microgram/ml), DLD-1 (ED50 = 0.005 microgram/ml) and HCT-15 (ED50 = 0.0009 microgram/ml] designated combretastatin A-4 (1c). The structure assigned by spectral techniques was confirmed by synthesis.

Animals↗
PubMed1989

Mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate: alkylation of sulfhydryl group(s) of beta-tubulin.

The compound, 2,4-dichlorobenzyl thiocyanate (DCBT) was previously shown to cause mitotic arrest, disruption of intracellular microtubules, and inhibition of tubulin polymerization, with resistance to the drug conferred by a mutation in a beta-tubulin gene (Abraham, I., Dion, R.L., Duanmu, C., Gottesman, M.M. and Hamel, E. (1986) Proc. Natl. Acad. Sci. USA 83, 6839-6843). We have now examined its mechanism of action in further detail and conclude that DCBT acts as a sulfhydryl alkylating reagent. A mixed disulfide forms between the 2,4-dichlorobenzyl mercaptan moiety of DCBT and protein sulfhydryl groups with release of cyanate anion to the medium. Gel filtration and dialysis of complexes of tubulin formed with either [nitrile-14C]DCBT, [35S]DCBT or [benzyl-3H]DCBT demonstrated persistent association of 35S and 3H with denatured tubulin, but no binding of 14C to the protein even under native conditions. With equimolar tubulin and DCBT, beta-tubulin is the predominant alkylated species. At high drug concentrations, superstoichiometric amounts of DCBT react with tubulin, and both subunits are alkylated almost equally. When extracts of drug-treated L1210 murine leukemia cells were examined by polyacrylamide gel electroporesis, we found that multiple proteins were alkylated by DCBT, but the most prominent radiolabeled band was that corresponding to beta-tubulin. Dithiothreitol partially reverses inhibition of tubulin polymerization by DCBT and removes almost all the 2,4-dichlorobenzyl mercaptan moiety covalently bound to tubulin. Mitotic arrest occurs with DCBT because tubulin is the cellular protein most sensitive to the agent, probably because of its high cysteine content (20/mol).

Alkylation↗
PubMed1989

Brain death criteria: third generation.

Hence, in the third generation of brain death criteria, the disappearance of evoked potentials is, in our opinion, the best of the new tests. There are, therefore, three tests alternatively available: ECS, EP-abolition and CCA, which permit a shortening of the observation time, before the determination of brain death. This is not valid for newborns. In these tests the difference between supratentorial and infratentorial primary brain lesions has to be considered carefully. In infratentorial lesions the apnea can precede the ECS by several hours.

Brain Death↗
PubMed1989

Acetylcholine and vasoactive intestinal peptide in cerebral blood vessels: effect of extirpation of the sphenopalatine ganglion.

The innervation of cerebral blood vessels by nerve fibers containing acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP) and the vasomotor effects of the two neurotransmitters have been analyzed in the rat following the uni- or bilateral removal of the sphenopalatine ganglion (SPG), which is thought to be the major origin of this innervation. Histochemistry of AChE-positive nerve fibers and the immunoreactivity toward VIP revealed only a 30% reduction in the innervation pattern of the rostral part of the cerebral circulation following the operation. At approximately 4 weeks postoperatively, the original nerve network was restored. Quantitative measurements of cholineacetyltransferase activity and VIP revealed similar reductions in the levels of collected large cerebral arteries at the base of the brain and in small pial vessels overlying the cerebral cortex at the various postoperative times following uni- or bilateral removal of the SPG. The two techniques thus complemented each other. Vasomotor reactivity to acetylcholine (ACh) and VIP was examined in proximal segments of the middle cerebral artery at the various postoperative times. Generally, the removal of the SPG had no effect on the responses to ACh or VIP. The evidence indicates that only approximately one-third of the cholinergic/VIP innervation of the rostral part of the cerebral circulation originates in the SPG.

Acetylcholine↗
PubMed1989

Time course of effects of unilateral lesions of the nucleus basalis of Meynert on glucose utilization by the cerebral cortex. Positron tomography in baboons.

In order to investigate the effects of a partial cholinergic deafferentation on the functional activity of the cortex, the cerebral metabolic rate of glucose (CMRGlu) was measured with positron emission tomography and 18F-2-fluoro-2-deoxy-D-glucose in 5 baboons (Papio anubis) both before and serially following stereotaxic electrocoagulation of the left nucleus basalis of Meynert (NbM). Four days postlesion, significant metabolic depression was present in the entire ipsilateral cerebral cortex, most marked in the frontotemporal region, and which slowly recovered close to normal within 6-13 weeks. Postmortem studies showed that the lesions were located largely in the NbM, and that a significant decrease in choline-acetyltransferase (ChAT) activity was present in the ipsilateral frontal, temporal and parietal cortices. The animal with the most limited histological lesion showed the least decrease in both ChAT activity and CMRGlu. There was a highly significant linear correlation between the regional cortical decreases in CMRGlu (early postlesion data) and in ChAT activity. These results indicate that cholinergic deafferentation induces a proportional metabolic depression in the cortex. However, compensatory mechanisms operate to restore the cortical metabolic activity gradually despite sustained cholinergic denervation, pointing to pre- and/or postsynaptic adaptation (plasticity). Moreover, unilateral NbM lesions also induced a significant reduction in contralateral CMRGlu, which also demonstrated recovery. Several mechanisms are discussed to explain this contralateral effect, but the most likely implicates a transcallosal depression of function as a result of ipsilateral effects of the NbM lesion. These metabolic effects of cholinergic deafferentation in the primate provide new insight into the mechanisms of cortical dysfunction, and the recovery thereof, following subcortical lesions. In addition, our findings have some relevance to the cortical consequences of cholinergic deafferentation observed in dementia of Alzheimer type.

Animals↗
PubMed1989

Omega 3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: an autoradiographic study with the photoaffinity ligand [3H]PK 14105.

The anatomical distribution of omega 3 (peripheral type benzodiazepine binding) sites in the immune system organs of the rat has been studied autoradiographically at both macroscopic and microscopic levels of resolution using either reversible or irreversible (UV irradiation) labeling with [3H]PK 14105. In thymus sections, [3H]PK 14105 labeled with high affinity (Kd, derived from saturation experiments = 10.8 nM) a single population of sites which possessed the pharmacological characteristics of omega 3 sites. In the thymus gland, higher omega 3 site densities were detected in the cortex than in the medulla; in these subregions, silver grains were associated to small (10-18 microns diameter) cells. In the spleen, omega 3 sites were more abundant in the white than in the red pulp. In the white pulp, silver grains were denser in the marginal zone than in the vicinity of the central artery and labeling was, as in the thymus, associated to small cytoplasm-poor cells. In the red pulp, omega 3 site associated silver grains were observed mainly in the Bilroth cords. In the lymph nodes, the medullary region showed a higher labeling than the surrounding follicles and paracortex. A significant accumulation of silver grains was observed in the lymph node medullary cords. In the intestine, Peyer patches were particularly enriched in omega 3 sites (especially in the periphery of the follicles). The distribution of omega 3 sites in the immune system organs suggests a preferential labeling of cells of T and monocytic lineages. This is consistent with the proposed immunoregulatory properties of some omega 3 site ligands.

Animals↗
PubMed1989

Pharmacological properties of the receptor(s) involved in the 5-hydroxytryptamine-induced contraction of the feline middle cerebral artery.

The characterization of the receptor(s) involved in the 5-hydroxytryptamine (5-HT)-induced contraction was studied in the cat middle cerebral artery. 5-HT agonists and antagonists were tested on isolated arterial segments and their vascular potency correlated with their affinity value for 5-HT receptor subtypes as defined in the literature. 5-Carboxamidotryptamine (5-CT) and RU 24969 (5-HT1A-1B agonists) were more potent and as potent as 5-HT, respectively. Agonists at the 5-HT1C-2 (alpha-methyl-5-HT) and 5-HT3 (2-methyl-5-HT) sites were significantly less potent than 5-HT. All these agents induced similar maximal effects. The selective 5-HT1A agonist, 8-hydroxy-2(di-n-propylamino)tetralin, was the least potent but elicited a more intense vasoconstriction. The 5-HT-induced contraction was potently inhibited by compounds with high affinity at the 5-HT2 site in an apparently noncompetitive manner. The order of potency was: pizotifen greater than ritanserin greater than or equal to dihydroergotamine greater than or equal to ketanserin greater than or equal to methysergide. The nonselective agent methiothepin inhibited strongly the vasoconstriction induced by both 5-HT and 5-CT although a decrease in the maximal responses was observed together with a shift of the dose-response curves. In contrast to 5-HT, the contraction induced by 5-CT was only slightly affected by micromolar concentrations of ketanserin. 5-HT1A-1B (propranolol and cyanopindolol), 5-HT1C (mesulergine) and 5-HT3 (MDL 7222 and GR 38032F) selective drugs were almost completely devoid of significant antagonistic activity. Thus, the pharmacology of the feline cerebrovascular receptor(s) shares agonist similarities with the 5-HT1B/5-HT1D receptor subtypes whereas its antagonist profile relates more to that of the 5-HT2 site described in mammalian brain. Although these results may suggest the presence of two receptors in this cerebrovascular smooth muscle, they do not exclude the possibility that a single 5-HT receptor, not yet described by radioligand binding techniques, mediates the contractions induced by 5-HT. A definitive classification of this site, however, appears premature and would require novel and more selective agents.

Animals↗
PubMed1988

Methylenedioxy-benzopyran analogs of podophyllotoxin, a new synthetic class of antimitotic agents that inhibit tubulin polymerization.

A new class of compounds was synthesized and, based on structural analogy to podophyllotoxin, examined as potential microtubule inhibitors and evaluated for in vivo antineoplastic activity. These agents are derivatives of methylenedioxy-benzopyran bearing a phenyl substituent at position 8. The hydrogen atoms at positions 7 and 8 are in a trans configuration, in contrast to the cis configuration of analogous hydrogen atoms at positions 1 and 2 in podophyllotoxin. Compounds with a variety of substituents at positions 6 and 7 were examined, as well as compounds with varying methoxy substituent patterns on the phenyl ring attached at position 8. The most active compounds inhibited tubulin polymerization at concentrations approximately stoichiometric with tubulin, competitively inhibited the binding of colchicine to tubulin, and caused mitotic arrest at cytotoxic drug concentrations. No structure-activity correlations were obvious for the substituents at positions 6 and 7, but optimal activity was only observed when the phenyl substituent at position 8 was a trimethoxybenzene ring identical to the analogous ring in podophyllotoxin (i.e. methoxy groups at positions 3', 4' and 5'). Despite their structural and functional similarities to podophyllotoxin, however, the methylenedioxy-benzopyran derivatives subtly differ from the natural product in their interaction with tubulin, for they stimulated rather than inhibited tubulin-dependent GTP hydrolysis.

Animals↗
PubMed1988

Modulation of cellular phorbol ester binding and/or protein kinase C activity by human placental fractions.

We describe two factors in human placenta that modulate the interaction of phorbol ester tumor promoters with cell membranes or with protein kinase C. One, phorbol ester binding inhibitory factor, can inhibit binding of [3H]phorbol-12,13-dibutyrate to cultured cells or to a membrane fraction but does not inhibit its binding to a homogeneous C kinase preparation (phorbol ester binding sites). The other, C kinase activating factor, stimulates C kinase activity in a calcium-dependent manner. We separated these two biochemical activities from a crude human placental fraction by gel filtration.

Animals↗
PubMed1988

Cornigerine, a potent antimitotic Colchicum alkaloid of unusual structure. Interactions with tubulin.

Cornigerine is a natural product analog of colchicine produced by Colchicum cornigerum in which the vicinal 2- and 3-methoxy groups are condensed into a methylenedioxy bridge. This produces a fourth ring and a structure which resembles a hybrid of colchicine, podophyllotoxin, and steganacin. Cornigerine was somewhat more toxic than colchicine with L1210 murine leukemia cells and caused them to accumulate in metaphase arrest. Cornigerine resembled colchicine in its interactions with tubulin in vitro, and it was also somewhat more potent than colchicine in these drug-tubulin interactions. Cornigerine inhibited tubulin polymerization both with and without microtubule-associated proteins, inhibited the binding of radiolabeled colchicine to tubulin, and stimulated tubulin-dependent GTP hydrolysis. Indirect evidence suggested that the binding of cornigerine to tubulin is relatively slow and temperature-dependent, like the binding of colchicine to the protein.

Animals↗