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PubMed1981

Effects of inhibitors of tubulin polymerization on GTP hydrolysis.

The effects of a number of antimitotic drugs on the GTPase activity of tubulin were examined. The previously reported stimulation with colchicine and inhibition with podophyllotoxin and vinblastine wee confirmed. Maytansine, which competes with vinblastine in binding to tubulin, was comparable to the latter in inhibiting GTP hydrolysis. Nocodazole, which competes with colchicine in binding to tubulin, was significantly superior to colchicine in enhancing GTP hydrolysis. This superiority arose from the more rapid bindng of nocodazole to tubulin, as the two drugs had comparable activity when drug and tubulin were preincubated prior to the addition of GTP. Both colchicine and podophyllotoxin contain a trimethoxybenzene ring, while the closest structural analogy of nocodazole to colchicine includes the trimethoxybenzene ring. To explore this apparent paradox, we examined a number of simpler colchicine analogs for their effects on tubulin-dependent GTP hydrolysis. While tropolone was without effect, 3,4,5-trimethoxybenzaldehyde and 2,3,4-trimethoxybenzaldehyde stimulated the reaction. We therefore conclude that the trimethoxybenzene ring of colchicine is primarily responsible for the drug's stimulation of the GTPase activity of tubulin and that the inhibitory effect of podophyllotoxin must derive from the latter's tetrahydronaphthol moiety.

Animals↗
PubMed1980

Colchicine binding to antibodies.

Antibodies to colchicine have been prepared by injecting rabbits with an antigen prepared by coupling deacetylcolchicine to bovine serum albumin with a water-soluble carbodiimide. Sera of high titer were obtained with apparent dissociation constants for colchicine ranging from 5 to 17 nM. Binding was rapid and occurred at low temperatures. The chemical specificity of the binding site(s) resembled that in tubulin for the A and B rings of colchicine. Unlike tubulin, the antibody binding site tolerated numerous changes in the tropolone moiety (C ring). In contrast to tubulin, binding of colchicine to the antibody site did not promote fluorescence. The colchicine binding capacity of the antibody was high enough to prevent colchicine fluorescence in the presence of tubulin and to restore polymerization in colchicine-inhibited tubulin preparations.

Animals↗
PubMed1995

Changes in platelet kinetics after a partial splenic arterial embolization in cirrhotic patients with hypersplenism.

We performed a partial splenic arterial embolization in 22 patients with cirrhosis associated with thrombocytopenia and then evaluated the changes in platelet kinetics after undergoing the procedure using 111In-tropolone-labeled platelets. The controls consisted of eight chronic hepatitis patients who showed a normal platelet count and normal spleen size. The mean splenic infarction ratio after the procedure was 54.9%. A platelet kinetics study was performed before and 2 months after the procedure. Before the procedure, the cirrhotic patients showed increases in the splenic volume and the spleen/liver uptake ratio of the 111In-labeled platelets on both the third and seventh days, and a decrease in the platelet recovery compared with the controls, which suggested an increased platelet pool in the spleen. In addition, the platelet survival time in cirrhotic patients was shortened, whereas the platelet-associated immunoglobulin G (PA-IgG) was higher than that of the controls, which suggested the involvement of immunologic mechanisms in the thrombocytopenia. With an increase of the platelet count after a partial splenic arterial embolization, the spleen/liver uptake ratio of the 111In-labeled platelets decreased, whereas the platelet recovery increased. Furthermore, the platelet survival time was prolonged, whereas the PA-IgG decreased. The platelet count showed a positive correlation with the platelet survival time and a negative correlation with PA-IgG before and after the procedure. These results suggest that a transcatheter splenic arterial embolization not only may reduce the increased platelet pool in the spleen but also may improve the thrombocytopenia induced by immunologic mechanisms in patients with cirrhosis.

Adult↗
PubMed1976

Oncogenic and tumor-promoting Spermatophytes and Pteridophytes and their active principles.

A survey and discussion are presented of plants classified as Spermatophyta and Pteridophyta, extracts of which have been shown to be oncogenic or tumor-promoting in animals. The active oncogenic and tumor-promoting principles, where known, have been identified. They represent tannins; pyrrolizidine, indole, tropolone, quinoline, purine, and benzophenanthridine alkaloids; nitroso compounds; triterpene glycosides; lignans; isoflavans; allyl benzenoids; simple (nu-pyrenes; and carbocyclic hydroxy acids. A total of 28 compounds of known structure have been identified as oncogens and several phorbol esters as tumor-promoters. Plants known to contain any of the 28 oncogens (excluding shikimic acid and caffeine) have been tabulated; they represent at least 454 species, 110 genera, and 34 families of Spermatophyta and Pteridophyta.

Animal Feed↗
PubMedDate not supplied

A higher blood flow window of reduced thrombogenicity and acceptable fragmentation in a hollow fiber hemodialyzer.

The effect of pulsatile blood flow on platelet thrombogenicity and platelet fragmentation (PF) in a hollow fiber hemodialyzer (HFD) was quantified with 111In labeled platelets and 125I labeled fibrinogen; 150 ml of blood was collected from Beagle dogs, Yorkshire pigs, and a human volunteer (non-smoker). Platelets were labeled with 111In tropolone (300 microCi) and fibrinogen was labeled with 125I. Sham dialysis (SHD) was performed with 120 HFDs (0.9 meter2) at 37 degrees C, with flow-rates of 150, 250, 500, and 950 ml/min.; after SHD, the washed HD radioactivity was measured with an ionization chamber. PF was measured by flow cytometry with GP IIb-IIIa murine monoclonal antibody. Platelet deposition decreased significantly for 3 species at higher flow; fibrinogen deposition (10-12%, 55-65 mg/m2), was not affected by flow. Adherent platelet thrombus decreased from (8.2 +/- 3.4) to (3.1 +/- 1.0) with human blood as flow rate increased from 150 to 950 ml/min; platelet thrombus level also decreased significantly (p < 0.005) from (20.3 +/- 6.2) to (4.5 +/- 1.9) with canine blood. Higher values were obtained for canine than human and porcine platelets. Platelet fragmentation, on the other hand, increased from 2.1-2.2% to 10.2-11.3% with increase of flow. Like platelets, deposition of canine fibrinogen was slightly higher than that of pig and human. The studies of adherent thrombus and platelet fragmentation identified an important flow-window of reduced thrombogenicity and acceptable fragmentation, encouraging extracorporeal circulation at higher blood flow.

Animals↗
PubMed1993

Competition between deferiprone, desferrioxamine and other chelators for iron and the effect of other metals.

Competition for iron between the leading oral iron chelator deferiprone (L1, 1,2-dimethyl-3-hydroxypyrid-4-one) and desferrioxamine (DF), and the effect of other metals has been studied. Visible spectroscopy was used to estimate the displacement of iron from DF-iron(III) and L1-iron(III) complexes, respectively, by varying quantities of up to 100 molar equivalent of a competing metal ion M+ (Mg2+, Al3+, Ca2+, Mn2+, Co2+, Cu2+, Zn2+ and Pb2+) at physiological pH. Copper and aluminium showed the greatest competition against iron, while magnesium, calcium and manganese had little or no effect and the other metals an intermediate effect. DF showed greater selectivity for iron than L1 under these conditions. An analogous series of experiments carried out using a competing chelator of up to 50 molar equivalent in a place of a competing metal ion showed that 8-hydroxyquinoline, diethylenetriamine-pentaacetic acid (DTPA) and tropolone were more effective at displacing iron from DF-Fe and (L1)3Fe complexes than maltol or omadine. Desferrioxamine was the most efficient competitor against L1. Stoichiometric studies of the L1 complexes of aluminium and copper at pH 7.4 using Job Plots, suggested the formation of (L1)3Al and (L1)2Cu complexes, respectively.

Aluminum↗
PubMed1996

[Diagnosis of intracardiac thrombi by various imaging techniques and activation of platelets and coagulation-fibrinolysis in patients with cardioembolic stroke].

Recent epidemiological studies have suggested that 15 to 30% of all ischemic stroke is comprised of cardioembolic stroke. The presence of intracardiac thrombi might prove to be the most reliable tool when making a diagnosis of cardioembolic stroke, although not always easy to determine even with recent advanced technique. In this study, sensitivities to detect intracardiac thrombi of transthoracic echocardiography (TTE), transesophageal echocardiography (TEE), cardiac-enhanced CT (CCT) and scintigraphy with indium-111-tropolone-labelled platelets (PSG) were compared, in order to provide a relevant guideline for the diagnosis of intracardiac thrombi in 83 patients suspected of cardioembolic stroke. Also studied was the correlation of intracardiac thrombi with activation of platelets and coagulation-fibrinolysis through performing various hemostatic tests in order to investigate their utility for the evaluation of in situ thrombosis or prothrombotic state in the heart chamber. Detection rates of intracardiac thrombi were 35% in TEE, 26% in CCT, 19% in PSG, and 11% in TTE. There was a significant difference in the sensitivity between TEE and TTE (p < 0.05). Left atrial thrombi were frequently detected in TEE (4 out of 5 patients) and CCT (7 out of 10), while they were found less in PSG (2 out of 4) an TTE (4 out of 10). Thrombi in the left appendage were visualized in 3 out of 3 by TEE, while only in 1 out of 3 by PSG, 1 out of 4 by TTE and 1 out of 4 by CCT. Left ventricular thrombi; CCT (3 out of 3), TTE (2 out of 3), PSG (1 out of 1); TEE was not performed since this technique could not be expected to provide high-quality images of left ventricular thrombi. Thus, left atrial thrombi were considered to be more sensitively detected by TEE and CCT, left appendage thrombi by TEE, and left ventricular thrombi by TTE and CCT. There was no patient in whom an intracardiac thrombus was visualized by PSG alone. On the basis of the results above, we propose the following guideline for the detection of intracardiac thrombi in patients presented with cardioembolic stroke. First, TTE and CCT appear to be relevant for screening tests because of simple and non-invasive techniques. These two tools might be sensitive enough to find left ventricular thrombi. Second, TEE should be recommended when a thrombus is suspected in the left atrium or appendage. Finally, PSG may be used to determine the activity of the thrombus, according to its necessity. Among the patients having intracardiac thrombi, frequently observed was the increase of beta-thromboglobulin, platelet factor 4, platelet lysis, thrombin-antithrombin III complex, D-dimer in 67%, 75%, 71%, 80% and 80%, respectively, as well as the shortening of platelet survival in 100%, while anrithrombin III was reduced in only 38%. In addition, when hemostatic abnormalities were compared between positive and negative groups of intracardiac thrombi, the shortening of platelet survival (p < 0.0001), the increase of platelet lysis, and the increase of D-dimer (p < 0.04) were more frequent in the positive group than in the negative group. These results indicate that the findings of activation of platelets and coagulation-fibrinolysis, except for the reduction of antithrombin III, especially the findings of platelet consumption and lysis as well as fibrinolysis activation are useful as sensitive parameters of in situ thrombosis or prothrombotic state, which may lead to the formation of intracardiac thrombi.

Adolescent↗
PubMed1997

Electrophoretic separation and identification of phenoloxidases in hemolymph and midgut of adult Anopheles stephensi mosquitoes.

Anopheles mosquitoes frequently respond to invading malaria parasites with a rejection mechanism consisting of phenoloxidase-mediated melanization of ookinetes in the mosquito midgut epithelium. The relative roles of hemolymph vs. midgut phenoloxidase in this rejection mechanism is unclear. We have separated and identified phenoloxidase isozymes from midgut and hemolymph of Anopheles stephensi by native gel electrophoresis followed by zymography. The isozymes from the 2 sites had distinctively different electrophoretic characteristics. Hemolymph possessed 2 phenoloxidase-positive bands, both of which were bifunctional molecules that oxidized monophenol as well as o-diphenol substrates. Midgut extract possessed 3 bands that migrated more rapidly than those of the hemolymph. None of these midgut bands had detectable monophenoloxidase activity; they possessed, however, a broad spectrum of diphenoloxidase activity in their ability to oxidize both o- and p-diphenol substrates, as well as the laccase substrate syringaldazine. The 2 most rapidly migrating midgut PO bands could be distinguished from the more slowly migrating band through their insensitivity to inhibition by the chelating agent tropolone. A question to be resolved in the future relates to the relative roles of hemolymph vs. midgut phenoloxidase in mosquito defense against invasive parasites.

Animals↗
PubMed2005

Oxidative stress in plant cell culture: a role in production of beta-thujaplicin by Cupresssus lusitanica suspension culture.

Oxidative stress is a common physiological stress that often challenges plants. Reactive oxygen species (ROS) are major factors in oxidative stress that significantly affect plant cell growth and secondary metabolism. Here we used beta-thujaplicin production by Cupressus lusitanica cell culture as an example to demonstrate the common occurrence of oxidative stress in cultivated plant cells and its effect on multiple aspects of cell culture process. C. lusitanica cells cultivated under Fe(2+) stress generate a significant level of ROS, and oxidative stress also occurs at late stages of C. lusitanica cell cultures under normal conditions. ROS production inhibited cell growth, induced lipid peroxidation and cell death, and enhanced ethylene and beta-thujaplicin production. It is demonstrated that Fe(2+) stress enhances ROS production via the Fenton reaction and promotes beta-thujaplicin production via ROS-induced lipid peroxidation that may activate cyclic oxylipin and ethylene pathways. Results further indicate that H(2)O(2) is a positive signal for beta-thujaplicin production, whereas superoxide anion radical (O(2) (- )) negatively affects beta-thujaplicin induction and strongly induces cell death. The study suggests that evaluating the oxidative stress and plant responses in a cell culture process is very necessary and important for understanding biochemical processes and for gaining the maximal productivity of target secondary metabolites.

Antioxidants↗
PubMed1987

Reversible inhibition of microtubules and cell growth by the bicyclic colchicine analogue MTC.

2-methoxy-5-(2,3,4-trimethoxyphenyl) 2,4,6-cycloheptatrien-1-one (MTC) is a synthetic colchicine analogue, lacking the B ring of the alkaloid (Fitzgerald: Biochem. Pharmacol. 25:1381-1387, 1976). MTC has been shown to bind reversibly to the colchicine binding site of tubulin and to inhibit microtubule assembly in vitro (Andreu et al: Biochemistry 23:1742-1752, 1984; Bane et al: J. Biol. Chem. 259:7391-7398, 1984). Its action on different cultured cell lines (PtK2, Pk15, and SV-3T3) has now been studied. 0.2 X 10(-6) M MTC stopped Pk15 and SV-3T3 cell growth, inducing an accumulation of mitoses in a few hours. Removal of MTC from the culture medium rapidly restored normal mitotic index and growth rates. Partial depolymerization of the cytoplasmic microtubules of PtK2 cells was observed at concentrations ranging from 2 to 5 X 10(-7) M. Maximal microtubule network depolymerization was obtained after 4 h of treatment with 2 to 5 X 10(-6) M MTC or at a higher MTC concentration (2 X 10(-5) M) for less than 2 h. Removal of 2 X 10(-5) M MTC (the highest MTC concentration used) from the culture medium resulted in almost complete microtubule polymerization after 10 min of drug recovery and a normal microtubule network in 20-30 min. MTC constitutes an antimitotic drug directed to the colchicine site. It is water-soluble, shows a fast and reversible action, and may therefore be employed as a convenient tool to study cellular microtubule-dependent functions.

Animals↗
PubMed1995

Comparative study of the colchicine binding site and the assembly of fish and mammalian microtubule proteins.

Isolated microtubules from cod (Gadus morhua) are apparently more stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cyclo heptatrien-1-one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 +/- 1.5) x 10(5)M-1 at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin-colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 x 10(-5) to 1 x 10(-3) M), MTC induced a large amount of cold-stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 10(-5) M colchicine no spirals were formed, while at 10(-4) M and 10(-3) M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2+ concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different tubulins.

Animals↗
PubMed1997

Differential effects of colchicine and its B-ring modified analog MTPT on the assembly-independent GTPase activity of purified beta-tubulin isoforms from bovine brain.

Tubulin occurs as several isoforms whose physiological significance is not clear. The binding of colchicine to tubulin inhibits its assembly and is associated with a stimulation of the intrinsic GTPase activity of tubulin. In an effort to study the conformational states of different beta-tubulin isoforms in the drug-bound form, the intrinsic GTPase activity was studied in the presence of colchicine and its B-ring-modified analog MTPT. The results show that the GTPase activities of alpha beta II, alpha beta III, and alpha beta IV are 3.1, 2.8, and 2.6 nmole/h/ml. In the presence of colchicine, the values were increased to 4.5, 9.6, and 3 nmole/h/ml for alpha beta II, alpha beta III, and alpha beta IV respectively. In the case of MTPT, which lacks the B-ring, the GTPase activities were 5.8, 11.5, and 7.3 nmole/h/ml, respectively, for alpha beta II, alpha beta III, and alpha beta IV. The results show that the B-ring of colchicine contributes differentially to the conformational changes in the beta-tubulin isoforms.

Animals↗
PubMed1999

Apoptosis during iron chelator-induced differentiation in F9 embryonal carcinoma cells.

We have previously demonstrated that three potent iron chelators, hinokitiol, dithizone and deferoxamine, induce differentiation of F9 embryonal carcinoma cells, as do other well-known morphogens such as retinoic acid (RA) and sodium butyrate (NaB). In this study, we compared the patterns of cell proliferation, cell death and cell cycle arrest during the process of differentiation induced by these five agents. When F9 cells were cultured with the agents at their individual differentiation-inducing concentrations, cell proliferation was rapidly inhibited by treatment with the iron chelators and NaB. In contrast, RA did not influence the rate of increase of cell number at the concentration of 1 microm. The three chelators also caused a marked reduction in cell viability, and the treated cells exhibited internucleosomal DNA fragmentation, whereas cells treated with NaB showed no apoptotic characteristics. RA induced apoptosis weakly at 1 microm and strongly at higher concentrations. In addition, all the iron chelators hindered cell cycle progression, resulting in an arrest at the G1-S interface or S phase. The phenomena observed in chelator-treated cells were considerably different from those in RA- or NaB-treated cells. It is concluded that the three iron chelators cause both severe apoptotic cell death and cell cycle arrest of proliferating F9 cells via cellular iron deprivation, and that this apoptotic change may be independent of the process of differentiation.

Animals↗
PubMed2000

The interaction of the B-ring of colchicine with alpha-tubulin: a novel footprinting approach.

Tubulin, the major structural component of the microtubules, participates actively in mitotic spindle formation and chromosomal organization during cell division. Tubulin is the major target for a variety of anti-mitotic drugs. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the binding sites on tubulin of these drugs is limited because of lack of a useful and appropriate tool. The photoaffinity labeling approach is the major technique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the binding site can be identified, namely, the residues which react with the photoaffinity label. Second, there are regions of tubulin which are not at the binding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perhaps most serious, limitation is that the traditional approach can detect areas which have nothing to do with the binding of the ligand but which are within a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the ligand. There has been a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site is on alpha and some supporting a binding site on beta. Colchicine also has significant effects on tubulin conformation, but the regions which are affected have not been identified. We have attempted here to address these questions by a novel "footprinting" method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational changes could be determined. Here, we report for the first time that the interaction of the B-ring of colchicine with the alpha-subunit affects a domain of tubulin which appears to be far from its binding site. This domain includes the cysteine residues at positions 295, 305, 315 and 316 on alpha-tubulin; these residues are located well away from the alpha/beta interface where colchicine appears to bind. This is correlated with the stabilizing effect of colchicine on the tubulin molecule. Furthermore, we also found that the B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, support a model whereby colchicine binds at the alpha/beta interface of tubulin with the B-ring on the alpha-subunit and the A and the C-rings on the beta-subunit.

Amino Acid Sequence↗