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Charge effect on the colchicine binding-site of tubulin.

Poly(L-lysine) was found to enhance colchicine binding activity of brain tubulin to a several folds. Bases of biological interests that were tested and found to be inactive were spermine, spermidine and even L-lysine. Part of this enhance binding is due to the increase in the affinity of colchicine-tubulin interaction in the presence of poly(L-lysine). Moreover, poly(L-lysine) stabilized the colchicine binding site of tubulin against thermal denaturation.

Animals↗

Dissociation of the inhibitory effect of dapsone on the generation of oxygen intermediates--in comparison with that of colchicine and various scavengers.

4,4'-Diaminodiphenyl sulfone (dapsone, DDS) is highly effective in dermatitis herpetiformis and immune complex disease in which polymorphonuclear leukocytes (PMN) play an important role. We studied the dose-response effect of DDS (10-1 mM) on the generation of oxygen intermediates (OI:O2-, H2O2, OH.) and chemiluminescence using PMN and the xanthine-xanthine oxidase system. The effect of colchicine and five oxygen radical scavengers on OI production in both systems was also examined and compared with that of DDS. We found that, except for O2- generation, which was slightly enhanced, DDS decreased the levels of OI measured as effectively as scavengers. Scavengers also capable of degrading OH. and 1O2 markedly reduced the levels of OH. and chemiluminescence while slightly increasing the level of O2-. Therefore, DDS was considered to exert effects similar to H2O2, and OH. and 1O2 scavengers. Colchicine did not decrease OI values produced in the xanthine-xanthine oxidase system but affected PMN-mediated OI generation, possibly by its cytoxic effect. Our results suggest that DDS, in contrast to colchicine brings about rapid clinical improvement in PMN-mediated autooxidative disorders by quenching reactive OI such as H2O2, OH. and chemiluminescence.

Colchicine↗

Neuronal transport in salamander nerves and its blockade by colchicine.

Neuronal transport and the effects of colchicine on it has been studied in salamander spinal nerves. Cholinesterase (ChE) accumulation above the cut region of a nerve at 12.5 degrees C was shown to depend upon two processes. One caused a transient increase which declined to zero by 24 h; the other was explained by axoplasmic transport. At 22 degrees C the transient change was not observed, but the rate of accumulation attributable to transport increased. The Q10 for this transport over the range 12.5 degrees C--22 degrees C is approximately three. The ChE accumulation in the sensory component of the mixed nerve was about equal to that in the motor. The rate of fast axoplasmic transport of labeled leucine was 56 mm/day at 22 degrees C; if ChE moves at the same rate, then only 7% of the total enzyme is carried by fast axoplasmic transport. The transport of ChE was reduced by at least 50% when nerves were bathed in a 75 mM solution of colchicine for 30 min; this treatment is known not to cause subsequent degeneration of these nerves. The rate of slow flow of labeled material after bathing the nerve trunk in tritiated colchicine was found to be approximately 0.5 mm/day.

Animals↗

Preferential blockade of the tubulin transport by colchicine.

L-[35S]Methionine was injected into the dorsal root ganglion (L5) of the adult rat, and migration of the neurofilament polypeptides (the triplet with molecular weights of 200,000, 160,000 and 68,000 daltons), alpha- and beta-tubulins and actin in the sciatic nerve and the dorsal root was quantitatively determined and also examined by fluorography. Colchicine (4 microgram) injected into the ganglion 10 min before methionine preferentially blocked the tubulin transport, with little if any blockade of the triplet and actin. Colchicine at this dose had no effects on the incorporation of L-[14C]leucine into the total protein and also into tubulins. In contrast to colchicine, vinblastine sulphate (4 microgram) injected into the ganglion in a similar way blocked the transport of all the triplet, tubulins and actin. Cytochalasin D (1 microgram) had no effect on the slow axoplasmic transport.

Animals↗

Intrahypothalamic colchicine infusions disrupt lordotic responsiveness in estrogen-treated female rats.

Since some estrogenic effects on lordotic responsiveness are mediated through hypothalamic protein synthesis, we conducted experiments to determine if axoplasmic transport in the hypothalamus is necessary for the induction and maintenance of this reflex by estrogen. Colchicine infusion into the hypothalamus, but not into the dorsal thalamus, of ovariectomized rats 24 h prior to administration of subcutaneous estrogen implants delayed the induction of lordotic responsiveness, as measured by the manual (cutaneous-pressure) method, by 2 days, as compared with vehicle-infused rats. In other experiments, colchicine infusion into the hypothalamus, but not into the dorsal thalamus, of conscious, ovariectomized, estrogen-implanted rats displaying maximal lordotic responsiveness resulted in a bimodal decline in lordotic responsiveness. An initial decline occurred 20-40 min after infusion, and was associated with general behavioral agitation and hyperactivity. A subsequent decline began 4 h after infusion and lasted for several days. Vehicle infusion did not decrease lordotic responsiveness. Colchicine infusion did not alter multiunit electrical activity recorded near hypothalamically directed cannulae tips over a period of several hours. Results suggest that axoplasmic transport within and/or from the hypothalamus is necessary for the estrogenic induction and maintenance of the lordosis reflex in rats.

Animals↗

Short-term effects of intrahypothalamic colchicine.

Colchicine was injected in the vicinity of the medial forebrain bundle to cause an accumulation of catecholamines in the lateral hypothalamus proximal to the injection site. Such injections caused severe deficits in consummatory behavior and motor performance within a few hours after injection, which were accompanied by the accumulation of catecholamines in the hypothalamus. Behavioural deficits occurring within 24 h after colchicine injection cannot be attributed to reduced synaptic transmission in the striatum or to dopamine depletion because these events do not commence until 24-48 h after colchicine administration. This study demonstrates the importance of considering all neurochemical changes which accompany catecholamine depletion when assessing the role of the catecholamine-containing system in the regulation of behaviour.

Animals↗

Colchicine-induced differential sprouting of the endplates on fast and slow muscle fibers in rat extensor digitorum longus, soleus and tibialis anterior muscles.

The patterns of sprouting of motor endplates were examined in fast extensor digitorum longus and slow soleus muscles and in tibialis anterior muscles containing fast and slow muscle fiber types. A histochemical technique combining nerve silver impregnation and endplate cholinesterase staining was developed for this task. Temporal examination of the innervation was conducted 3, 7 and 10 days after either a 45 or 90 min application of the ipsilateral sciatic nerve with 5 mM colchicine. This dosage of drug did not cause detectable axon or muscle fiber degeneration, unlike 60 mM which was highly neurotoxic. At 3 days following treatment with the lower concentration, there were no significant differences in the percentages of intranodal, preterminal and ultraterminal sprouts between the normal (non-treated), sham-treated, contralateral systemic-control and drug-treated groups of muscles. By 7 and 10 days, the muscles on the drug-treated side exhibited significant increases in the 3 types of sprouts. Collateral sprouting was uncommon: most outgrowths remained on the muscle fibers innervated by the parent axons. Endplates in the tibialis anterior muscles of the control and drug-treated groups were classified Complex, Intermediate or Simple according to the relative degrees of branching of the terminal arbors. The occurrence of endplate classes and muscle fiber types was correlated in the superficial and deep regions of this muscle. Complex endplates innervated fast glycolytic fibers, Intermediate endplates supplied fast oxidative glycolytic fibers, and Simple endplates served slow oxidative fibers. In response to colchicine, the endplates of the slow muscles sprouted more than those of fast muscles while the innervation of slow fiber types sprouted less than that of fast fiber types. Furthermore, intranodal sprouts were more prevalent in slow muscles and ultraterminal sprouts more numerous in fast muscles whereas intranodal sprouts predominated on fast fiber types and ultraterminal sprouts were characteristic of slow fiber types. These apparently contradictory results were reconciled when it was noted that soleus endplates were mostly Complex and Intermediate, and the extensor digitorum longus contained more Simple endplates. Thus, consistency of sprouting patterns among endplate types of the 3 muscles was recognized when the pre-existing branching patterns were considered. This indicated that the patterns of sprouting were determined by the motor neurons rather than the muscle fibers. The observed sprouting responses supported the hypothesis that colchicine treatment of motor axons caused muscle fibers to elaborate a diffusible sprout-inducing factor.

Aging↗

Fetal hippocampal cell suspensions ameliorate behavioral effects of intradentate colchicine in the rat.

Colchicine, a neurotoxin that preferentially destroys dentate gyrus granule cells and mossy fibers, was injected into the hippocampus of adult rats. Three weeks later, the rats were tested for colchicine-induced hypermotility after which they received fetal hippocampal explants. Locomotor activity was retested three weeks later, after which the rats were trained over a period of four weeks on a food-reinforced, spatial, working memory task in an 8-arm radial maze. Fetal hippocampal explants were found to attenuate significantly the colchicine-induced hypermotility and spatial learning deficits. Histological observations showed the presence of surviving hippocampal explants in both the lesioned and the control rat brains, suggesting that the presence of viable implants facilitates the recovery of behavioral function in rats with spatial memory deficits.

Animals↗

Enhancement of neurotrophic activity in cholinergic cells by hippocampal extract prepared from colchicine-lesioned rats.

The effect of hippocampal extract on neurotrophic activity for cultured cholinergic cells was determined. Extract prepared from lesioned hippocampus caused by intrahippocampal infusion of colchicine promoted neuron survival of chick ciliary ganglion cells. The maximal level of survival-promoting activity was reached at 12 days postlesion. The biochemical differentiation of NG108-15 cells was assessed by measuring the activity of choline acetyltransferase (ChAT). After 3 days in culture, hippocampal extract was found to stimulate ChAT activity in a concentration-dependent manner. Hippocampal extract prepared after colchicine lesions elicited a large increase in ChAT-enhancing activity. The effect of hippocampal extract was additive to that of dibutyryl cyclic AMP (db-cAMP). The neurite outgrowth from NG108-15 cells was also potentiated when cells were cultured with hippocampal extract plus db-cAMP. The results suggest that one or several factors in colchicine-lesioned hippocampus promote neurotrophic activity, and the enhancing effect of hippocampal extract on cellular differentiation may act, at least in part, through a mechanism distinct from that for db-cAMP.

Acetylcholinesterase↗

Effect of optic nerve lesions and intraocular colchicine on cell proliferation in the germinal zone of the optic tectum and in the torus longitudinalis in the goldfish.

Postembryonic development of the optic tectum occurs in part through proliferation of cells in the germinal zone located at the caudal edges of each lobe. Autoradiography experiments by others have shown that [3H]thymidine labeling in the germinal zone is decreased following optic nerve crush or enucleation and restored above normal levels during optic nerve regeneration. The present autoradiography experiments examined the relationship between retinal innervation and the rate of mitotic activity in the tectum germinal zone and in the torus longitudinalis. The fish received optic nerve crush to temporarily deafferent the tectum, enucleation for permanent deafferentation, or an intraocular injection of 0.01-1.0 microgram of colchicine to reversibly inhibit axonal transport in the optic nerve. Thymidine labeling in the tectum germinal zone showed that nerve crush resulted in decreased mitotic activity in most fish within 6 days followed by recovery by 21 days; enucleation decreased mitotic activity more uniformly and for more than 42 days with recovery by 84 days postaxotomy; colchicine produced a dose-dependent inhibition of mitotic activity which was reversed by 42 days postinjection. Axonal transport was restored by 42 days postinjection. In the torus longitudinalis, nerve crush produced a brief increase in mitotic activity followed by a return to normal; enucleation and colchicine resulted in a lasting decrease in mitotic activity and atrophy indicating a loss of cells or neuropil. The data are consistent with the proposal that cell proliferation in the tectum germinal zone is stimulated by the accretion of fibers from developing retinal ganglion cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Entorhinal cortex lesion or intrahippocampal colchicine injection increases peripheral type benzodiazepine binding sites in rat hippocampus.

The peripheral type benzodiazepine binding site (PTBBS) has been proposed to be a good marker for reactive glial cells following brain insults. In the present study, homogenate binding of 3H-Ro5-4864 and quantitative autoradiography of 3H-PK-11195 binding (two ligands for the PTBBS) were used to assess the distribution, time-course and extent of reactive gliosis in the hippocampus following deafferentation by unilateral entorhinal cortex lesion or neuronal death produced by intrahippocampal colchicine injection. Intrahippocampal colchicine injections produced a 3-fold increase in 3H-Ro5-4864 binding in the dentate gyrus within 2 days. This effect was doubled in animals pretreated with the lysosomal inhibitor chloroquine. Quantitative autoradiography of 3H-PK-11195 binding 1 or 2 weeks after colchicine injection indicated that the increase in binding was restricted to the dorsal hippocampus both rostrally and caudally and was present in the dentate gyrus and CA1. Following a unilateral electrolytic lesion of the entorhinal cortex, the binding of 3H-Ro5-4864 to homogenates of the dentate gyrus was doubled 18 h after the lesion, reached a maximum at 4 days post-lesion, and returned to control values by 2 months after the lesion. A transient increase in binding was also observed 2 and 4 days post-lesion in the dentate gyrus contralateral to the lesion side. Autoradiography of 3H-PK-11195 binding indicated that the increase in PTBBS following entorhinal cortex lesion was restricted to the molecular layer of the dentate gyrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effects of actinomycin D and puromycin on colchicine-induced endoreduplication in Chinese hamster cells in vitro.

Experiments were carried out to study the events through which the process of endoreduplication occurs after treatment with colchicine in Chinese hamster cells cultivated in vitro, and to analyse the differences between the processes leading to normal mitosis and those leading to endoreduplication. Chinese hamster cells (line C-125) were treated with colchicine (10(-4) M) for 1 h, in order to induce endoreduplication. Before and after colchicine treatment the cultures were treated at various intervals with actinomycin D and puromycin. For preparation of mitosis specific RNA synthesis is required, and also synthesis of a specific protein, which is not required for endoreduplication. Moreover this suggests not only that the cells that endoreduplicate do not need new RNA and protein synthesis, but that inhibition of their synthesis apparently favours the processes of endoreduplication.

Cell Division↗

Serum bile acid in the evaluation of colchicine treatment of carbon tetrachloride-induced liver injury.

The usefulness of measuring serum-conjugated bile acid concentrations by radioimmunoassay in colchicine-modified carbon tetrachloride-induced liver lesions in rats was assessed by comparing the concentrations with the results of some routinely employed liver function tests such as serum aspartate transaminase, alanine transaminase, alkaline phosphatase, and serum total protein. The serum cholylglycine levels were significantly (P less than 0.003) raised along with the serum aspartate transaminase (P less than 0.01) and alanine transaminase (P less than 0.01) activity levels in the carbon tetrachloride-treated group of rats when compared with the group treated with carbon tetrachloride plus colchicine. Colchicine prevented the increase in serum cholylglycine, aspartate transaminase, alanine transaminase, and alkaline phosphatase induced by carbon tetrachloride but had no effect on serum total protein levels. This study suggests that radioimmunoassay of serum cholyglycine is a sensitive and specific indicator of liver injury and it is a useful tool in monitoring the treatment provided.

Alanine Transaminase↗

A quantitative freeze-etching study on the effects of cytochalasin B, colchicine and concanavalin A on the formation of intercellular contacts in vitro.

The effects of concanavalin A (conA), cytochalasin B (CB), and colchicine acting on cell membrane-associated structures, such as surface receptors or submembranal cytoskeletal components, were studied in trypsin-isolated plexus cells reaggregated in vitro. Following reaggregation, these cells form intercellular contacts, i.e. a zonula occludens (ZO) and integrated nexus (gap junctions, GJ). In the control cultures the GJ occupied 4.7%, the tight junctions (TJ) occupied 14 microns/microns2 of the surface of the zonula occludens (ZO). All the drugs/lectins studied inhibit contact formation in a characteristic manner. ConA inhibits the formation of GJ (1% of the ZO) and causes 5 times more defective TJ strands than the controls. CB inhibits the assembly of the TJ (75% of the controls) and the GJ (1% of the ZO) when it is administered during the period of contract formation. Colchicine inhibits the formation of GJ (1% of the ZO) and produces 2.5 times more open-end strands of TJ than the controls. All effects are reversible with the exception that integrated GJ are not re-formed after colchicine administration. It is suggested that an intact interaction between surface receptors, intramembranous particles and associated cytoskeletal structures is necessary for remodelling cellular contacts.

Animals↗

Synthesis and secretion of proteoglycans by cultured chondrocytes. Effects of monensin, colchicine and beta-D-xyloside.

Chondrocytes, isolated from elastic ear cartilage of young rabbits, were grown in monolayer cultures in Ham's F-12 medium. Synthesis and secretion of macromolecules were monitored by labelling with radioactive precursors and the effect of monensin and other experimental agents was investigated. Monensin caused an inhibition of the incorporation of precursors into macromolecular material and a moderate intracellular accumulation when used in higher concentrations. The effect was more pronounced for 35SO4 than for 3H-labelled glucose or proline. p-Nitrophenyl-beta-D-xyloside alleviated this inhibition to some extent, but there was a concomitant increase in the amount of intracellular labelled material. Colchicine and monensin together caused a severe inhibition of the incorporation of 35SO4 and a marked shift of the label to the intracellular compartment. Colchicine also increased the sensitivity of the cells to monensin, lowering the minimal effective concentration about one order of magnitude. The latter results are consistent with the idea that cytoplasmic microtubules have a stabilizing function on the secretory pathways and, that their removal by colchicine, causing a 'randomizing' of the Golgi complex, makes these pathways more vulnerable to monensin.

Animals↗

Immunofluorescence demonstration of tubulin and actin in estrogen-induced rat prolactinoma cells in vitro. Alteration of their distribution after bromocriptine, colchicine and cytochalasin B treatments.

Cultured cells in vitro from estrogen-induced rat prolactin-secreting adenomas (prolactinomas) were examined by indirect immunofluorescence microscopy for the distribution of cytoskeletal proteins and alterations of cytoskeleton after treatment with bromocriptine, colchicine and cytochalasin B (CB). After 8 days in culture, prolactinoma cells were well expanded and developed cytoplasmic processes were seen. The cytoplasmic microtubules were observed as fine reticular networks radiating from perinuclear portions toward the cell periphery when decorated with an antibody against tubulin. On the other hand, the actin filaments showed diffuse and spotty distribution when detected with an anti-actin antibody. Contaminated fibroblasts showed a reticular distribution of microtubules and a parallel array of actin cables which corresponds to "stress fibers" throughout the cytoplasm. After treatment with bromocriptine, the reticular distribution of microtubules in prolactinoma cells changed into a coarse and sparse pattern, which was identical with the changes in the distribution of tubulin after treatment with colchicine. On the other hand, distribution of actin was not affected by bromocriptine. Bromocriptine treatment did not alter the distribution of microtubules and actin filaments in fibroblasts, whereas colchicine changed the distribution of microtubules in both prolactinoma cells and fibroblasts. CB treatment changed the localization of actin filaments in both kinds of cells. These in vitro studies indicated bromocriptine would selectively affect the cytoplasmic microtubular system of prolactinoma cells.

Actins↗

Effects of colchicine on cardiac cell function indicate possible role for membrane surface tubulin.

The effects of the tubulin-binding drug colchicine on cultured neonate cardiac cell function were investigated. Application of low doses of colchicine (but not lumicolchicine) caused an early reversible increase in beating rate with a concomitant decrease in amplitude. Treatment of the cells with trypsin at a dose that removes surface tubulin but does not inhibit spontaneous beating, diminished the colchicine effect. Surface radio-iodination of the live cultures followed by two-dimensional gel electrophoresis and radioautography revealed that two spots were heavily labeled. These spots co-migrated with purified brain tubulin. Fibroblasts derived from the cardiac cultures did not label over the tubulin spots. Trypsin treatment removed the presumptive tubulin from the radioautographs but only removed the most basic portion of the alpha-tubulin spot from the stained gel. These results are consistent with a surface membrane role for an iso-form of tubulin in neonate cardiac cells.

Animals↗

The effect of colchicine on synaptonemal complex formation in Allium ursinum.

Interference of colchicine with meiotic chromosome pairing in the wild garlic, Allium ursinum, was studied using a whole-mount spreading technique for synaptonemal complexes. Colchicine was found to cause (i) pairing suppression (arrest of leptotene) and (ii) deficient pairing initiation at zygotene in connection with morphologically anomalous, malfunctioning pairing initiation sites. Both of these phenomena could be responsible for the reduction of chiasma frequency by colchicine previously reported in the literature.

Allium↗