Application Of Colchicine Research Articles

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.

Intergeneric hybrids between Enarthrocarpus lyratus, a wild species, and crop brassicas

Attempts were made to produce intergeneric hybrids between Enarthrocarpus lyratus, a wild species, and several species of crop brassicas: B. campestris, B. nigra, B. oleracea, B. juncea, B. napus and B. Carinata. Hybrids using E. lyratus as female parent were realized by means of embryo rescue in four combinations - E. lyratus x B. campestris, E. lyratus x B. oleracea, E. lyratus x B. napus and E. lyratus x B. carinata. Reciprocal crosses showed strong pre-fertilization barriers and yielded no hybrids except in one combination - B. Juncea x E. Lyratus - in which a single hybrid could be realized. All of the hybrids were multiplied in vitro through the multiplication of axillary shoots. Morphological and cytological studies confirmed hybridity. All hybrids were completely pollen sterile except for E. lyratus x B. carinata, which showed 2% pollen fertility. Attempts to double the chromosome number through the in vitro application of colchicine to axillary meristems of F1 hybrids were successful in only one hybrid, E. lyratus x B. oleracea. Cytological studies of the hybrids indicated the presence of a partial homology between the genomes of E. lyratus and crop brassicas. Backcross progenies were raised from all of the five F1 hybrids to develop malesterile alloplasmic lines.

Polyploidy and Aneuploidy in the Tamarillo, Cyphomandra betacea (Cav.) Sendt. (Solanaceae)

AbstractThe successful induction of tetraploids in the tamarillo (Cyphomandra, betacea [Cav.] Sendt.) by the application of colchicine to the germinating seed is described. Chimeric and solid tetraploids were produced although the proportion of the chimeric types could be minimized by optimizing the treatment time. There were reciprocal differences in all interploidy crosses, with triploids being ineffective in crosses when used as the paternal parent. The analysis of aniline blue stained gynoecia showed that incompatibilities in interploidy crosses occurred largely at the post‐zygotic stage of the reproductive process. Triploids and aneuploids were produced from interploidy crosses. The features of some aneuploids were described. Most aneuploids produced were primary trisomics (2n = 2x + 1 = 25) but some had 26 chromosomes and two others were hyperpolyploids. Pollen fertility in these plants varied from 10% to 90%. Morphological traits did not differ between the aneuploids or between the aneuploids and diploids. Fruit and seed size in the aneuploids were comparable to those of the diploids and were thus suitable for potential commercial exploitation.

Substance Pimmunoreactivfty in the superficial laminae of the hamster olfactory bulb

The direct application of colchicine to the hamster olfactory bulb has engendered the appearance of a broader distribution of substance P-like immunoreactivity (SP-LI) than has been previously reported. In the glomerular layer, both external tufted and periglomerular cells were strongly immunopositive. These two classes could be identified by somal size and the presence and branching pattern of intraglomerular dendrites. SP-LI neurons were also identified in the external plexiform layer and many were similar to middle tufted cells. These findings demonstrate that substance P expression is not uniquely associated with classes of projection neurons, but is also found in periglomerular cells, a class of inhibitory local circuit neurons.

Regulation of Aberrant Neurofilament Phosphorylation in Neuronal Perikarya. I. Production Following Colchicine Application to the Sciatic Nerve

Neurofilament (NF) triplet proteins are normally poorly phosphorylated in neuronal perikarya, the two high molecular weight polypeptides becoming extensively phosphorylated once the NF enters the axon. Abnormal expression of phosphorylated NF (pNF) epitopes in neuronal perikarya has been revealed using monoclonal antibodies in a variety of human and experimental conditions. In the present study, we asked whether pNF epitopes are expressed in sensory neurons in the L4 and L5 dorsal root ganglia (DRG) following blockade of fast axonal transport in a model producing few (less than 1%) degenerating fibers. Colchicine (5 mM) was briefly (45 minutes) applied to the sciatic nerve at mid-thigh twice (once weekly) and the animals studied two weeks following the first colchicine application; contralateral nerves were either treated with saline or crushed. Modest to intense immunoreactivity was found with antibody 07-05 (directed against pNF epitopes on the two high molecular weight NF polypeptides) in 30.4% and 45.1% of DRG neurons from colchicine-treated and crushed nerves, respectively; only a rare cell body demonstrated immunostaining from the contralateral saline-treated nerves. Immunoreactivity was not observed with antibody 07-05 at two and five days following single colchicine application. In a separate study, colchicine or saline was applied (as above) 1-2 cm proximal to a nerve crush. Colchicine application did not influence the extent of DRG neurons expressing pNF epitopes; immunostaining with antibody 07-05 was present in 44.7% and 43.8% of DRG neurons from saline-treated and colchicine-treated crushed nerves, respectively. The results indicate that structural interruption of nerve-target contact is not necessary to induce aberrant NF phosphorylation in neuronal perikarya. It is suggested that loss of a retrogradely transported "trophic" signal(s) triggers this response.

In vitro multiplication of a sterile interspecific hybrid, Brassica fruticulosa x B. campestris

In vitro methods for plant multiplication of a sterile interspecific hybrid between Brassica fruticulosa and B. campestris through either micropropagation or callus regeneration is described. Shoot-tip, single-node and leaf explants, obtained from in vitro-grown hybrids, regenerated on media containing NAA and BA. In vitro application of colchicine induced chromosome doubling in in vitro-regenerated shoots resulting in the production of fertile amphidiploids. Comparative studies on regeneration potential of the hybrid and its parents were also carried out using callus from leaf explants. The explants of B. fruticulosa and the hybrid were capable of shoot and root formation while those of B. campestris failed to form shoots but produced profuse roots. The results demonstrate the efficacy of an in vitro method in producing a large number of hybrid plants and fertile amphidiploids from incompatible crosses that yield very few hybrid seeds/seedlings.

An in vitro chromosome doubling method for clovers (Trifolium spp.)

This research reports a new technique for chromosome doubling of clover (Trifolium sp.) axillary meristems via in vitro colchicine application. Plant material utilized included T. pratense (red clover) cv. Kenstar clones, and three interspecific hybrids: T. ambiguum (kura clover) × T. repens (white clover); T. alpestre × T. pratense; and T. sarosiense × T. pratense. Vegetative axillary meristems were excised from plants, surface sterilized, and trimmed to a length of 0.5–1 mm. Meristems were placed on the surface of a shoot proliferation medium (ML8) containing colchicine (0.1%) for 48 or 72 h and then transferred back to ML8. Alternative treatments were to preculture meristems on ML8 for 7 days prior to colchicine treatment. Plantlets with two or three trifoliolate leaves were induced to root on CR2 or RL rooting media. Preculturing of meristems on ML8 prior to colchicine exposure resulted in the highest chromosome doubling frequencies among the different genotypes, although there was apparent genotype × treatment interaction. Chromosome doubling frequencies were as high as 81 and 44% for initial root tips and mature shoots, respectively. To make rapid assessments of ploidy level of flowering plants, pollen shape was examined. Chromosome doubling increased the pollen stainability of the T. ambiguum × T. repens hybrid from 2.5 to 33.6%, but did not result in fertility in the other two interspecific hybrids.Key words: Trifolium, colchicine, chromosome doubling, interspecific hybrids.

Expression of c-JUN, JUN B and JUN D proteins in rat nervous system following transection of vagus nerve and cervical sympathetic trunk

Expression of c-JUN, JUN B and JUN D proteins was investigated in axotomized neurons following transection of the vagus nerve and the cervical sympathetic trunk in the rat. Vagotomy induced the expression of c-JUN and JUN D in the nodose ganglion, dorsal motor nucleus of the vagus nerve and nucleus ambiguus, whereas JUN B was not expressed in these areas. c-JUN and JUN D appeared after 10 h in the nodose ganglion and after 24 h in the dorsal motor nucleus of the vagus nerve with a maximum of immunoreactivity after 48 h. The c-JUN protein remained expressed at an increased level up to 100 days, whereas the immunoreactivity of JUN D declined after five days. Crush of the vagus nerve initially evoked an intense expression of c-JUN and JUN D, but in the course of regeneration the expression of c-JUN and JUN D had returned to more basal levels after 100 days. Similar to vagotomy, application of colchicine and vinblastine on to the intact vagus nerve induced expression of c-JUN and JUN D. On the other hand, application of lidocaine prior to vagotomy did not prevent the expression of these proteins. Transection of the cervical sympathetic trunk induced expression of c-JUN and JUN D, but not of JUN B, in the preganglionic sympathetic neurons of thoracic spinal cord. In these neurons, expression of c-JUN was still enhanced after 60 days whereas JUN D had returned to basal level. One hour after vagotomy, c-JUN and JUN B were transynaptically expressed in the area of central termination of sensory vagal neurons and declined within 10 h to basal levels. JUN D showed a late onset of expression, it appeared after 5 h and persisted for 60 days in this area. We postulate that the expression of c-JUN and JUN D in axotomized neurons is induced by deprivation of a target-derived suppressor.

Evidence for involvement of dynein-tubulin system in pigment aggregation within tilapia melanophores

1. 1. Using cultured melanophores of the tilapia, Oreochromis niloticus, and Brij 58-permeabilized cells, we investigated the mechanism that drives the aggregation of pigment granules. 2. 2. Although norepinephrine-induced aggregation of melanosomes was not significantly inhibited by pretreatment of cells with cytochalasin B, application of colchicine or vinblastine, both of which are antimitotic poisons, impaired the response of melanophores to the amine in a dose-dependent manner. 3. 3. Indirect immunofluorescence staining showed that the radial arrangement of microtubules within tilapia melanophores disappeared after melanophores had been exposed to colchicine or vinblastine. A normal distribution of microtubules was observed in cytochalasin-treated cells that responded to norepinephrine by pigment aggregation. 4. 4. Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) and vanadate, inhibitors of dynein ATPase, were also effective in blocking the norepinephrine-induced aggregation of melanosomes. 5. 5. From these results, we conclude that cytoplasmic microtubules are essential for the centripetal translocation of melanosomes, and that the motive force involved in the process may be generated by interactions between dynein and tubulin.

Cytogenetics and reproductive behavior of induced and natural tetraploid guayule (Parthenium argentatum Gray)

Tetraploid guayule (Parthenium argentatum Gray) plants with 72 chromosomes were obtained by colchicine application to the shoot apices of the diploid (2n = 36) seedlings. Interspecific hybridization of guayule as female with Parthenium rollinsianum Rzedowski (2n = 36) was used to compare the mode of reproduction of the induced tetraploids with that of natural tetraploids. Plants produced from crosses of induced tetraploid guayule and P. rollinsianum were all hybrids. Hybrid plants showed 2n = 54 chromosomes, indicating that the artificially produced tetraploids reproduce sexually. Interspecific crosses between natural tetraploids and P. rollinsianum produced predominantly maternal type progeny. This suggests that the apomictic mode of reproduction in guayule is a consequence of mutations which accumulated in the natural polyploids. Diploid guayule plants showed regular meiosis with an average of 14.86 rod and 3.14 ring bivalents. Induced tetraploids produced 0.25 I, 29.21 II, 0.13 III, and 3.24 IV. In comparison, natural tetraploids showed 1.20 I, 30.85 II, 0.06 III, and 2.23 IV. The frequency of multivalents, which was limited in the induced tetraploids, showed further reduction in natural polyploid guayule, which may indicate diploidization in these plants. Percent pollen stainability was 98.5 ± 1.03, 83.7 ± 12.24, and 89.5 ± 3.41 for diploids, induced tetraploids, and natural tetraploids, respectively. Both induced and natural tetraploids had larger capitula, achenes, and pollen and thicker leaves than the diploids.Key words: guayule, natural rubber, reproductive behavior, meiosis, polyploid induction.

Organization of oligodendroglial membrane sheets. I: Association of myelin basic protein and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase with cytoskeleton

Membrane sheets elaborated by cultured murine oligodendroglia provide a unique system for examining associations between myelin proteins and cytoskeletal elements. Interactions can be observed and manipulated more readily than in the multilamellar myelin membrane in vivo. Immunocytochemical staining of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) shows that it is distributed diffusely in some regions of membrane sheets, but colocalized with tubulin in lacy networks and major veins in other regions. Staining with phalloidin also reveals two distributions of F-actin: 1) small aggregates within the diffuse CNPase regions and 2) filaments colocalized with tubulin and CNPase in the lacy networks and veins. Application of colchicine at 10 micrograms/ml for 4 hr disrupts microtubular structures in the lacy network, while those in major veins remain intact. This suggests that microtubules in the lacy network are treadmilling more rapidly than those in the major veins. The distribution of CNPase and F-actin is not altered under these conditions. In contrast, cytochalasin B disrupts F-actin, microtubules, and CNPase in the lacy networks, indicating that cross-linking between these three proteins is disrupted. Both colchicine and cytochalasin B cause fusion of myelin basic protein (MBP) domains in membrane sheets. This appears to be a consequence of disruption of microtubules in the lacy networks, which normally outline the MBP domains. In summary, these results provide evidence for 1) direct association of CNPase with F-actin and tubulin in cytoskeletal structures and 2) organization of MBP into domains via association with microtubules in the lacy networks.

Topical application of colchicine, vinblastine and vincristine prevents strychnine-enhanced transsynaptic degeneration in the medullary dorsal horn following transection of the inferior alveolar nerve in adult rats

The effect of topical application of axonal transport blockers to the transected peripheral nerve was assessed by quantitating the strychnine-enhanced transsynaptic degeneration following transection of the inferior alveolar nerve in adult rats. Systemic administration of strychnine (1 mg/kg/day) for 7 days at the postoperational interval of 23 days proved to be suitable for quantitating the transsynaptic degeneration at the light microscopic level. When the proximal stump of transected nerve was treated with 2% colchicine immediately after transection, 5.8 +/- 6.8 dark neurons in a single section of the medullary dorsal horn, ipsilateral to the nerve transection, were observed. Following similar treatment with 0.4% vinblastine and 0.2% vincristine, 24.4 +/- 10.5 and 9.4 +/- 7.0 dark neurons were seen, respectively. When compared with 43.0 +/- 9.4 dark neurons, which were seen in animals without alkaloid treatment, colchicine, vinblastine and vincristine significantly reduced the transsynaptic degeneration by 86, 43, and 78%, respectively. Possible mechanisms involved in prevention of transsynaptic degeneration by the alkaloids are discussed.

Proliferative activity of endotheliocytes of growing capillaries of the rabbit cornea

The authors studied the intensity of DNA synthesis by cells of newly formed capillaries, growing in the rabbit cornea, after infliction of a silver nitrate burn and local application of colchicine. The intensity of capillary growth was investigated during stimulation and a combination of the burn with colchicine. Changes in activity of DNA synthesis by the endotheliocytes of newly formed capillaries during exposure throughout growth were also investigated. The intensity of cell proliferation was studied by measuring the incorporation of tritium-labelled thymidine into the endotheoiocyte nuclei.

The induction of hexaploidy in cherry rootstocks using in vitro regeneration techniques

An in vitro regeneration technique using root callus has been combined with colchicine application to produce hexaploid plants from the sterile triploid cherry rootstock Colt. Colchicine (50 mg/l) was applied together with auxin (IBA) (3 mg/l) either before or after root primordium formation in single excised micropropagating shoots. In both cases shoots were regenerated in vitro from the putative hexaploid roots. Both methods produced hexaploid plants but more than 70% of the regenerated clones were still triploid. The hexaploid plants were distinguishable from the triploids on morphological as well as cytogenetic grounds. Quantitative characters such as leaf length/breadth ratio, stomatal length and density in the hexaploids were significantly different from both regenerated and non-regenerated triploids.

Induction of polyploidy and cytological characterization of autotetraploids of Trifolium riograndense Burkart (Leguminosae)

Nine polyploids (including two mixaploids) were obtained in Trifolium riograndense with three methods of colchicine application to seedlings: immersion, dropping and cotton. The best method showed to be immersion for 6 h in 0.30% colchicine. Leaves with more than 3 folioles were frequently observed in some (but not all) polyploids. The number of pollen grain pores increased from 3 to 4 in tetraploids. Stomatal cell size was not a reliable criterium to distinguish ploidy levels. Two percent of aneuploidy occurred in the C1 generation. Meiotic behavior was characterized by high bivalent and low quadrivalent numbers with suggestions of genetic control of chromosome pairing. Meiotic index ranged from 70% to 98% and pollen viability from 66 to 90%. An apparent tendency to decrease the number of quadrivalents and to increase mean meiotic index and pollen viability from C0 to C1 was detected. The use of these autopolyploids in basic and applied research as well as in genetic breeding is promising.

Chromosome doubling of mature haploid Brussels sprout plants by colchicine treatment

The effect of colchicine concentration, and the mode and time of colchicine application in doubling the chromosomes of haploid Brussels sprout plants was assessed by scoring treated plants for the presence of diploid flowers and seed set after self-pollination.

Effect of hormonal and neurotrophic factors on expression of fast myosin in slow muscle

Myosin ATPase and succinic dehydrogenase activity and fast myosin (indirect immunochemical test) were assayed in m. soleus of guinea-pigs after the administration of T4 (200 micrograms/kg) to animals every day for 3 weeks. This was followed by the application of 10 mM colchicine solution to sciatic nerve for 6 min. Fast muscle fibers and the line of precipitation with antiserum to fast myosin were revealed in soleus muscle of experimental animals after the application of colchicine and T4 injection.

Topical application of colchicine, vinblastine and vincristine prevents strychnine-enhanced transsynaptic degeneration in the medullary dorsal horn following transection of the inferior alveolar nerve in adult rats

The effect of topical application of axonal transport blockers to the transected peripheral nerve was assessed by quantitating the strychnine-enhanced transsynaptic degeneration following transection of the inferior alveolar nerve in adult rats. Systemic administration of strychnine (1 mg/kg/day) for 7 days at the postoperational interval of 23 days proved to be suitable for quantitating the transsynaptic degeneration at the light microscopic level. When the proximal stump of transected nerve was treated with 2% colchicine immediately after transection, 5.8 ± 6.8 dark neurons in a single section of the medullary dorsal horn, ipsilateral to the nerve transection, were observed. Following similar treatment with 0.4% vinblastine and 0.2% vincristine, 24.4 ± 10.5 and 9.4 ± 7.0 dark neurons were seen, respectively. When compared with 43.0 ± 9.4 dark neurons, which were seen in animals without alkaloid treatment, colchicine, vinblastine and vincristine significantly reduced the transsynaptic degeneration by 86, 43, and 78%, respectively. Possible mechanisms involved in prevention of transsynaptic degeneration by the alkaloids are discussed.

Induction of the action potential in innervated slow muscle fibers of the frog. Effects of tetrodoxin, vincristine and colchicine

Experiments were made to induce action potentials in innervated slow muscle fibers of Rana temporaria. Drugs were applied to the sciatic nerve or its spinal roots by one of the following methods: (a) implantation of a silastic cuff containing tetrodotoxin (TTX); (b) subepineural injection of TTX combined with subcutaneous injections of vincristine; and (c) epineural application of colchicine or vincristine. Twelve to 50 days later slow fibers responded to direct or indirect stimulation with an action potential. A substantial number of these fibers were innervated by 2 or 3 slow motor axons. It is concluded that these effects of TTX and colchicine (vincristine) are due to inhibition of the axoplasmic transport of an unidentified substance. The results support the hypothesis that the inexcitability of normal slow fibers is due to a ‘trophic’ effect of their motor axons.

Tip cell growth and the frequency and distribution of particle rosettes in the plasmalemma: Experimental studies inFunaria protonema cells

ComparingFunaria protonema tip cells of different age and of experimentally modified growth rate (by changing the light-dark-regime, by application of colchicine and of D2O and by plasmolysis) we found that the site and intensity of growth are related closely to the distribution and frequency of particle rosettes in the PF of the plasma membrane. The results confirm previous suggestions that the rosettes are involved in cellulose fibril formation and that they have a rather short life time (about 10–15 minutes,Reiss et al. 1984). The appearance of rosettes seems to depend on the exocytosis of Golgi vesicle containing wall matrix material. Morphometric calculations suggest that each Golgi vesicle may incorporate one rosette into the plasmalemma in caulonema tip cells.