Intrastriatal injection of colchicine induces striatonigral degeneration in mice

Recent studies from environmental toxicology and molecular genetics demonstrate that midbrain dopamine (DA) neurons are particularly vulnerable to microtubule depolymerizing agents, indicating the involvement of microtubule dysfunction in the pathogenesis of Parkinson's disease. Here we show that intrastriatal injection of colchicine (COL), a well-known microtubule disruptor, induced degeneration of striatonigral pathway. Microtubule disruption caused by unilateral injection of COL blocked the retrograde axonal transport of fluorogold previously injected into striatum and induced substantial death of striatal and DA neurons in substantia nigra pars compacta. Furthermore, COL-induced pathologic changes were associated with robust glial reaction, which may be conducive to the degeneration of striatonigral pathway. We also found that intrastriatal injection of COL resulted in side bias in spontaneous turning activities and apomorphine-induced rotational behavior. Together, our results provide in vivo data lending support to the concept that microtubule dysfunction may play a significant role in the death of DA neurons, though glial reaction may be involved and contribute to the degenerative process. Moreover, intrastriatal COL may serve as another experimental model of striatonigral degeneration (Parkinson's variant of multiple system atrophy), given the concurrent loss of both striatal and DA neurons.     

利用染色体加倍技术创建油菜非整倍体

采用秋水仙素染色体加倍技术,获得了纯合黑籽甘蓝型油菜品系NJ230(Brassica napus)的染色体加倍材料(2n=76).对加倍材料连续自交3代,观察加倍材料后代染色体数目及性状变异,结果表明:(1)加倍材料当代(D1)植株矮化;(2)加倍材料自交1代群体(D2)呈现雄性不育、种子颜色等性状变异;(3)加倍材料自交2代后(D3、D4),多数植株染色体数为2n≤38;(4)获得了1个2n=32的黄籽非整倍体系.本实验为油菜非整倍体创建提供了一种可行的方法.     

Effects of Cycloheximide,Aminoglutethimide, Cytochalasin B,and Colchicine on Ovulation and the Ultrastructure of the Ovarian Follicle Wall in the Stellate Sturgeon Acipenser stellatus Pall

The inhibitor of protein synthesis cycloheximide, inhibitor of steroidogenesis aminoglutethimide, and inhibitor of prostaglandin synthesis indomethacin, as well as the drugs affecting the cell cytoskeleton, such as cytochalasin B and colchicine, were used for studying the mechanisms of ovulation in the stellate sturgeon Acipenser stellatus Pall. Follicles were isolated from the body cavity within certain time intervals after the injection of pituitary suspension to a female and cultivated in media with the inhibitors. In the case of follicles isolated in the middle of the period from hormonal injection until ovulation, cycloheximide, cytochalasin B, and aminoglutethimide suppressed ovulation most effectively, while in the case of oocytes isolated during the last quarter of this period, aminoglutethimide and cytochalasin B were the most effective. It was shown using TEM and SEM that cycloheximide suppressed all processes related to the preparation for ovulation, except the initial ones: contraction of follicle cells and their processes and secondary flattening of these cells. In the presence of aminoglutethimide, the follicle cells underwent pathological changes. Incubation in the media containing indomethacin and colchicine prevented degradation of the outer theca layer at the follicle apex. In the presence of cytochalasin B affecting the cytoskeleton, the drawing of follicle cell processes from the jelly coat channels was blocked, the outer theca cells were strongly contracted, but the cell layer integrity was affected and it was divided in separate fragments. A relationship is discussed between the metabolic processes and morphological changes that lead to ovulation. It was proposed on the basis of the present and previous data that the preovulatory preparation of the follicle tissues comprises two contractile and two apoptotic processes distinctly coordinated in time and space.     

In vitro production and identification of autotetraploids of Scutellaria baicalensis

The roots of Scutellaria baicalensis are a major traditional Chinese medicine. We report research on induction, characteristics and chemical analysis of polyploid plants of S. baicalensis. Immersing calluses in 0.2% colchicine solution for 12 h prior to culture induced a high number of tetraploid plants. The induction rate reached as high as 40% of treated calluses. More than 50 lines of tetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Seven elite lines have been selected for further selection and breeding into new varieties for commercial production.     

Evidence in favour of a role for peripheral-type benzodiazepine receptor ligands in amplification of neuronal apoptosis

The mitochondrial peripheral benzodiazepine receptor (PBR) is involved in a functional structure designated as the mitochondrial permeability transition (MPT) pore, which controls apoptosis. PBR expression in nervous system has been reported in glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand fluo-FGIN-1-27 in mitochondria of rat cerebellar granule cells (CGCs). Additionally, the effect of PBR ligands on colchicine-induced apoptosis was investigated. Colchicine-induced neurotoxicity in CGCs was measured at 24 h. We show that, in vitro, PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4- benzodiazepin-2-one (Ro5-4864) and diazepam (25– 50 M) enhanced apoptosis induced by colchicine, as demonstrated by viability experiments, flow cytometry and nuclear chromatin condensation. Enhancement of colchicine-induced apoptosis was characterized by an increase in mitochondrial release of cytochrome c and AIF proteins and an enhanced activation of caspase-3, suggesting mitochondrion dependent mechanism that is involved in apoptotic process. Our results indicate that exposure of neural cells to PBR ligands generates an amplification of apoptotic process induced by colchicine and that the MPT pore may be involved in this process.     

<Emphasis Type="Italic">In vitro</Emphasis> induction of polyploidy in annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis>)

The present work aims to establish a protocol for in vitro polyploidization using hypocotyl segments or cotyledonary nodes from in vitro grown annatto seedlings. The culture medium used to induce polyploidization was supplemented with MS salts, B5 vitamin complex, 100 mg l^– myo-inositol, 3% (w/v) sucrose, 2.28 M ZEA and 0.30 M IAA (hypocotyl segments) or 4.56 M ZEA (cotyledonary nodes), 0.8% (w/v) agar, and different concentrations of microtubule depolymerising agents, namely colchicine (0, 25, 250 and 1250 M) and oryzalin (0, 5, 15 and 30 M). To determine the optimum duration of either colchicine or oryzalin treatment for the induction of tetraploids, explants were treated for 15 or 30 days on regeneration medium. High frequencies of polyploidy in regenerated shoots from cotyledonary nodes were achieved in culture medium supplemented with 15 M oryzalin, for 15 days. Ploidy determination was based on chromosome counting in metaphasic cells from apical buds, and in the number of pairs of heterochromatic markers on the biggest chromosome, as visualized in interphasic nuclei, detection being easier in the latter. Among the characteristics evaluated, the measurements based on stomata length, width, area and frequency enabled greater discrimination between diploid and polyploid regenerated shoots.     

In vitro propagation of Iphigenia indica,an alternative source of colchicine

The present study involves in vitro propagation of Iphigenia indica (Kunth.) through multiplication of whole corms and corm buds. The whole corms produced very small micro-corms, which developed plants individually whereas corm buds multiplied to produce numerous shoots at variable rates in presence of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The best response in corm and bud multiplication was obtained in Murashige and Skoog's basal medium (MS) supplemented with 2.69 M NAA and 8.88 M BAP. The shoots regenerated were further cultured on MS medium containing NAA and indole-3-butyric acid (IBA) for initiation of roots. MS medium with 5.38 M NAA and 4.92 M IBA induced highest percentage of roots (81%) within 2 weeks in culture.     

Effect of polyploidy induction on biomass and ginsenoside accumulations in adventitious roots of ginseng

Adventitious roots ofPanax ginseng C.A. Meyer (a natural tetraploid) were treated with 50 or 100 mg L^-1 colchicine for 12, 24,36, 48, or 60 h to induce polyploid (octoploid) roots. The largest number of octoploid roots was obtained with a 100 mg L^-1 colchicine treatment over 60 h. To verify that ginsenoside was being accumulated in the developing tissues, the tetraploid (control) and octoploid roots were cultured for 40 d in Murashige and Skoog media that lacked NH₄NO₃ but was supplemented with 2 mg L^-1 naphthaleneacetic acid and 50 g L_-1 sucrose. Levels of fresh and dry biomass were greater in the octoploid roots. Although total ginsenoside and Rb-group ginsenoside contents were less in the octoploid roots than in the tetraploids, the former had a higher amount of Rg-group ginsenosides (especially Rg1). These results demonstrate the benefit that polyploid adventitious roots provide in enhancing the production of secondary metabolites in ginseng.     

The ultrastructure of contractile tubules in the heliozoon Actinophrys sol and their possible involvement in rapid axopodial contraction

We employed an improved fixation procedure for electron microscopy using ruthenium red, and found a bundle of contractile tubules inside the axopodia of the heliozoon Actinophrys sol. Upon food uptake, the tubules shorten and transform into a mass of small granules when rapid axopodial contraction occurs, suggesting that these structures are involved in the process of axopodial contraction. The relationship between transformation of the contractile tubules and accompanying disassembly of the axonemal microtubules was studied by examining the ultrastructure of the contractile tubules after disassembly of the microtubules was artificially induced by cold or colchicine treatment. Granulation of the contractile tubules was induced by cold but not by colchicine treatment. During recovery from cold treatment, granular forms of the contractile tubules became re-elongated and their initial tubular appearance was restored. These results suggest that the contractile tubules in heliozoon axopodia play a role in repetitive cytoplasmic contraction.     

Air drying method using nitrous oxide for chromosome counting in maize

Nitrous oxide (N₂O), colchicine, trifluralin, amiprophos-methyl, 8-hydroxyquinoline, and temperature pretreatments (cold and cold-hot-cold) were compared for chromosome counting in maize (Zea mays L.). Pretreated root tips were prepared by enzymatic maceration and air drying, and the number of countable figures and mitotic indexes were recorded. N₂O treatment at 10 atm for 3 hr produced the largest number of countable chromosome figures (266.5 per preparation) and an average of 44.2 nonoverlapped chromosome figures per preparation. Treatment with 0.04% 8-hydroxyquinoline for 3 hr exhibited a moderate number of countable chromosome figures (53.9 per preparation). The effects of colchicine, trifluralin, amiprophos-methyl and temperature pretreatments were limited.