Influence of colchicine on the addition of a sugar to the enamel protein in secretory ameloblasts of cultured germs of rat molar tooth by 3H-galactose radioautography

Summary The influence of colchicine on the addition of ³H-galactose to the enamel protein in secretory amelloblasts of cultured germs of rat molar tooth was investigated by light- and electron-microscopic radioautography. In tooth germs cultured without colchicine, the reaction products of ³H-galactose were observed over Golgi cisternae at early chase times and then localized over the enamel with time. In tooth germs cultured with colchicine, the silver grains were seen over the Golgi cisternae, condensing granules and accumulated secretory granules. Some grains also appeared with time over the pale granular material precipitated in the intercellular space with colchicine treatment. In quantitative analysis with light microscopic radioautography, values of silver grain counts over the unit area (100 m²) on ameloblasts and enamel of colchicine-treated tooth germs were significantly lower at both 0 min and 30 min chase after 30 min pulse than those of control tooth germs, respectively. This finding indicates that colchicine diminished the incorporation of ³H-galactose into the secretory ameloblast of cultured tooth germs. It is suggested that colchicine decreases the activity of the Golgi apparatus with regared to the addition of sugar to the synthesizing glycoprotein in the secretory ameloblast.     

Schistosoma mansoni: Radioautography of colchicine's effect on [3H]proline incorporation into adults in vitro

Adult Schistosoma mansoni were studied radioautographically in order to ascertain the effect of exposures to a fixed concentration of colchicine (5 × 10^?4M) for varying time intervals upon the incorporation of [³H]proline in the tegument. Additionally, a study was made on the effect of varying time exposures of colchicine on the cytochemical localization of alkaline phosphatase (EC 3.1.3.1) in the tegumental invaginations. Worms exposed to colchicine for more than 2 hr preceding addition of the labeled amino acid displayed significant changes in the pattern of distribution. The most profound change was noted in the male tegument where a statistically significant decrease was observed in treated worms. Female worms, on the other hand, failed to display any effect of the drug on the distribution pattern for the times utilized. The distribution of alkaline phosphatase activity was much reduced in the teguments of both sexes. Morphological effects of the drug included disappearance of microtubules from the cytoplasmic connectives, a stacking of RER in the subtegumental cells, and accumulation of discoid granules and membranous bodies in the subtegumental cells. It is hypothesized that the amino acid is associated with the discoid granule at the subtegumental cell level and is ultimately translocated, with the aid of microtubules in the cytoplasmic connectives, to the tegument. Alkaline phosphatase activity is assumed to be associated with the membranous body.     

Microtubules,protoplasts and plant cell shape

Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10^-3 M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules—which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface—act as an essential template or scaffolding for the orientated deposition of cellulose.     

Effects of Monensin and Colchicine on Myelin Galactolipids

Monensin and colchicine have been used in a variety of systems to disrupt functioning of the Golgi apparatus and transport of Golgi-derived vesicles to the plasma membrane. In this study the effects of monensin and colchicine on the synthesis of cerebroside and sulfatide and their appearance in myelin were examined to determine whether these myelin components are processed through the Golgi apparatus. Brain slices from rats 17 days old were incubated with [3H]galactose and [35S]-sulfate to label cerebroside and sulfatide. Myelin was isolated on sucrose density gradients. Fractions highly enriched in cerebroside and sulfatide were prepared from homogenates and myelin fractions by lipid extraction, alkaline methanolysis, and in some cases TLC. Monensin at 0.1 microM had no significant effect on synthesis of these galactolipids as measured by incorporation of [3H]-galactose into cerebroside or [35S]sulfate into sulfatide in homogenates. However, appearance of [35S]sulfatide in the myelin fraction was reduced to 49% of control, while appearance of [3H]cerebroside was not significantly reduced. Colchicine from 1 mM to 0.1 microM had effects similar to monensin, that is, appearance of [35S]sulfatide in myelin was depressed, but again [3H]cerebroside was not affected. Incorporation of [35S]sulfate into sulfatide in homogenate was 93% of control, while appearance of [35S]sulfatide in the myelin fraction was depressed to 58% of control. The inhibition of appearance of sulfatide in myelin by colchicine and monensin is consistent with the view that sulfation of cerebroside occurs in the Golgi and that sulfatide is transported via Golgi-derived vesicles to the forming myelin membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of secretion in thymic epithelial cells: Ultrastructural studies of the effect of blockage at various levels

Summary The observation of secretory phenomena in mouse thymic epithelial cells is disappointing since no real secretion image is found. An adequate technique for such a study is to block the secretion pathway and to observe by electron microscopy cells accumulating secretory products. For this purpose, we used three means of blocking secretion: Firstly, since the thymic epithelial cell is regulated by a feedback phenomenon, secretion was blocked by antibodies against thymulin, one of the hormones secreted by these cells. Secondly, colchicine was used to modify the intracellular transport of the secretory product. In both of these types of experiments, electron microscopy showed a great increase in the number of clear vacuoles and their granular contents in epithelial cells. In a third series of experiments, we used monensin at a concentration that blocks the intracellular transport of secretory proteins at the various levels of the Golgi apparatus. In this series, only an increased number of vacuoles was observed, but they appeared devoid of all granular content. It can be concluded that in the thymic epithelial cell, a discrete system of secretion directs the passage of the product, originating in the cisternae of the endoplasmic reticulum, into clear vacuoles, the terminal element of the cellular secretory apparatus.     

Early embryonic development of the dipteran insect Heteropeza pygmaea in the presence of cytoskeleton-affecting drugs

Summary Embryos of the paedogenetically reproducing gall midge Heteropeza pygmaea develop floating in the haemocoel of a so-called mother larva. The egg membranes remain permeable and the embryos increase in size during embryonic development by taking up nutrients from the haemolymph. Such embryos can be cultured in vitro, i.e. in haemolymph drops obtained from mother larvae. We tested the effects of several drugs known to interact with cytoskeletal elements on different stages of embryonic development, including cleavage and gastrulation. The drugs were added to the in vitro cultures and the effects were studied with time-lapse cine-micrography. Colchicine and vinblastine blocked cleaving eggs in metaphase stage and arrested yolk globule oscillation. In spite of such a block blastoderms once formed continued development through germ band formation and extension and also increased in size. Cytochalasin B did not affect the stage of cleavage; however, it inhibited gastrulation and subsequent morphogenetic processes and also prevented size increase. We conclude that (1) the functioning of microtubules is needed for yolk globule oscillation during cleavage interphases but not for the gastrulation processes subsequent to blastoderm formation and (2) microfilaments do not play an important role in cleavage, at least not for the orderly succession of the cleavage divisions, but are essential for the morphogenetic movements associated with gastrulation. We suggest that during cleavage a limited stock of microtubules and their precursors is responsible for both transport of chromosomes during mitoses and translocation of organelles during interphase. Yolk oscillation seems to be a secondary effect and of minor or no importance for the normal course of embryonic development.Dedicated to Professor Gerhard Krause on the occasion of his 80th birthday     

The effect of colchicine on protein secretion by differentiating odontoblasts and ameloblasts in the hamster tooth in vitro as shown by radioautography with 3H-proline

Summary We have examined radioautographically the protein synthetic and secretory activity of differentiating odontoblasts and ameloblasts, exposed for 9 h in vitro to various concentrations of colchicine in the presence of ³H-proline. Colchicine impairs the cytodifferentiation of the dental epithelium into ameloblasts and of the dental mesenchyme into odontoblasts; the effects depend on the dose. However, denial epithelial cells are more sensitive to the drug than dental mesenchymal cells. In stages prior to odontoblast differentiation, colchicine enhances the number of radioautographic grains over the dental epithelium without changing the grain counts over the dental basement membrane area: This suggests that in vitro the dental epithelium synthesizes and secretes proline-containing components that are not constituents of the dental basement membrane. Also, during the subsequent stages of ameloblast differentiation colchicine increases the number of radioautographic grains over the preameloblasts. The present data suggest that the primary in vitro target of colchicine is not the dental mesenchyme, but the dental epithelium. The data also indicate that differentiating ameloblasts synthesize and secrete significant amounts of proteins in vitro prior to the first deposition of enamel.     

海马结构与大白鼠避暗行为的学习和记忆 Ⅳ.微量秋水仙素注入海马结构各部的影响

在成年的雌性Wistar大白鼠双侧海马结构的各部,分别注以0.5微升含3微克秋水仙素的生理盐水,观察能否影响避暗行为的学习和记忆,并作相关的组织学检查。结果表明:双侧背部或后部背侧的齿状回颗粒细胞的损毁,对大白鼠避暗行为的学习和记忆无明显的影响;若损毁Krieg大白鼠脑图谱的H6.0毫米水平面腹侧的齿状回颗粒细胞,则非常显著地削弱(但未能完全阻断)避暗行为的学习和记忆。     

Immunohistochemical localization of TRH in rat CNS: comparison with RIA studies

The localization of thyrotropin releasing hormone (TRH) in rat brain determined by use of avidin-biotin immunoperoxidase histochemistry was compared with the distribution and quantitation by radioimmunoassay (RIA). Male Sprague-Dawley rats received intracisternal injections of 100 micrograms of colchicine or saline and were sacrificed 24 hours later. Brains were either perfused with lysine-periodate fixative and processed for TRH immunohistochemistry or were dissected into 9 brain regions for TRH RIA. In colchicine pretreated rats. TRH immunoreactive perikarya were observed only in nuclei of the hypothalamus and brain stem. No cell body staining was observable in non-colchicine treated rats. With the exception of the olfactory bulb, brain regions exhibiting dense TRH staining contained high concentrations of TRH as measured by RIA. Colchicine pretreatment did not alter the concentration of TRH in most brain regions, however, there was a significant increase in brain stem TRH content 24 hours following colchicine administration. These findings indicate that immunohistochemical localization of TRH corresponds well with endogenous concentrations of TRH determined by RIA.