Effect of colchicine on the Golgi complex of rat pancreatic acinar cells.

Colchicine administered to adult rats at a dosage of 0.5 mg/100 g of body weight effected a disorganization of the Golgi apparatus in pancreatic acinar cells. The results obtained after various periods of treatment (10 min to 6 h) showed (a) changes in all components of the Golgi complex, and (b) occurrence of large vacuoles that predominated in cytoplasmic areas outside the Golgi region. The alterations in Golgi stacks concerned elements of the proximal and distal side: (a) accumulation of transport vesicles, (b) formation of small, polymorphic secretion granules, and (c) alterations in the cytochemical localization of enzymes and reaction product after osmification. Transport vesicles accumulated and accompanied short, dilated cisternae, which lack mostly the reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase, and osmium deposits after prolonged osmification. After 4 to 6 h of treatment, accumulated transport vesicles occupied extensive cellular areas; stacked cisternae were not demonstrable in these regions. The changes on the distal Golgi side included GERL elements: condensing vacuoles were diminished; they were substituted by small, polymorphic zymogen granules, which appeared to be formed by distal Golgi cisternae and by rigid lamellae. Unusually extended coated regions covered condensing vacuoles, rigid lamellae, and polymorphic secretion granules. A cytochemical distinction between Golgi components and GERL was possible neither in controls nor after colchicine treatment. The cytochemical alterations in Golgi components were demonstrable 20-30 min following administration of colchicine; at 45 min, initial morphological changes--augmentation of transport vesicles and formation of polymorphic zymogen granules--became apparent. 20 min after administration of colchicine, conspicuous groups of large vacuoles occurred. They were located mostly in distinct fields between cisternae of the endoplasmic reticulum, and were accompanied by small osmium--reactive vesicles. Stacked cisternae were not demonstrable in these fields. Vacuoles and vesicles were devoid of reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. The results provide evidence that formation of stacked Golgi cisternae is impaired after colchicine treatment. The colchicine--induced disintegration of the Golgi complex suggests a regulatory function of microtubules in the organization of the Golgi apparatus.

Étude ultrastructurale des coiffes des racines de chêne (Quercus robur). I. Les pivots

Three zones may be distinguished in rootcaps of orthogeotropic taproots of oak seedlings.(1) The meristematic zone: cells have no distinctive characteristics except that they always have small rounded vacuoles.(2) The statocytes have amyloplastids accumulating in the lower cell portion. The nucleus is near the upper end. Unlike the herbaceous species studied, these cells have large central vacuoles. This may be a characteristic of woody species. This hypothesis is discussed.After a 90 or 135° rotation in a vertical plane, amyloplastids become progressively concentrated in the new lower end of the statocyte. Orientation of the endoplasmic reticulum also changes, and the vacuolar system is reorganized. At room temperature root curvature begins to appear more than 2 h after the rotation.(3) The outside cell layers are composed of mucilage secreting cells with dictyosomes producing large vesicles containing polysaccharides. These accumulate in the periplasmic area. A strong inosine diphosphatase activity is shown in the vesicles.

ANALYTICAL FRACTIONATION OF HOMOGENATES FROM CULTURED RAT EMBRYO FIBROBLASTS

Homogenates of cultured rat embryo fibroblasts have been assayed for acid phosphatase, N-acetyl-ß-glucosaminidase, cathepsin D, acid deoxyribonuclease, cytochrome oxidase, NADH cytochrome c reductase, 5'-nucleotidase, inosine diphosphatase, acid pyrophosphatase, neutral pyrophosphatase, esterase, catalase, cholesterol, and RNA. The validity of the assay conditions was checked. Neutral pyrophosphatase is a readily soluble enzyme. Acid hydrolases, except acid pyrophosphatase, are particle-bound enzymes, which exhibit a high degree of structural latency. They are activated and solubilized in a parallel fashion by mechanical treatments and tensio-active agents. Catalase is also particle-bound and latent; activating conditions stronger than those for hydrolases are required to activate the enzyme. Acid pyrophosphatase, 5'-nucleotidase and inosine diphosphatase are firmly particle-bound, but not latent; they are not easily solubilized. In differential and isopycnic centrifugation, the latent hydrolases, cytochrome oxidase and catalase dissociate largely from each other; this suggests the occurrence of lysosomes and peroxisome-like structures besides mitochondria. The distribution patterns of 5'-nucleotidase and cholesterol are largely similar; digitonin influences their equilibrium density to the same extent; these two constituents are thought to be related to the plasma membrane. Inosine diphosphatase and acid pyrophosphatase are also partially associated with the plasma membrane, although some part of these enzymic activities probably belongs to other structures. NADH cytochrome c reductase is associated partly with the endoplasmic reticulum, partly with mitochondria.

Glycosyltransferases in the Golgi membranes of onion stem

Cell fractions consisting largely of Golgi membranes were prepared from the meristematic region of the onion. Several enzyme activities were found to be localized in these fractions: inosine diphosphatase, galactosyltransferases and glucosyltransferases. The fractions catalysed the transfer of [14C]galactose from UDP-galactose to endogenous and cell-sap acceptors, to N-acetylglucosamine and to ovalbumin. In the presence of bovine α-lactalbumin, transfer to glucose (lactose synthesis) was catalysed. [14C]Glucose was transferred from UDP-glucose to endogenous and cell-sap acceptors, to cellobiose and to fructose (sucrose synthesis). All these activities were latent, being potentiated by detergents (Triton X-100 or sodium deoxycholate). The characteristics of some of these enzyme activities are described and their biological significance is discussed.

A CHARACTERIZATION OF NUCLEOSIDE DIPHOSPHATASE IN THE ONION ROOT TIP

Inosine diphosphatase of a Golgi-enriched fraction of the onion root tip was characterized. Peak enzyme activity occurred at pH 4.8 and 7.0, although considerable activity was present between the peaks. The activity at neutral pH approximately doubled during a 4-day cold storage; no such increase of the pH 4.8 activity occurred under similar conditions. Treatment with deoxycholate or Triton X-l00 also activated the pH 7.0 enzyme. Although magnesium, manganese and calcium all supported inosine diphosphatase activity, 2 mM manganese supported maximal activity. Uridine diphosphate, guanosine diphosphate and inosine diphosphate were hydrolyzed much more rapidly than the other substrates tested. Sodium fluoride, uranyl nitrate, potassium chloride and heat all partially inhibited the enzyme. Glutaraldehyde and lead nitrate, two reagents to which nucleoside diphosphatase is exposed when studied cytochemically, greatly reduced enzyme activity.

Phosphatases and Differentiation of the Golgi Apparatus

Cytochemical techniques for the electron microscopic localization of inosine diphosphatase, thiamine pyrophosphatase, and acid phosphatase have been applied to the developing root tip of Zea mays. Following formaldehyde fixation the Golgi apparatus of most of the cells showed reaction specificity for IDPase and TPPase. Following glutaraldehyde fixation marked localization of IDPase reactivity in the Golgi apparatus was limited to the root cap, the epidermis, and the phloem. A parallelism was apparent between the sequential morphological development of the apparatus for the secretion of a polysaccharide product, the fairly direct incorporation of tritiated glucose into the apparatus to become a component of this product and the development of the enzyme reactivity. Acid phosphatase, generally accepted as a lysosomal marker, was found in association with the Golgi apparatus in only a few cell types near the apex of the root. The localization was usually in a single cisterna at the face of the apparatus toward which the production of secretory vesicles builds up and associated regions of what may be smooth endoplasmic reticulum. Since the cell types involved were limited regions of the cap and epidermis and some initial cells, no functional correlates of the reactivity were apparent. Despite the presence of this lysosomal marker, no structures clearly identifiable as ‘lysosomes’ were found and the lack of reaction specificity in the vacuoles did not allow them to be so defined.

ULTRASTRUCTURAL AND CYTOCHEMICAL OBSERVATIONS ON B-16 AND HARDING-PASSEY MOUSE MELANOMAS THE ORIGIN OF PREMELANOSOMES AND COMPOUND MELANOSOMES

The B-16 and Harding-Passey mouse melanomas were studied by light microscopy (tyrosinase, acid phosphatase, aryl sulfatase, thiamine pyrophosphatase and inosine diphosphatase activities) and electron microscopy (morphology and tyrosinase and acid phosphatase activities). Lysosomal enzyme activity is present in individual premelanosomes and melanosomes as well as in compound melanosomes. Acid phosphatase and tyrosinase activities are present in a Golgi-associated system of smooth endoplasmic reticulum (GERL) and small vesicles related to it. The acid phosphatase and tyrosinase activities of premelanosomes, and morphologic appearances, support the hypothesis that the granules arise from GERL. On the basis of the evidence presented, it is suggested that compound melanosomes arise within melanoma cells by autophagy.