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PubMed1985

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus.

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.

Animals↗
PubMed1985

Phosphoproteins and protein kinases of the Golgi apparatus membrane.

Incubation of a highly purified fraction derived from rat liver Golgi apparatus with [gamma-32P]ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on Mg2+, independent of Ca2+, calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on Ca2+ and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.

Adenosine Triphosphate↗
PubMed1976

Ganglioside biosynthesis. Characterization of uridine diphosphate galactose: GM2 galactosyltransferase in golgi apparatus from rat liver.

An enzyme that transfers galactose from UDP-Gal to ganglioside GM2 (Tay-Sachs ganglioside) was concentrated 50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents or phospholipids as dispersing agents. Of the numerous detergents tested, sodium taurocholate and Triton CF-54 were most effective in stimulating the reaction. Cardiolipin alone was more effective than any of the detergents tested in stimulating enzyme activity. The pH optimum for the reaction varied with the nature of the dispersing agent. With sodium taurocholate, Triton CF-54 and cardiolipin, the pH optima were 6.2, 5.9, and 5.6, respectively. The enzyme had a nearly absolute requirement for Mn2+, with maximum activity being attained at a concentration of 15 mM Mn2+. Other divalent or trivalent cations were either less effective than Mn2+ or inhibited the transferase reaction. The Km values calculated for UDP-Gal and GM2 were 1.1 X 10(-4) M and 9.9 X 10(-5) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane and not part of the luminal contents. The newly synthesized GM2, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.

Animals↗
PubMed1981

Effect of colchicine on the Golgi apparatus and on GERL of rat jejunal absorptive cells. Ultrastructural localization of thiamine pyrophosphatase and acid phosphatase activity.

Ultrastructural localization of thiamine pyrophosphatase (TTP) and acid phosphatase (AcPase) activity was performed on jejunal absorptive cells of rats pretreated with the antimicrotubular agent colchicine and of control animals. Demonstration of TPP activity showed that most of the dislocated Golgi stacks after colchicine application lacked positively staining cisternae of the mature side. This cytochemical finding is in agreement with the morphologically demonstrable changes of the Golgi stacks resulting in a loss of polarity and give evidence for a colchicine-induced deficiency of the Golgi apparatus. The cytochemical localization of AcPase activity showed deposits of reaction product over lysosomes and GERL and demonstrated a dislocation of GERL occurring concomitantly with the changes of the Golgi apparatus. The antimicrotubular effect of colchicine is well documented; thus the morphological and cytochemical changes of the Golgi apparatus and of GERL might be due to a disturbed microtubular function after application of this agent suggesting an influence of microtubules in the maintenance of the integrity of these organelles. This hypothesis includes the possibility of an involvement of microtubules in formation and differentiation of Golgi stacks and GERL as well as a kind of "skeletal"function being responsible for their characteristic structure and fashion.

Acid Phosphatase↗
PubMed1984

[Ultrastructure of the mitotic apparatus of metaphase cells in a pig embryo kidney tissue culture after stopping the action of 2-mercaptoethanol].

The ultrastructure of the metaphase mitotic apparatus has been studied in the KEPV cells during 6 hours after the removal of 2-mercaptoethanol (0.001 M). Starting from the analysis of chromosome disposition, the structures of the kinetochore regions and of the mitotic spindle poles, and the degree of integrity of the mitotic spindle microtubules, six types of metaphase cells were revealed. A comparison of the results of the present paper with those of the earlier studies enabled us to present the dynamics of the metaphase mitotic apparatus reconstruction. Four basic stages are revealed in this process. At the first stage, the K-metaphase centrioles form diplosomes again, the number and extent of kinetochore microtubules increase too. At the second stage, the metaphase plate forms, but interpolar and astral microtubules are absent. At the third stage, the structure of the kinetochore regions becomes normal. Thus, the metaphase plate may have formed before the orientation of kinetochores to the poles took place. At the fourth stage, the interpolar and astral microtubules appear; the mitotic spindle reestablishes completely. It is supposed that the formation and functioning of the mitotic apparatus is not confined to the interaction of microtubules of different types.

Animals↗
PubMed1984

Monensin-induced swelling of Golgi apparatus cisternae mediated by a proton gradient.

Monensin, a monovalent ionophore, caused swelling of mature cisternae of plant Golgi apparatus. The appearance of swollen cisternae was time-dependent and linear over a period of 1 h with an estimated maximum rate of production of one swollen cisterna every 3 to 4 min. Implicit in these observations was a need for the uptake of osmotically active monovalent cations to have occurred accompanied by a concomitant efflux of H+ and the entry of water. Furthermore, to sustain the H+ efflux, a source of H+ influx also would be required. To test for the latter, cisternal swelling, as visualized by electron microscopy, was monitored by treatment of wild carrot cells in suspension culture with drugs and inhibitors known to interfere with proton gradients. Swelling was inhibited by the protonophore, FCCP, by the inhibitor of lysosomal acidification, quercetin, and by the lysosomotropic amines, chloroquine and ammonia. While antimycin A, an inhibitor of mitochondrial oxidative phosphorylation, was ineffective, cyanide dramatically decreased swelling. The numbers of swollen cisternae produced could be reduced by prolonged treatment with arsenate, such that an ATP requirement is indicated, at least, for cisternal formation. Swelling was promoted by citrate, representative of a permeant organic anion. Reductions in numbers of monensin-induced swollen cisternae in the presence of quercetin, vanadate, and chloroquine could be compensated for by the addition of citrate. We conclude that the monensin-induced swelling of Golgi apparatus cisternae may involve a mechanism generating a proton gradient at or near the mature Golgi apparatus face.

Arsenates↗
PubMed1982

Golgi apparatus and acid phosphatase in mammary gland and kidney cells under the influence of diethylstilbestrol (DES).

It is commonly accepted that hormonal disturbances in experimental animals produce pathological changes in several organs namely in mammary glands and kidney. Taking into consideration the close relationship between acid phosphatase and Golgi apparatus, the present study was undertaken, to study the relationship in kidney and mammary gland cells under the influence of DES. The observation indicate, that acid phosphatase and Golgi apparatus were localized in the cytoplasm of mammary gland and kidney cells. It has also been found a parallel change in the activity of acid phosphatase and the degree of development of Golgi apparatus after DES administration.

Acid Phosphatase↗
PubMed1978

Cytochemical demonstration of glutaraldehyde-resistant NADH-ferricyanide oxido-reductase activities in rat-liver plasma membranes and Golgi apparatus.

NADH-ferricyanide reductase activity was demonstrated in rat liver endomembranes by cytochemical procedures. The activity observed in plasma membrane and mature portions of the Golgi apparatus resisted fixation in 0.1% glutaraldehyde, a characteristic which permitted differentiation of the NADH-ferricyanide reductase of plasma membranes and mature Golgi apparatus elements from those of mitochondria, microbodies, endoplasmic reticulum and nuclear envelope. With the latter membranes, activity could be demonstrated only with unfixed material or following brief glutaraldehyde fixation and was greatest with broken cells or isolated fractions due to problems of penetration of reagents. Biochemical studies paralleled cytochemical findings with respect to glutaraldehyde fixation and sensitivity to other metabolic inhibitors. The findings provide evidence that a NADH-ferricyanide reductase may be among the membrane constituents conserved and/or modified during flow differentiation of membranes. The basis for a method to evaluate plasma membrane contamination of endoplasmic reticulum fractions and to differentiate among mature and immature secretory vesicles of the Golgi apparatus is also indicated.

Animals↗
PubMed1995

[Corticosteroid responsive inflammatory myopathy with autoantibody against Golgi apparatus].

A 51-year-old woman of progressive myopathy predominantly involving proximal groups of limb-muscle was reported. Serum enzymes originating from the skeletal muscle always remained within normal range, and a positive autoantibody against Golgi apparatus and the SS-A (Ro) antibody in serum were noted. In muscle biopsy performed twice, extensive degenerating/regenerating fibers and sparce inflammatory cells between connective tissue elements in light microscopy, and disrupted lamellar structure of Golgi apparatus in electron microscopy were observed. Corticosteroid therapy was markedly effective. In the present case of a "so-called" limb-girdle syndrome, humoral autoantibodies, especially against Golgi apparatus, could induce a metabolic disturbance in the muscle fiber.

Autoantibodies↗
PubMed1994

Protein kinase C zeta is associated with the mitotic apparatus in primary cell cultures of the shark rectal gland.

Protein kinase C zeta (PKC zeta) is an atypical PKC isoform that has recently been implicated in cell division and cell growth. However, there has been no morphologic evidence for the involvement of PKC zeta in mitogenic signal transduction. Here we use immunocytochemistry to demonstrate that PKC zeta co-localizes with microtubules in both interphase and metaphase cells of the shark rectal gland in primary culture. This co-localization is present after non-ionic detergent treatment and is disrupted by nocodazole. During mitosis, PKC zeta is associated with the mitotic apparatus and co-localizes with beta-tubulin in spindle microtubules, while entirely sparing astral microtubules. These findings provide the first evidence that PKC zeta is associated with the mitotic apparatus. The striking presence of PKC zeta in the central portion of the mitotic apparatus suggests a functional role for this kinase isoform in cell division.

Animals↗
PubMed1996

Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus.

Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis (TSC), a human genetic syndrome characterized by the development of tumors in a variety of tissues. We have previously shown that tuberin is a widely expressed 180 kDa protein which exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. In this study we have used affinity-purified antibodies against tuberin to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression was detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin were found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines revealed a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) revealed that the staining of tuberin was consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolished the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrated co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tumor suppressor function of tuberin.

Animals↗
PubMed1996

Role of the Golgi apparatus in the phosphorylation of apolipoprotein B.

Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.

Adenosine Triphosphate↗
PubMed1996

Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus.

Several studies have demonstrated that lipid-free apolipoproteins can promote cholesterol and phospholipid efflux from cells; however, the mechanisms and the role of cell-mediated pathways involved remain incompletely elucidated. We have recently demonstrated that brefeldin A or monensin, agents that disrupt Golgi apparatus structure and function, inhibit intracellular cholesterol efflux from cells to high density lipoproteins. In the present study we examined the effects of those agents on cell cholesterol and phospholipid efflux to purified apolipoprotein A-I (apoA-I) and apolipoprotein-depleted acceptors from cholesterol-loaded fibroblasts. Brefeldin A or monensin treatment of cells during incubation with apoA-I inhibited efflux of cellular cholesterol by greater than 80% compared with control cells, measured by changes in cellular cholesterol radioactivity, mass, and the substrate pool of cholesterol available for esterification by acyl coenzyme A:cholesterol acyltransferase. Inhibition of cholesterol efflux by these agents could not be overcome by increasing the apoA-I concentration and persisted during incubations up to 24 h. Similarly, brefeldin A and monensin inhibited up to 80% of apoA-I-mediated efflux of labeled phospholipids from cholesterol-loaded cells relative to controls. In contrast, lipid efflux mediated by apolipoprotein-depleted acceptors (trypsin-modified HDL and sonicated phospholipid vesicles) was not sensitive to these drugs. On the basis the known effects of brefeldin A and monensin on Golgi apparatus structure and function, these results are consistent with the notion that efflux of cell lipids by apolipoprotein-dependent mechanisms, but not by apolipoprotein-independent mechanisms, require active cellular processes involving an intact and functional Golgi apparatus.

Apolipoproteins↗
PubMed1998

[Current concept of structure and function of the Golgi apparatus. On the 100-anniversary of the discovery by Camillo Golgi].

The paper is a brief review of the current data on the structure and function of the Golgi apparatus since its discovery till the recent investigations, including the works published in 1997. Apart from reviewing the electron microscopy level of the structure of the Golgi apparatus, the data are considered on its molecular and supramolecular organization. The paper analyses critically the proposed mechanisms of the intracellular transport of proteins and their processing and modifications in the Golgi apparatus both in terms of the vesicular theory and in a model based on the gradual maturation of the cis-cistern and its transformation to the trans-cistern (i.e. its propagation from one pole to the other, a so-called "progression"). Experimental data are described, which disagree with the current models of the intracellular transport. Based on the literature and authors' own data, a modified model of the intracellular transport is proposed. This model eliminates, to a degree, the contradictions present in the models discussed above.

Cell Biology↗
PubMed1998

Influence of bis(maltolato)oxovanadium(IV) on activity of galactosyltransferase (GalT) and morphology of rat liver Golgi apparatus in control and streptozotocin diabetes.

The relation between bis(maltolato)oxovanadium(IV) (BMOV) influencing the biochemical activity of rat liver Golgi apparatus and the morphology of this organelle was studied in normal and streptozotocin-diabetic rat livers. Ultrastructural examinations revealed marked differences in the morphology of Golgi apparatus in three groups of animals. In the control rats treated only with 0.5% NaCl we did not find any biochemical and morphological changes. Marked changes were found in the rat liver after 1.8 mmol BMOV in 0.5% NaCl (as drinking solution) applied for 7 days, so-called "control" group for vanadium. In this group Golgi apparatus seemed shorter than in the diabetic animals. Finally, the same treatment of rats with previously induced SZ-diabetes, showed relatively small morphological alterations. The ultrastructural observation was compatible with the activity of galactosyltransferase (GalT), the Golgi marker enzyme. In diabetic rats treated with BMOV the activity of this enzyme was almost the same as in controls. Summing up dramatic alterations, previously found in diabetic-untreated rats [22], normalized after orally applied BMOV solution, even after a short time.

Animals↗
PubMed2000

Actin-associated protein synaptopodin in the rat hippocampal formation: localization in the spine neck and close association with the spine apparatus of principal neurons.

Dendritic spines are sites of synaptic plasticity in the brain and are capable of remodeling their shape and size. However, little is known about the cellular mechanisms that regulate spine morphology and motility. Synaptopodin is a recently described actin-associated protein found in renal podocytes and dendritic spines (Mundel et al. J Cell Biol. [1997] 139:193-204), which is believed to play a role in spine plasticity. The present study analyzed the distribution of synaptopodin in the hippocampal formation. In situ hybridization histochemistry revealed a high constitutive expression of synaptopodin mRNA in the principal cell layers. Light microscopic immunohistochemistry showed that the protein is distributed throughout the hippocampal formation in a region- and lamina-specific manner. Postembedding immunogold histochemistry demonstrated that synaptopodin is exclusively present in dendrites and spines, specifically in the spine neck in close association with the spine apparatus. Spines lacking a spine apparatus are not immunoreactive for synaptopodin. These data suggest that synaptopodin links the spine apparatus to actin and may thus be involved in the actin-based plasticity of spines.

Actins↗
PubMedDate not supplied

Tripartite and bipartite models of the photochemical apparatus of photosynthesis.

Tripartite and bipartite models for the photochemical apparatus of photosynthesis are presented and examined. It is shown that the equations for the yields of fluorescence from the different parts of the photochemical apparatus of the tripartite model transform into the simple equations of the bipartite formulation when the probability for energy transfer from the light-harvesting chlorophyll a/b complex to photosystem II is unity. The nature of the 695 and 735 nm fluorescence bands which appear in the emission spectrum of chloroplasts at low temperature is examined. It is proposed that these bands are due to fluorescence from energy-trapping centres which form in the antenna chlorophyll of photosystem II and photosystem I on cooling to low temperature. Even though these fluorescence emissions can be regarded as low temperature artifacts since they are not present at physiological temperatures, they nevertheless are proportional to the excitation energy in the two photosystems and can be used to monitor energy distribution in the photochemical apparatus. However, the question of their artifactual nature is crucial to the interpretation of fluorescence-lifetime measurements at low temperature.

Chlorophyll↗
PubMed2006

DiGeorge syndrome and pharyngeal apparatus development.

DiGeorge syndrome is the most frequent microdeletion syndrome in humans, and is characterized by cardiovascular, thymic and parathyroid, and craniofacial anomalies. The underlying cause is disturbed formation of the pharyngeal apparatus, a transient structure present during vertebrate development that gives rise to endocrine glands, craniofacial tissue, and the cardiac outflow tract. The pharyngeal apparatus is composed of derivatives of ectoderm, endoderm, mesoderm and the neural crest. Thus, complex interactions between cell types from different origins have to be orchestrated in the correct spatiotemporal manner to establish proper formation of the pharyngeal system. The analysis of engineered mouse mutants developing a phenotype resembling DiGeorge syndrome has revealed genes and signalling pathways crucial for this process. Intriguingly, these mouse models reveal that interference with either of two distinct phases of pharyngeal apparatus development can contribute to the aetiology of DiGeorge syndrome.

Animals↗