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Role of the Golgi apparatus in the early glycosylations of glycosphingolipids
118
Cell Biology International
Reports, Vol. 74, Abstracts Supplement
1990
ROLR OF THB GOLGI APPARATUS IR THR RARLY GLYCOSYLATIORS OF GLYCOSPHIRGOLIPIDS Riccardo Ghidoni, Uarco Trinchera, Dario Carrettoni and Biagio Leone. Department of Medical Chemistry and Biochemistry, and H S. Raffaele Scientific Institute, University of Milan, Milano, Italy. The present study addresses the subcellular aspects of the initial assembly of the oligosaccharide chain of glycosphingolipids. To elucidate this point, we prepared different subcellular fractions from rat liver, including an extremely purified Golgi apparatus fraction, which contains intact with a 150-fold cisternal stacks gangliosideenrichment of and glycoprotein glycosyltransferases. Then characterixed we UDP-Gal: glucosylceramide Rl~lgalactosyltransferase lactosyl(GalT-21, which produces ceramide, a key intermediate in the of glycosphingolipids, and biosynthesis estabilished its subcellular Moreover, by the in vivo localization. of different administration to rats labelled exogenous glycosphingolipids, specific position of the molecule ztermined the subcellular traffic' 1% netabolites involved ' the early R::ults indicate glycosylation steps. that GalT-2 is localized in the Golgi where glucosylceranide is apparatus, made available from different metabolic and where the products of origin, glycosylations are formed. further
THEODDNTOeLASTSOFRATMDLARS AFTER PRIMARY DSNTINDGSNESIS Paolo Romagnol i,Flavio Galeotti,Giovanni Mancini, El isabetta Francini. Department of Human Anatomy and Histology, University of Florence, Italy This studywas aimed at defining the evolution with time of the organelles of odontoblasts after primary dentinoganesis, in teeth with limited eruption. The odontoblastsofratfirstlowermolarswere anaIysedwith morphometrical methods applied to electron microscopy, since primary dentinogeneois to complete eruption, i.e., since 10 to 40 d of age. All the organelles underwent atrophy, but at dif ferent rates among each other; in particular, the endoplasmic reticulum decreased in size progressivel y from 10 to 40 d, whereas the Gol gi apparatus decreased markedly between 10 and 14 d and then remained practical1 y unchanged in size. Autophagocytosis was never enhanced above what occurring during primary dentinogenesis. The degree of POlarization towards dentine was reduced progressivelyandat4Odthecentriolesof some odontoblasts were located towards pulp, instead of dentine. The distribution of the extension of the endoplasmic reticulum,assumedasan estimate of that of the secretory activity, was such that in ca. 90% cells at 40 d this organelle was smaller than in anycell at 10 d. These results suggest that cell atrophy may occur without an increase in autophagocytosis, that the organelles along the secretory pathway may be regulated independently of each other, and that in the odontoblasts, as in several types of epithelial secretory cells, the fraction of cells engaged into appreciable secretory activity is correlated with thedegreeofactivationof the whole cell population.
THE EFFECT OF ESTRADIOL 17(3 ON ENDOMEMBRANES AND PEROXISOMES IN FISH HEPATOCYTES Nada Pipan,Peter VeraniE.Institute of Human Biology,Medical Faculty,Ljubljana,LipiEeva 2, Yugoslavia The known stimulating effect of estradiol 17fi (E2) on- the synthesis of vitellogenin (1) was supplemented by the stereological and cytochemical analysis of endoplasmic reGolgi apparatus (GA) and peroticulum (ER), xisomes (PO) in hepatocytes of treated male zebrafish (Brachydanio rerio). The hormon (1,ug E2/1 1 water) was applied for 6, 20, 30 and 40 days. Cis cisternae of GA were stained by prolonged osmification and trans part by tiamino pyrophosphatase (TPP) and For the identification of acid phosphatase. PO diaminobenzidine (DAB) method was used. The results showed the enlargement of all Maximal membranes analysed in present study. increase of the surface density of ER was reached after 30 days. The treatment did not change the size of individual dictyosomes of GA but induced the increase of their nuThe osmiofility of cis part became mber. much stronger and in some cases the trans cisternae were also modified. The PO responded on the stimulation by the decrease of their mean radius and multiplication of organelles that resulted in an increase of the surface of PO membranes per volume unit ob hepatocytes. The processes is reversible; in fish kept 4 days in pure water after 40 days of treatment the initial stage was reestablished. 1. Peute J, Huiskamp R, van Oordt PGWJ, Cell Tiss. Res. 242:377-382, 1985
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DEGSUDATION OF I@l MUTANTS IN TEIEENDOPLABILMC-UM. AnnaMaria Fra+, Cristina Alberini”, Paola Casiello”, Dario FinazziA and Roberto Sitia*O. *Iatituto San Raffaele, Milano, “University of Brescia, Brescia, “I&it&o Nazionale per la Ricerca sul Cancro, Genova,Italy. B lymphocytes do not secrete IgM and plasmacells only secrete polymeric IgM molecules. Extensive intracellular degradation of secretory IgM occurs in both cell types (virtually 100% in B cells, about 25% in plasma cells). We have previously shown that retention of secretory IgM by B cells and the failure of plasmacells to secrete monomeric IgM are both attributable to the cyst&e residue (cys 575) found at the carboxi-terminus of secretory IgM heavy chains (pa). Thus, cys 575 is involved both in polymerisation of IgM and in intracellular retention of unassembled intermediates. Retention takes place within the endoplasmic reticulum (ER), and, in general, correlates with binding to BiP. Substitution of cys 575 with alanine results in increased rate of secretion and inhibition of degradation. The inverse correlation between rate of transport and ER degradation might eimply reflect the time the protein spends in the ER. However, differences in the degradation rates for wild type andmutentpa were observed also in the presence of brefeldin A, when no transport beyond the ER takes place. We propose that, in the case of IgM, the signals required for retention may also be utiheed to target unassembled molecules to ER degradation. SupportedbyCNRandAIRC.
Cell Biology International
Reports, Vol. 74, Abstracts Supplement
1990
ROLR OF THB GOLGI APPARATUS IR THR RARLY GLYCOSYLATIORS OF GLYCOSPHIRGOLIPIDS Riccardo Ghidoni, Uarco Trinchera, Dario Carrettoni and Biagio Leone. Department of Medical Chemistry and Biochemistry, and H S. Raffaele Scientific Institute, University of Milan, Milano, Italy. The present study addresses the subcellular aspects of the initial assembly of the oligosaccharide chain of glycosphingolipids. To elucidate this point, we prepared different subcellular fractions from rat liver, including an extremely purified Golgi apparatus fraction, which contains intact with a 150-fold cisternal stacks gangliosideenrichment of and glycoprotein glycosyltransferases. Then characterixed we UDP-Gal: glucosylceramide Rl~lgalactosyltransferase lactosyl(GalT-21, which produces ceramide, a key intermediate in the of glycosphingolipids, and biosynthesis estabilished its subcellular Moreover, by the in vivo localization. of different administration to rats labelled exogenous glycosphingolipids, specific position of the molecule ztermined the subcellular traffic' 1% netabolites involved ' the early R::ults indicate glycosylation steps. that GalT-2 is localized in the Golgi where glucosylceranide is apparatus, made available from different metabolic and where the products of origin, glycosylations are formed. further
THEODDNTOeLASTSOFRATMDLARS AFTER PRIMARY DSNTINDGSNESIS Paolo Romagnol i,Flavio Galeotti,Giovanni Mancini, El isabetta Francini. Department of Human Anatomy and Histology, University of Florence, Italy This studywas aimed at defining the evolution with time of the organelles of odontoblasts after primary dentinoganesis, in teeth with limited eruption. The odontoblastsofratfirstlowermolarswere anaIysedwith morphometrical methods applied to electron microscopy, since primary dentinogeneois to complete eruption, i.e., since 10 to 40 d of age. All the organelles underwent atrophy, but at dif ferent rates among each other; in particular, the endoplasmic reticulum decreased in size progressivel y from 10 to 40 d, whereas the Gol gi apparatus decreased markedly between 10 and 14 d and then remained practical1 y unchanged in size. Autophagocytosis was never enhanced above what occurring during primary dentinogenesis. The degree of POlarization towards dentine was reduced progressivelyandat4Odthecentriolesof some odontoblasts were located towards pulp, instead of dentine. The distribution of the extension of the endoplasmic reticulum,assumedasan estimate of that of the secretory activity, was such that in ca. 90% cells at 40 d this organelle was smaller than in anycell at 10 d. These results suggest that cell atrophy may occur without an increase in autophagocytosis, that the organelles along the secretory pathway may be regulated independently of each other, and that in the odontoblasts, as in several types of epithelial secretory cells, the fraction of cells engaged into appreciable secretory activity is correlated with thedegreeofactivationof the whole cell population.
THE EFFECT OF ESTRADIOL 17(3 ON ENDOMEMBRANES AND PEROXISOMES IN FISH HEPATOCYTES Nada Pipan,Peter VeraniE.Institute of Human Biology,Medical Faculty,Ljubljana,LipiEeva 2, Yugoslavia The known stimulating effect of estradiol 17fi (E2) on- the synthesis of vitellogenin (1) was supplemented by the stereological and cytochemical analysis of endoplasmic reGolgi apparatus (GA) and peroticulum (ER), xisomes (PO) in hepatocytes of treated male zebrafish (Brachydanio rerio). The hormon (1,ug E2/1 1 water) was applied for 6, 20, 30 and 40 days. Cis cisternae of GA were stained by prolonged osmification and trans part by tiamino pyrophosphatase (TPP) and For the identification of acid phosphatase. PO diaminobenzidine (DAB) method was used. The results showed the enlargement of all Maximal membranes analysed in present study. increase of the surface density of ER was reached after 30 days. The treatment did not change the size of individual dictyosomes of GA but induced the increase of their nuThe osmiofility of cis part became mber. much stronger and in some cases the trans cisternae were also modified. The PO responded on the stimulation by the decrease of their mean radius and multiplication of organelles that resulted in an increase of the surface of PO membranes per volume unit ob hepatocytes. The processes is reversible; in fish kept 4 days in pure water after 40 days of treatment the initial stage was reestablished. 1. Peute J, Huiskamp R, van Oordt PGWJ, Cell Tiss. Res. 242:377-382, 1985
P160
P157
P159
P158
DEGSUDATION OF I@l MUTANTS IN TEIEENDOPLABILMC-UM. AnnaMaria Fra+, Cristina Alberini”, Paola Casiello”, Dario FinazziA and Roberto Sitia*O. *Iatituto San Raffaele, Milano, “University of Brescia, Brescia, “I&it&o Nazionale per la Ricerca sul Cancro, Genova,Italy. B lymphocytes do not secrete IgM and plasmacells only secrete polymeric IgM molecules. Extensive intracellular degradation of secretory IgM occurs in both cell types (virtually 100% in B cells, about 25% in plasma cells). We have previously shown that retention of secretory IgM by B cells and the failure of plasmacells to secrete monomeric IgM are both attributable to the cyst&e residue (cys 575) found at the carboxi-terminus of secretory IgM heavy chains (pa). Thus, cys 575 is involved both in polymerisation of IgM and in intracellular retention of unassembled intermediates. Retention takes place within the endoplasmic reticulum (ER), and, in general, correlates with binding to BiP. Substitution of cys 575 with alanine results in increased rate of secretion and inhibition of degradation. The inverse correlation between rate of transport and ER degradation might eimply reflect the time the protein spends in the ER. However, differences in the degradation rates for wild type andmutentpa were observed also in the presence of brefeldin A, when no transport beyond the ER takes place. We propose that, in the case of IgM, the signals required for retention may also be utiheed to target unassembled molecules to ER degradation. SupportedbyCNRandAIRC.