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J W Slot

Publications and source records attributed to J W Slot.

At least 109 records · Page 6Linked to original sources
PubMed1983

Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

Affinity-purified monospecific antibodies against human fibrinogen and the platelet-specific proteins platelet factor 4 and beta thromboglobulin were used to localize these antigens in thin and ultra-thin frozen sections of mildly fixed, washed human blood platelets. By immunofluorescent double-labeling experiments the distribution of fibrinogen was compared to that of platelet factor 4 and beta thromboglobulin. All three antigens occurred in virtually all platelets and showed and identical, dotlike distribution. For immunoelectron microscopy we used protein A-colloidal gold on ultra-thin frozen sections to visualize the specific reaction indirectly. The staining for platelet factor 4, beta thromboglobulin, and fibrinogen localized exclusively over alpha-granules of washed platelets. Within the granules, platelet factor 4 was localized preferentially in the electron dense, alpha-granule nucleoid, whereas fibrinogen was more predominant in the electron-lucent granule periphery. Beta thromboglobulin localization did not show a preferential intragranular distribution.

Antigens↗
PubMed1983

Immunoelectron microscopic exploration of the Golgi complex.

Using immunogold labeling of ultrathin cryosections, we have studied the localization of secretory, lysosomal, and membrane proteins in the Golgi complexes of several cell types. All proteins were present in the stacks of Golgi cisternae, illustrating that the cisternae comprise a ubiquitous way station for proteins with multiple destinations. The labeling patterns support the concept that peripheral Golgi vesicles represent the main site of secretory protein concentration. Of the membrane proteins studied, the Golgi enzyme galactosyltransferase was confined to the trans-most few cisternae, whereas the receptors for asialoglycoproteins and for polymeric immunoglobulin A occurred in most cisternae, with increasing concentration approaching the trans side. The findings are discussed in relation to a cisternal cis to trans progression of Golgi cisternae and membrane specificity.

Animals↗
PubMed1983

The pathway of the asialoglycoprotein-ligand during receptor-mediated endocytosis: a morphological study with colloidal gold/ligand in the human hepatoma cell line, Hep G2.

The human hepatoma cell line, Hep G2, was used as a model system to delineate the morphological aspects of receptor-mediated endocytosis of asialoglycoproteins (ASGP). These hepatoma cells, which have several structural features in common with liver parenchymal cells, contain a well-differentiated endocytotic apparatus which includes coated vesicles and CURL (compartment of uncoupling receptor and ligand) similar to that found in rat liver cells in vivo (Geuze et al., 1983). In order to precisely identify the route of ligand from the cell surface to the CURL sorting vesicles, Hep G2 cells were allowed to endocytose in synchrony ASGP bound to 12 nm colloidal gold for 5 to 60 min. Thereafter the subcellular distribution of the gold particles was examined in ultrathin cryosections. This study demonstrates that, once internalized, ASGP ligand enters a system of CURL tubules prior to delivery to the lumenal contents of attached endocytotic vesicles. These vesicles then in turn transform into multivesicular bodies and secondary lysosomes. Thus, the tubular portions of CURL provide the entry for ASGP ligand into the sorting compartment.

Asialoglycoprotein Receptor↗
PubMed1983

Albumin localization in rat liver parenchymal cells.

In this study, we re-investigated the localization of albumin in hepatocytes using immunocytochemistry (ICC) on ultrathin cryo-sections of rat livers, which were immersion or perfusion-fixed with paraformaldehyde or a mixture of paraformaldehyde and glutaraldehyde. The sections were indirectly labelled with rhodamine-conjugated IgG or with 8 nm protein A-colloidal gold particles. We demonstrate that perfusion-fixation is obligatory for a proper localization of albumin. After perfusion fixation, all hepatocytes of fed and fasted rats were uniformly labelled. At the subcellular level albumin was localized in hepatocytes in the membrane-bound compartments of the entire secretory pathway, i.e. the rough and smooth ER, peripheral Golgi vesicles, all Golgi cisternae and the secretory granules. Concentration of albumin appeared mainly to occur at the cis-Golgi side.

Albumins↗
PubMed1982

Immunocytochemical localization of the receptor for asialoglycoprotein in rat liver cells.

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.

Animals↗
PubMed1982

Immunocytochemical localization of beta-glucuronidase in the male rat preputial gland.

Present knowledge of the in situ intracellular localization of acid hydrolases is mainly based on enzyme-cytochemical observations. In the preputial gland cells beta-glucuronidase and acid phosphatase were thus demonstrated in lysosome-like secretory granules and the GERL system. We applied immunocytochemistry to localize beta-glucuronidase at the earliest sites of biosynthesis. Lysosomal beta-glucuronidase was purified from preputial gland by column chromatography and SDS gel electrophoresis. Antibodies were raised in rabbit and affinity-purified preparations were used for immunocytochemistry on thin frozen sections of perfusion fixed preputial glands. Indirect procedures were applied with a second antibody labelled with rhodamine for fluorescence, and 5 or 8 nm protein A-gold probes for electron microscopy. beta-Glucuronidase occurred in all cells, except for the precursor cells, and was localized throughout the endoplasmic reticulum and perinuclear space, in all Golgi cisternae and storage granules, and in autophagic vacuoles. Thus in the preputial gland cell, beta-glucuronidase is present in both the lysosomal and the secretory system.

Animals↗
PubMed1981

Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections.

Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au5 and PA/Au16) are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue. The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i.e., antibody 1 - PA/Au5 - antibody 2 - PA/Au16. When this was done, no significant interference between PA/Au5 and PA/Au16 occurred. Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations. We prepared two rabbit antibodies against GP-2. One antibody (R x ZGM) was obtained by immunizing with native membrane material. The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone. THe immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules. On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina. Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al., 1980, Eur. J. Cell Biol. 23:122-128). Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas.

Amylases↗
PubMed1981

Sizing of protein A-colloidal gold probes for immunoelectron microscopy.

Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25-35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.

Animals↗
PubMed1980

The subcellular localization of immunoglobulin in mouse plasma cells, as studied with immunoferritin cytochemistry on ultrathin frozen sections.

Cells of mineral oil-induced plasmacytomas (MOPC) and normal plasma cells from mouse lymph nodes were used to study the intracellular localization of IgG by means of immunoferritin cytochemistry on ultrathin frozen sections IgG was demonstrated in the rough endoplasmic reticulum, in virus-containing smooth ER of the tumor cells, in peripheral elements at the cis-side of the Golgi complex, and in all Golgi cisternae. It is suggested that the peripheral elements transfer IgG molecules from the RER to the Golgi complex. Vacuoles showing a strong immunoreaction occurred at the trans-side of the Golgi complex. These vacuoles were normal in lymph node plasma cells and were occasionally seen in the tumor plasma cells. It is proposed that these vacuoles carry IgG from the Golgi complex to the cell membrane and, hence, can be considered as secretory vacuoles.

Animals↗
PubMed1980

Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study.

Amylase (Am) and chymotrypsinogen (Chtg) were demonstrated in rat and guinea pig exocrine pancreatic cells by immunofluorescence and immunoferritin cytochemistry on thin and ultrathin frozen sections. We describe two observations indicating that Am and Chtg may behave differently in the pre-Golgi phase of their intracellular transport. Firstly, aggregates of material within the RER cisternae of the guinea pig (so-called intracisternal granules) reacted strongly with anti-Chtg, but showed no affinity for anti-Am. Secondly, in both rat and guinea pig, the increase in labeling intensity from cytoplasm (RER) to secretory granules was larger for Chtg than for Am. We hypothesize that the two proteins do not travel in-parallel towards the Golgi complex. Compared with Chtg, Am would lag behind in the RER cisternae.

Amylases↗
PubMed1980

Fate of the major zymogen granule membrane-associated glycoproteins from rat pancreas. A biochemical and immunocytochemical study.

A glycoprotein resembling the major zymogen granules membrane-associated glycoprotein (GP-2) from the exocrine rat pancreas was detected in a 200 000 g sedimentable subfraction from pancreatic secretion. This glycoprotein had a slightly smaller molecular weight than GP-2 (70 000 D versus 75 000 D), gave a line of identity with GP-2 in a double immunodiffusion test against anti GP-2, and tryptic peptide charts of both glycoproteins were largely similar. Immunofluorescence cytochemistry showed that GP-2 antigens were exclusively located in the exocrine pancreas, where they were preferentially found in the perimeter of secretory granules and in the acinar lumina. These results suggest the possibility of loss of GP-2 from exocrine cells during secretion and raise doubts as to the status of GP-2 as a true membrane glycoprotein.

Animals↗
PubMed1979

A morphometrical study of the exocrine pancreatic cell in fasted and fed frogs.

The influence of feeding on the ultrastruct of the frog exocrine pancreatic cell was studied by morphometrical procedures. Volume and surface of various cell structures were measured and expressed per unit cell volume. The average cellular size was not influenced by feeding. Though protein synthesis changes 5-to 10-fold (van Venrooij, W. J., and C. Poort. 1971. Biochim. Biophys. Acta. 247:468-470), no significant differences were observed in the amount of membrane that constitutes the rough endoplasmic reticulum (RER) and that represented the major part of total cellular membranes. The appearance of the RER changed. When fasted, most of its membrane was arranged in stacks of tightly packed, narrow cisternae. Within 4 h after feeding, these cisternae were separated and irregularly dilated, and ribosomes became ordered in typical rosettes on their surface. The total volume of the Golgi system increased twofold after feeding. The vesicular and tubular elements at the Golgi periphery did not change, but the volumes of the Golgi cisternae and the condensing vacuoles increased 2.5- and 6-fold, respectively. The increased in the amount of membrane present in these structures was only 1.6- and 3.5-fold, which reflects the more distended appearance of the cisternae and the rounded shape of the condensing vacuoles after feeding. Feeding halved the number of secretory granules per cell, and signs of exocytosis were more common than in fasted animals. These findings suggest that, in the frog pancreatic cell, fluctuations in the production of secretory proteins are not accompanied by an important breakdown and renewal of cellular membranes. This may favor a rapid and strong response of the cell to feeding.

Animals↗
PubMed1979

Effect of fasting and feeding on synthesis and intracellular transport of proteins in the frog exocrine pancreas.

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.

Animals↗
PubMed1979

Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex.

Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.

Amylases↗