scholarly journals Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes.

1983 ◽  
Vol 97 (5) ◽  
pp. 1582-1591 ◽  
Author(s):  
E S Sztul ◽  
K E Howell ◽  
G E Palade
Keyword(s):  
Membrane Proteins ◽  
Golgi Complex ◽  
Rat Hepatocytes ◽  
Secretory Component ◽  
Bile Canaliculus ◽  
Sds Page ◽  
Iga Antibody ◽  
Co Precipitation ◽  
Kinetics Of

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.

scholarly journals Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies.

1985 ◽  
Vol 100 (4) ◽  
pp. 1255-1261 ◽  
Author(s):  
E S Sztul ◽  
K E Howell ◽  
G E Palade
Keyword(s):  
Golgi Complex ◽  
Rat Hepatocytes ◽  
Companion Paper ◽  
Secretory Component ◽  
Cell Fractionation ◽  
Vesicular Carriers ◽  
Hepatic Proteins ◽  
Intact Rats ◽  
Er Membrane

In the companion paper (Sztul, E. S., K. E. Howell, and G. E. Palade, J. Cell Biol., 100:1248-1254), we have shown that pulse labeling of hepatic proteins with [35S]cysteine can be obtained in vivo in intact rats. Soluble label clears the plasma in approximately 5 min, and incorporated label reaches peak values in the liver approximately 20 min after injection. In the present study, we show that the 105,000-mol-wt protein (105K), kinetically the earliest intracellular form of secretory component (SC), is the predominant form found, between 5 and 20 min postinjection, in homogeneous rough microsomal fractions. The second kinetically defined form, i.e., 116K, is the predominant species present in relatively homogeneous, light Golgi fractions in which it appears at approximately 15 min, and peaks at approximately 25 min, postinjection. The third kinetically defined form, 120K, is found 30 min after injection as the major SC species (albeit still accompanied by its immediate precursor, 116K), in a sinusoidal plasmalemmal fraction isolated by immunoadsorption to anti-SC-coated Sepharose beads. These findings lead to the following conclusions: (a) SC is synthesized on polysomes attached to the rough endoplasmic reticulum (ER) membrane; (b) it is partially translocated across the ER membrane and core glycosylated co-translationally to give a 105K peptide; (c) 105K moves from the ER to the Golgi complex where it is terminally glycosylated to give the 116K form; (d) the latter moves to the sinusoidal plasmalemma where it appears together with the final mature form, 120K. Kinetic evidence indicates that the vesicular carriers involved in the transport of SC from the Golgi complex to the sinusoidal plasmalemma, and from the latter to the biliary front of the hepatocytes, are present in a Golgi heavy fraction and a crude carrier vesicle fraction from which they remain to be isolated, purified, and characterized.

Analysis of Hepatocyte Distribution and Survival in Vascular Beds with Cells Marked by 99mTC or Endogenous Dipeptidyl Peptidase IV Activity

1997 ◽  
Vol 6 (4) ◽  
pp. 377-386 ◽  
Author(s):  
Sanjeev Gupta ◽  
Srinivasa Rao G. Vasa ◽  
Pankaj Rajvanshi ◽  
Lionel S. Zuckier ◽  
Christopher J. Palestro ◽  
...  
Keyword(s):  
Rat Hepatocytes ◽  
Liver Parenchyma ◽  
Histochemical Staining ◽  
Time Activity ◽  
Liver Sinusoids ◽  
Pulmonary Capillaries ◽  
Vascular Beds ◽  
Kinetics Of ◽  
Cell Fragments

Knowledge of the kinetics of cell distribution in vascular beds will help optimize engraftment of transplanted hepatocytes. To noninvasively localize transplanted cells in vivo, we developed conditions for labeling rat hepatocytes with 99mTc–pertechnetate. The incorporated 99mTc was bound to intracellular proteins and did not impair cell viability. When 99mTc hepatocytes were intrasplenically injected into normal rats, cells entered liver sinusoids with time–activity curves demonstrating instantaneous cell translocations. 99mTc activity in removed organs was in liver or spleen, and lungs showed little activity. However, when cells were intrasplenically transplanted into rats with portasystemic collaterals, 99mTc appeared in both liver sinusoids and pulmonary alveolar capillaries. To further localize cells, we transplanted DPPIV+ F344 rat hepatocytes into syngeneic DPPIV – recipients. Histochemical staining for DPPIV activity demonstrated engraftment of intrasplenically transplanted cells in liver parenchyma. In contrast, when 99mTc hepatocytes were injected into a peripheral vein, cells were entrapped in pulmonary capillaries but were subsequently broken down with redistribution of 99mTc activity elsewhere. Intact DPPIV+ hepatocytes were identified in lungs, whereas only cell fragments were present in liver, spleen, or kidneys. These findings indicate that although the pulmonary vascular bed offers advantages of easy accessibility and a relatively large capacity, significant early cell destruction is an important limitation.

scholarly journals Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

1997 ◽  
Vol 8 (10) ◽  
pp. 1911-1931 ◽  
Author(s):  
Randall S. Taylor ◽  
Steven M. Jones ◽  
Rolf H. Dahl ◽  
Mark H. Nordeen ◽  
Kathryn E. Howell
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Calcium Uptake ◽  
Rat Hepatocytes ◽  
Calcium Atpase ◽  
Endoplasmic Reticulum Calcium ◽  
Calcium Atpases ◽  
In Transit

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

scholarly journals In vivo digestion of infant formula in piglets: protein digestion kinetics and release of bioactive peptides

2012 ◽  
Vol 108 (12) ◽  
pp. 2105-2114 ◽  
Author(s):  
Karima Bouzerzour ◽  
François Morgan ◽  
Isabelle Cuinet ◽  
Cécile Bonhomme ◽  
Julien Jardin ◽  
...  
Keyword(s):  
Infant Formula ◽  
Human Life ◽  
Milk Proteins ◽  
Protein Digestion ◽  
Sds Page ◽  
Newborn Child ◽  
Digestion Kinetics ◽  
Β Lactoglobulin ◽  
Kinetics Of

The first months of life correspond to a key period in human life where dramatic physiological changes (establishment of microbiota, development of the immune system, etc.) occur. In order to better control these changes it is necessary to understand the behaviour of food in the gastrointestinal tract of the newborn. Infant formula is the only food for the newborn when breast-feeding is impossible. The kinetics of digestion of milk proteins and the nature of the peptides liberated in the small intestine throughout infant formula digestion have never been extensively investigated so far and were therefore studied using the piglet as a model of the newborn child. Piglets were fed infant formula by an automatic delivery system during 28 d, and slaughtered 30, 90 and 210 min after the last meal. Contents of stomach, proximal and median jejunum and ileum were collected and characterised. The extent of β-lactoglobulin (β-lg), α-lactalbumin (α-la) and casein proteolysis was monitored by inhibition ELISA, SDS-PAGE, immunoblotting and MS. At 30 min after the last meal, caseins were shown to be extensively hydrolysed in the stomach. Nevertheless, peptides originating mainly from β-caseins (from 509 to 2510 Da) were identified in the jejunum and ileum of the piglets. β-Lg partially resisted gastric digestion but completely disappeared in the stomach after 210 min. α-La had a similar behaviour to that of β-lg. Two large peptides (4276 and 2674 Da) generated from β-lg were present in the ileum after 30 and 210 min and only one (2674 Da) after 90 min.

Transcytotic vesicular carriers for polymeric IgA receptors accumulate in rat hepatocytes after bile duct ligation

1991 ◽  
Vol 98 (2) ◽  
pp. 205-216 ◽  
Author(s):  
J.M. Larkin ◽  
G.E. Palade
Keyword(s):  
Bile Duct ◽  
Serum Proteins ◽  
Bile Duct Ligation ◽  
Rat Hepatocytes ◽  
Frozen Sections ◽  
Transmission Electron ◽  
Duct Ligation ◽  
Sds Page ◽  
Vesicular Carriers

In rat hepatocytes, transcytotic vesicular carriers transport the mature 120 × 10(3) Mr form of the polymeric IgA receptor (pIgA-R), with or without its ligand, pIgA, from the sinusoidal to the biliary plasmalemma, where the ectodomain of the receptor is cleaved to produce an 80 × 10(3) Mr fragment that is secreted into the bile. Here we show that cholestasis induced by bile duct ligation results in the accumulation of transcytotic carriers, identified by the 120 × 10(3) Mr pIgA-R and pIgA, in the pericanalicular cytoplasm of hepatocytes. To determine the extent of pIgA-R accumulation, hepatic total microsomes (TM) were prepared from control and cholestatic rats. Solubilized TM proteins were separated by SDS-PAGE and receptor forms were detected by immunoblotting and autoradiography. Quantitative densitometry of these autoradiograms showed that after duct ligation the 120 × 10(3) Mr receptor accumulated to a level approximately threefold higher than the control. Concomitantly, immunologically related, novel 124, 90 and 80 × 10(3) Mr proteins (cholestatic antigens) became detectable. Immunoblot analyses of biliary and serum proteins showed that cholestasis resulted in: (1) a marked decrease in the concentrations of the 80 × 10(3) Mr receptor and pIgA in the bile, whereas albumin concentrations remained at control levels; and (2) a marked increase in the concentration of the 80 × 10(3) Mr receptor in the serum. Positive sites for pIgA-R were localized to the pericanalicular cytoplasm of hepatocytes by indirect immunofluorescence on semithin frozen sections in cholestatic hepatocytes. The sites were more numerous and the positive signal stronger than in controls. One day post-ligation, pIgA-positive sites were located to the same pericanalicular cytoplasm of hepatocytes; by three days, however, most pIgA appeared in sinusoidal endothelia and Kupffer cells. To validate the vesicular character of the receptor-positive sites, sham-operated and cholestatic livers were processed for either transmission electron microscopy (TEM) or immunogold localization of receptors on thin frozen sections. TEM verified the accumulation of pericanalicular vesicles in cholestatic hepatocytes. Immunogold tests localized pIgA-R to pleiomorphic, pericanalicular vesicles, which were increased in number, size and concentration of antigenic sites in cholestatic hepatocytes. These findings indicate that bile duct ligation provides a method for manipulating the in vivo transcytotic pathway and for accumulating previously unstudied transcytotic carriers in hepatocytes.

scholarly journals Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers.

1994 ◽  
Vol 125 (4) ◽  
pp. 733-741 ◽  
Author(s):  
L Saucan ◽  
G E Palade
Keyword(s):  
Golgi Complex ◽  
Magnetic Beads ◽  
Glucose Transporter ◽  
Sucrose Density Gradient ◽  
Secretory Proteins ◽  
Sds Page ◽  
Vesicular Carriers ◽  
Glandular Cells ◽  
Rat Livers

From rat livers labeled in vivo for 30 min with [35S] cys-met, we have isolated two classes of vesicular carriers operating between the Golgi complex and the basolateral (sinusoidal) plasmalemma. The starting preparation is a Golgi light fraction (GLF) isolated by flotation in a discontinuous sucrose density gradient and processed through immunoisolation on magnetic beads coated with an antibody against the last 11 aa. of the pIgA-R tail. GLF and the ensuing subfractions (bound vs nonbound) were lysed, and the lysates processed through immunoprecipitation with anti-pIgA-R and anti-albumin antibodies followed by radioactivity counting, SDS-PAGE, and fluorography. The recovery of newly synthesized pIgA-R was > 90% and the distribution was 90% vs 10% in the bound vs nonbound subfractions, respectively. Albumin radioactivity was recovered to approximately 80%, with 20% and 80% in bound vs nonbound subfractions, respectively. Other proteins studied were: (a) secretory-apolipoprotein-B, prothrombin, C3 component of the complement, and caeruloplasmin; (b) membrane-transferrin receptor, EGR-receptor, asialoglycoprotein receptor, and the glucose transporter. In all the experiments we have performed, the secretory proteins distributed up to 85% in the nonbound subfraction (large secretory vacuoles), whereas the membrane proteins were segregated up to 95% in the bound subfraction (small vesicular carriers). These results suggest that in hepatocytes, membrane and secretory proteins are transported from the Golgi to the basolateral plasmalemma by separate vesicular carriers as in glandular cells capable of constitutive and regulated secretion.

Origin, kinetics of circulation and fate in vivo of the major excretory–secretory product of Acanthocheilonema viteae

Parasitology ◽  
1989 ◽  
Vol 99 (2) ◽  
pp. 229-239 ◽  
Author(s):  
W. Harnett ◽  
M. J. Worms ◽  
A. Kapil ◽  
M. Grainger ◽  
R. M. E. Parkhouse
Keyword(s):  
Molecular Weight ◽  
Life Cycle ◽  
Half Life ◽  
Secretory Product ◽  
Parasite Life Cycle ◽  
Acanthocheilonema Viteae ◽  
Sds Page ◽  
Kinetics Of ◽  
Immunohistological Analysis

SummaryThe excretions-secretions (E-S) of Acanthocheilonema viteae consist mainly of one product, molecular weight 62 kDa. This molecule is synthesized during the vertebrate phase of the parasite life-cycle and is first detectable in the E-S of L4 parasites. It is cross-reactive with E-S of human filarial parasites as a consequence of possessing a phosphorylcholine (PC) moiety. The 62 kDa molecule has been employed as a model for the study of the origin and fate of filarial E-S. Immunohistological analysis has shown the molecule to be located predominantly in the parasite gut. Transplantation of adult female [S] methionine pulsed worms into uninfected jirds resulted in the radio-labelled secreted 62 kDa antigen being detected in the bloodstream within 4 h by SDS–PAGE/immunoprecipitation analysis. The systemic half-life of the molecule as estimated by clearance of injected, purified I-labelled material was measured in naive and infected jird hosts.

scholarly journals Biogenesis of the polymeric IgA receptor in rat hepatocytes. I. Kinetic studies of its intracellular forms.

1985 ◽  
Vol 100 (4) ◽  
pp. 1248-1254 ◽  
Author(s):  
E S Sztul ◽  
K E Howell ◽  
G E Palade
Keyword(s):  
Single Gene ◽  
Kinetic Studies ◽  
Rat Hepatocytes ◽  
Secretory Protein ◽  
Secretory Component ◽  
Major Band ◽  
Iga Antibodies ◽  
Definition Of ◽  
Endoglycosidase H

The polymeric IgA receptor (or secretory component [SC]) is a major biliary secretory protein in the rat. It was identified as an 80,000-mol-wt (80 K) glycoprotein by coprecipitation (with IgA) by anti-IgA antibodies (Sztul, E. S., K. E. Howell, and G. E. Palade, 1983, J. Cell Biol., 97:1582-1591) and was used as antigen to raise anti-SC antibodies in rabbits. Pulse labeling with [35S]cysteine in vivo, followed by the immunoprecipitation of solubilized total microsomal fractions with anti-SC sera, made possible the identification of three intracellular forms of SC (all apparently membrane proteins) and the definition of their kinetic and structural interrelations. At 5 min postinjection of [35S]cysteine, a major band of Mr 105,000 was maximally labeled. This peptide lost radioactivity concomitantly with the appearance of a radioactive doublet of Mr 116,000 and 120,000 at 15-30 min postinjection. Loss of radioactivity from 116K paralleled increased labeling of the 120K peptide which appears to be the mature form of the receptor. The 105K form was sensitive to endoglycosidase H which converted it to a 96K peptide. The 116K and 120K forms were resistant to endoglycosidase H but sensitive to endoglycosidase F which converts them to 96K and 100K forms, respectively. Taken together, these findings support the following conclusions: (a) All rat hepatic SC forms are the products of a single gene; (b) all SC forms are N-glycosylated; (c) the 116K form is the result of the terminal glycosylation of the 105K form; and (d) the 120K peptide is probably produced by modifications at other sites than its complex oligosaccharide chains.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

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