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A L Hubbard

Publications and source records attributed to A L Hubbard.

At least 73 records · Page 4Linked to original sources
PubMed1986

A study into possible deviation from the Hardy-Weinberg equilibrium by the alleles of the hypervariable sequence in the region of the human insulin gene.

A number of studies have reported on possible relationships between a hypervariable sequence near the 5' end of the insulin gene and some common diseases. Control populations within these studies appear to disobey the Hardy-Weinberg equilibrium. In this study we have ascertained the allele frequencies in 181 random individuals. The chi-squared statistic comparing this population with a theoretical Hardy-Weinberg population was 0.61. This makes it unlikely that in the general population the Hardy-Weinberg rule is disobeyed. Possible reasons for the discrepancy between our study and the studies of others are discussed.

Adolescent↗
PubMed1985

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane.

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. We identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). We also developed 125I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125I-wheat germ agglutinin. 125I-labeled Ricinus communis agglutinin I and 125I-peanut agglutinin blotting of the desialylated proteins revealed few if any conventional O-linked oligosaccharides, suggesting that the sialyl residues represent termini of N-linked complex-type oligosaccharides. Depending upon the protein, we estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains. The labeling of these three proteins by 125I-concanavalin A was sensitive to treatment with endoglycosidase H, and each exhibited a quantitative reduction in Mr after the treatment, as assessed independently by 125I-wheat germ agglutinin blotting. At this level of analysis, we were unable to discern differences in the types of oligosaccharides present on these seven glycoproteins that correlate with their patterns of expression within the plasma membrane domains of this polarized epithelial cell.

Animals↗
PubMed1985

The production and assessment of monoclonal antibodies to cortisol.

In an extensive series of experiments, Balb/C mice and Lou rats were immunised with 3-O-(carboxymethyl)oximinocortisol conjugated to bovine serum albumin. The spleen cells from selected animals were fused with cells from mouse or rat plasmacytoma lines. Out of many hundreds of hybridomas screened, more than seventy produced antibody that bound 125I-labeled cortisol. These cultures were investigated further for stability of antibody production, affinity for cortisol and cross-reactivity with other steroids. An unexpected but consistent finding was that immunised rats produced antibody which cross-reacted with 11-deoxycortisol to a level greater than 100% and this characteristic was reproduced by rat-rat hybridomas. Strategies designed to improve the chances of generating non-cross-reactive anti-cortisol monoclonal antibodies did not appear to be successful. Nevertheless, several monoclonals were identified with properties that suggest they may be useful for the development of sensitive and specific cortisol assays.

Animals↗
PubMed1985

Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies.

We have localized and identified five rat hepatocyte plasma membrane proteins using hybridoma technology in combination with morphological and biochemical methods. Three different membrane preparations were used as immunogens: isolated hepatocytes, a preparation of plasma membrane sheets that contained all three recognizable surface domains of the intact hepatocyte (sinusoidal, lateral, and bile canalicular), and a glycoprotein subfraction of that plasma membrane preparation. We selected monoclonal IgGs that were hepatocyte specific and localized them using both immunofluorescence on 0.5-micron sections of frozen liver and immunoperoxidase at the ultrastructural level. One antigen (HA 4) was localized predominantly to the bile canalicular surface, whereas three (CE 9, HA 21, and HA 116) were localized predominantly to the lateral and sinusoidal surfaces. One antigen (HA 16) was present in all three domains. Only one antigen (HA 116) could be detected in intracellular structures both in the periphery of the cell and in the Golgi region. The antigens were all integral membrane proteins as judged by their stability to alkaline extraction and solubility in detergents. The apparent molecular weights of the antigens were established by immunoprecipitation and/or immunoblotting. In a related study (Bartles, J.R., L.T. Braiterman, and A.L. Hubbard, 1985, J. Cell. Biol., 100:1126-1138), we present biochemical confirmation of the domain-specific localizations for two of the antigens, HA 4 and CE 9, and demonstrate their suitability as endogenous domain markers for monitoring the separation of bile canalicular and sinusoidal lateral membrane on sucrose density gradients.

Animals↗
PubMed1985

Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane.

We have used a combined biochemical and morphological approach to establish the suitability of certain endogenous and exogenous domain markers for monitoring the separation of rat hepatocyte plasma membrane domains in sucrose density gradients. As endogenous domain markers, we employed two of the integral plasma membrane protein antigens, HA 4 and CE 9, localized to the bile canalicular and sinusoidal/lateral domains, respectively, of the hepatocyte plasma membrane in rat liver tissue (Hubbard, A. L., J. R. Bartles, and L. T. Braiterman, 1985, J. Cell Biol., 100:1115-1125). We used immunoelectron microscopy with a colloidal gold probe to demonstrate that HA 4 and CE 9 retained their domain-specific localizations on isolated hepatocyte plasma membrane sheets. When the plasma membrane sheets were vesiculated by sonication and the resulting vesicles were centrifuged to equilibrium in sucrose density gradients, quantitative immunoblotting revealed that the vesicles containing HA 4 and those containing CE 9 exhibited distinct density profiles. The density profile for the bile canalicular vesicles (marked by HA 4) was characterized by a single peak at a density of 1.10 g/cm3. The density profile for the sinusoidal/lateral vesicles (marked by CE 9) was bimodal, with a peak in the body of the gradient at a density of 1.14 g/cm3 and a smaller amount in the pellet (density greater than or equal to 1.17 g/cm3). We used this sucrose gradient fractionation as a diagnostic procedure to assign domain localizations for several other hepatocyte plasma membrane antigens and enzyme activities. In addition, we used the technique to demonstrate that 125I-wheat germ agglutinin, introduced during isolated liver perfusion at 4 degrees C, can serve as an exogenous domain marker for the sinusoidal domain of the rat hepatocyte plasma membrane.

5'-Nucleotidase↗
PubMed1985

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments.

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin.

Animals↗
PubMed1985

Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway.

Polymeric IgA (pIgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeloma pIgA. When from 1 microgram to 1 mg 125I-pIgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 125I-pIgA was maximal in bile 30-60 min after injection, and approximately 80% of the total 125I-pIgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-pIgA conjugate (125I-pIgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 125I-pIgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after 125I-pIgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection approximately 30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained 125I-pIgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 125I-pIgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.

Animals↗
PubMed1984

125I-wheat germ agglutinin blotting: increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination.

Two substantial improvements in sensitivity in the identification of 125I-wheat germ agglutinin-binding glycoproteins on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels are reported. The major improvement in sensitivity (about 30-fold) derives from the use of 2% (w/v) polyvinylpyrrolidone (average Mr 40,000) instead of bovine serum albumin or denatured hemoglobin as the quenching agent (or carrier) during incubation with 125I-wheat germ agglutinin in detergent-free, phosphate-buffered saline. Under these conditions, specific labeling with 125I-wheat germ agglutinin is observed for orosomucoid derivatives that display N-acetylglucosamine or sialic acid residues at the nonreducing termini of their oligosaccharides, as well as for a number of glycoprotein components of a rat hepatocyte plasma membrane fraction. An additional improvement in sensitivity (up to 10-fold) results from an increase in the binding of 125I-wheat germ agglutinin to sialic acid-containing glycoproteins after treatment of the blots with 5 mM sodium metaperiodate followed by 5 mM aniline in the presence of 30 mM sodium cyanoborohydride. This treatment appears to cause the sequential oxidation and reductive phenylamination of the side chain of glycoprotein sialic acid residues.

Amination↗
PubMed1984

Production of high affinity monoclonal antibodies to deoxycorticosterone.

Monoclonal antibodies to deoxycorticosterone were produced. Mice were immunised with deoxycorticosterone-3-mono-oxime-BSA conjugate and spleen cells were then hybridised with NS1/1Ag4-1 mouse myeloma cells using 1500 mol. wt polyethylene glycol. The hybrids were grown in RPMI 1640 medium containing HAT to facilitate selection of positive clones. The clones and subclones were screened by using deoxycorticosterone-3-mono-oxime-[125I]iodohistamine. Dextran-coated charcoal was used for separation of antibody bound and free fractions. Two independent clones producing antibody which specifically binds labelled deoxycorticosterone were obtained. Cells from the two best sub-clones were used to raise ascites fluid. Comparison of these antibodies with one of the best conventional antisera previously raised in rabbits showed that the affinity constants were almost comparable (0.49-1.4 X 10(10) l/mol). Cross-reactivity of monoclonal antibodies with cortisol, corticosterone, testosterone and pregnenolone was lower than for polyclonal antisera, but for progesterone the cross-reactivity was similar in both cases. The assay sensitivity obtained with ascites fluid was comparable to that of conventional antibody (2.5 pg/ml). The dilution of ascites fluid which produced 50% binding of the label was 1:4,000,000. These results confirm that it is possible to produce monoclonal antibodies to corticosteroids.

Animals↗
PubMed1984

Rapid release of galactose-terminated ligands after endocytosis by hepatic parenchymal cells: evidence for a role of carbohydrate structure in the release of internalized ligand from receptor.

We have compared the behavior of 125I-labeled asialoorosomucoid (ASOR) and 125I-labeled asialotriantennary glycopeptide (TRI) from alpha-1-protease inhibitor in the perfused rat liver. These two ligands are recognized and internalized by the Gal/GalNAc receptor system of hepatocytes. We found that both ligands could be efficiently degraded when continuously perfused through a liver at 37 degrees C. However, as much as 51% of the internalized 125I-labeled TRI was rapidly (t1/2 = 3 min) released intact into the perfusion medium under conditions that permitted ligand-receptor association. In contrast, ASOR was totally degraded under these conditions. When dissociation was promoted (i.e., in the presence of GalNAc or EGTA), as much as 57% of the internalized ASOR was rapidly released (also intact). In each case, the shorter the initial internalization phase, the greater the proportion of ligand that was subsequently released. Neither the degree of receptor occupancy nor the temperature (16-37 degrees C) during the initial internalization phase influenced the rate or extent of the release process. Electron microscopic localization of internal ASOR-horseradish peroxidase conjugate demonstrated that peripheral endosomes were the major compartment labeled during exocytosis. We conclude that substantial fractions of both ligands rapidly return to the cell surface after internalization, ASOR bound to receptor and most of TRI free from receptor. The remaining ligand molecules enter the lysosomal pathway.

Animals↗
PubMed1984

A domain-specific marker for the hepatocyte plasma membrane. II. Ultrastructural localization of leucine aminopeptidase to the bile canalicular domain of isolated rat liver plasma membranes.

Leucine aminopeptidase (LAP) is an integral membrane glycoprotein localized to the apical membrane domain of intestinal and kidney epithelial cells. By indirect immunofluorescence, we have shown that antibodies raised against rat intestinal LAP recognized a similar protein concentrated in the bile canalicular (BC) domain of the hepatocyte in situ (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558). We have extended this localization to the ultrastructural level. When a saponin-permeabilized, agarose-embedded plasma membrane (PM) fraction was incubated with affinity-purified anti-LAP, 85% of the protein A-gold particles associated with the three recognizable PM domains were present in the BC. The levels of labeling on the other two domains (sinusoidal and lateral) did not exceed that observed with nonimmune controls. The concentration of LAP in the BC domain in isolated PM sheets prompted us to use this antigen for the affinity isolation of BC membrane (Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1497-1504, companion paper).

Animals↗
PubMed1984

A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption.

Previous immunolabeling studies (Roman, L.M., and A.L. Hubbard, 1983, J. Cell Biol., 96:1548-1558; Roman, L.M., and A.L. Hubbard, 1984, J. Cell Biol., 98:1488-1496, companion paper) established leucine aminopeptidase (LAP) as a specific marker for the bile canalicular (BC) domain of the rat hepatocyte plasma membrane (PM). In this study, we have isolated membrane from a sonicated PM vesicle fraction using anti-LAP-coated Staphylococcus aureus cells as a solid-phase immunoadsorbent. The extent and specificity of the immunoadsorption were assessed by following the behavior of LAP (the BC marker) and 32P-labeled membrane phospholipids (a uniform membrane marker). The BC fraction obtained was significantly enriched in LAP (yield: greater than 70% of PM-LAP). Alkaline phosphatase, 5'-nucleotidase, and a 110,000-dalton glycoprotein, HA-4, were enriched in the BC fraction to the same extent as LAP (enzyme or antigen/LAP = 1.0). However, alkaline phosphodiesterase I was not enriched to the same degree (enzyme/LAP = 0.5). Contamination of this BC fraction by membrane derived from the sinusoidal domain and endoplasmic reticulum, as determined from the distribution of the asialoglycoprotein receptor and NADH cytochrome c reductase, respectively, was small (less than 13%).

Animals↗
PubMed1984

Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics.

We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes.

Animals↗
PubMed1983

Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains.

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.

Animals↗
PubMed1983

Isolation of rat hepatocyte plasma membranes. II. Identification of membrane-associated cytoskeletal proteins.

Rat liver plasma membranes were isolated as presented in the preceding paper (Hubbard, A. L., D. A. Wall, and A. Ma., 1983, J. Cell Biol., 96: 217-229) and found to contain many filaments associated both with desmosomes along the lateral surface and with the cytoplasmic aspects of membranes comprising each of the three domains (lateral [LS], bile canalicular [BC] and sinusoidal [SF] ). Exposure of the plasma membranes to alkaline media (up to pH 11) resulted in loss of recognizable filaments without loss of domain morphology or membrane enzyme activities. Electrophoretic analysis of solubilized components from control and alkaline-extracted plasma membranes revealed that three major polypeptides present at 43, 52, and 56 kdaltons in the control had been released by alkaline treatment (pH 11) and could be quantitatively recovered in the supernate. The 43-kdalton component was identified as cytoplasmic actin by comparison of its tryptic 125I-peptide map to those of muscle (alpha) and brush border (beta, gamma) actins. The 52- and 56-kdalton polypeptides were identified as tonofilament components by their solubility properties and their ability to reassemble into 9.5-nm filaments from monomers present in an alkaline extract.

Actins↗
PubMed1983

A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain.

Indirect immunofluorescence was used to establish a domain-specific marker for hepatocyte plasma membranes. In frozen sections of fixed rat liver (0.5-4 microns), antibodies directed against rat intestinal leucine aminopeptidase (LAP) recognized an antigen that was restricted to the bile canalicular plasma membrane. Fluorescence was not observed on the sinusoidal or lateral membranes, and intracellular staining was not detected. The liver antigen was identified as LAP, based on its chemical similarity to intestinal LAP. First, immunoprecipitation experiments using trypsin-solubilized intestinal LAP (G-200 fraction, 91% pure) established a correlation between the loss of LAP enzyme activity from the soluble fraction and the appearance in the specific immunoprecipitates of polypeptides migrating on SDS PAGE between 110,000 and 130,000 daltons. The antigen precipitated from a detergent extract of liver plasma membranes had the same electrophoretic mobility. Second, the chymotryptic map of the major band in the liver immunoprecipitate was similar to that of purified intestinal LAP.

Acid Anhydride Hydrolases↗