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P M Novikoff

Publications and source records attributed to P M Novikoff.

At least 19 recordsLinked to original sources
PubMed1992

A role for microtubules in sorting endocytic vesicles in rat hepatocytes.

The vectorial nature of hepatocyte receptor-mediated endocytosis (RME) and its susceptibility to cytoskeletal disruptors has suggested that a polarized network of microtubules plays a vital role in directed movement during sorting. Using as markers a well-known ligand, asialoorosomucoid, and its receptor, we have isolated endocytic vesicles that bind directly to and interact with stabilized endogenous hepatocyte microtubules at specific times during a synchronous, experimentally initiated, single wave of RME. Both ligand- and receptor-containing vesicles copelleted with microtubules in the absence of ATP but did not pellet under similar conditions when microtubules were not polymerized. When 5 mM ATP was added to preparations of microtubule-bound vesicles, ligand-containing vesicles were released into the supernatant, while receptor-containing vesicles remained immobilized on the microtubules. Release of ligand-containing vesicles from microtubules was prevented by monensin treatment during the endocytic wave. Several proteins, including the microtubule motor protein cytoplasmic dynein, were present in these preparations and were released from microtubule pellets by ATP addition concomitantly with ligand. These results suggest that receptor domains within the endosome can be immobilized by attachment to microtubules so that, following monensin-sensitive dissociation of ligand from receptor, ligand-containing vesicles can be pulled along microtubules away from the receptor domains by a motor molecule, such as cytoplasmic dynein, thereby delineating sorting.

Adenosine Triphosphate↗
PubMed1991

Expression of specific UDP-glucuronosyltransferase isoforms in carcinogen-induced preneoplastic rat liver nodules.

The expression of specific UDP-glucuronosyltransferase isoforms in 2-acetylaminofluorane-induced rat liver preneoplastic nodules was studied; livers from pair-fed littermates were used as controls. For comparison, liver and kidney from 3-methylcholanthrene-treated or untreated (control) rats were used. Steady-state UDP-glucuronosyltransferase mRNA levels were determined by Northern blot analysis or in situ hybridization of tissue sections using a 30-mer oligonucleotide specific for the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase (which is active toward 4-nitrophenol) or a double-stranded cDNA probe specific for androsterone-UDP-glucuronosyltransferase. For 3-methylcholanthrene-inducible UDP-glucuronosyltransferase, the mRNA level was very low in control liver; there was a 15-fold increase after 3-methylcholanthrene treatment. This mRNA was present at relatively high concentration in the kidney and there was a threefold increase after 3-methylcholanthrene administration. In livers with preneoplastic nodules 1 mo after cessation of carcinogen administration, this mRNA concentration was approximately 15 times greater than in control liver. Similar changes in the level of the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase were also observed by in situ hybridization of tissue sections. Immunocytochemical studies using an antiserum that recognizes the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase showed a marked increase in the concentration of this isoform in preneoplastic nodules compared with the adjacent nonnodular liver.

2-Acetylaminofluorene↗
PubMed1991

Organic anion transport in HepG2 cells: absence of the high-affinity, chloride-dependent transporter.

In previous studies, we identified a 55 kD organic anion-binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity Cl(-)-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin-binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Characterizations of and interactions between bile ductule cells and hepatocytes in early stages of rat hepatocarcinogenesis induced by ethionine.

Numerous hepatic cell lineage pathways have been proposed for the development of hepatocarcinogensis induced by chemical carcinogens in rats. The roles of bile ductule cells and hepatocytes in the development of carcinogenesis were investigated using light and electron microscopic procedures to detect differences in morphology and in the phenotypic expression of antigens that are associated with each cell type. In early stages of hepatocarcinogenesis (4-10 weeks after initiation of feeding of a choline-deficient ethionine containing diet), both bile ductulelike (BDL) cells and hepatocytes were seen in mitosis. At the light microscope level, BDL cells showed intense cytoplasmic pyronin (RNA) staining and were positive for the antigens defined by monoclonal antibody 270.38 (bile ductule cells and "oval" cell marker) and glutathione-S-transferase (Yp isoform), whereas hepatocytes were positive for the antigens defined by monoclonal antibodies 270.26 and 258.26 (liver parenchymal cell markers), catalase activity (peroxisome marker) and adenosine triphospatase activity (bile canalicular marker). The authors frequently encountered BDL cells and hepatocytes in close proximity. Ultrastructural examination showed extensive plasma membrane appositions between a subset of BDL cells and hepatocytes. Desmosome structures, tight junctions, microvilli interdigitations and ATPase-positive bile canalicularlike structures were present along the contiguous plasma membrane domains of BDL cells and hepatocytes. Many of the BDL cells attached to hepatocytes were also attached to other BDL cells that had retained a basal lamina. In many cases, BDL cells connected to both hepatocytes and other BDL cells were no longer completely surrounded by basal lamina and had acquired a dual polarity as a consequence of their sharing apical and lateral membrane domains with both BDL cells and hepatocytes. BDL cells showed increased numbers of microperoxisomes (catalase positive organelles) and numerous free ribosomes. Hepatocytes showed a prominent development of the smooth endoplasmic reticulum, a feature prominent in hepatocytes within hyperplastic nodules. Since BDL cells and hepatocytes proliferate and BDL cells and hepatocytes develop intercellular junction sites, the authors propose that both cell types in early stages of carcinogenesis have the capacity to enter the cell lineage pathway leading to the development of hepatocarcinoma. Furthermore, the finding that BDL cells and hepatocytes form multiple attachment sites at the level of the plasma membrane, suggests the possibility that at some stage convergence of separate hepatic cell pathways may occur.

Animals↗
PubMed1990

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Affinity Labels↗
PubMed1990

Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes.

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.

Albumins↗
PubMed1990

Hybridomas using athymic nude mouse injected with Crohn's disease (CD) tissue filtrate. Immunoreactivity of the hybridomas with CD sera.

Injections of Crohn's disease (CD) tissue filtrates produce lymphoma and hyperplastic lymph nodes from plasma cell hyperplasia (PCH) in athymic nude (nu/nu) mice; these lymphoid tissue contain an antigen(s) recognized by CD serum/gamma G immunoglobulin (IgG). To immortalize the "CD-reactive antigen(s)," the authors fused the lymphoid cells from a CD tissue filtrate primed nu/nu mouse with nonsecretory mouse myeloma cells. Hybrids were screened and selected based on their reactivity with CD serum IgG, but not with control serum IgG in an indirect immunofluorescence assay (IF). Two CD-positive hybridomas were examined by IF with sera from 47 CD, 38 ulcerative colitis (UC), 13 controls with other gastrointestinal diseases, 19 with autoimmune diseases, and 21 normal subjects. Sera from 16 CD patients (34%) reacted with the two hybridomas, but only one of 38 UC sera and none of the 53 other disease or normal control sera reacted. The immunoreactivity of CD sera was significantly higher than UC sera (P less than 0.01) and each of the other groups (P less than 0.007). Using immunoperoxidase techniques at light and electron microscopic levels, the authors localized CD-associated antigen(s) in the plasma membrane of the two hybridomas. Further characterization of these hybridomas and the immunoreactive protein(s) may provide an important probe(s) for the diagnosis and the understanding of the pathogenesis of CD.

Animals↗
PubMed1988

Retrovirus-mediated transduction of adult hepatocytes.

Retrovirus-mediated gene transfer was used to develop a method for introducing genes into primary cultures of adult rat hepatocytes. Subconfluent monolayers of hepatocytes, cultured in hormonally defined media on different matrix substrata, were infected with helper-free stocks of a replication-defective retrovirus that constitutively expresses high levels of beta-galactosidase. Retrovirus-mediated transduction was measured by two methods: (i) an in situ cytochemical stain that specifically detects the expression of viral expressed beta-galactosidase, and (ii) Southern blot analysis, which measures the relative copy number of integrated provirus. Maximal transduction efficiency of approximately equal to 25% was achieved when the cells were infected after 3 days in culture; matrix had little effect on transduction efficiency. Enzyme cytochemical (catalase and glucose 6-phosphatase) and peroxidase immunocytochemical (asialoglycoprotein and UDP-glucuronosyltransferase) analyses of the cultures indicated that greater than 95% of cells were hepatocytes. The demonstration of hepatocyte-specific organelles in cells expressing the viral-directed beta-galactosidase provided unambiguous evidence for the transduction of hepatocytes. These methods should be useful in the development of liver-directed somatic gene therapy and in the study of liver-specific gene regulation.

Animals↗
PubMed1988

Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid.

Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.

Animals↗
PubMed1987

Development of a monoclonal antibody specifically reactive to gastrointestinal goblet cells.

A mouse monoclonal antibody (7E6A5) of IgG isotype, reacting specifically with mucin-producing goblet cells of the human gastrointestinal tract, has been developed. 7E6A5 reacts by an ELISA with colonic protein eluted from a DEAE column. A screening by immunoperoxidase assay of 76 specimens from 19 different human tissues showed that the immunoreactivity of 7E6A5 was confined exclusively in the globules of goblet cells in the colon, the appendix, and the small intestine. Nongoblet small and large intestinal epithelial cells did not react. Immunoelectron microscopy demonstrated the reactivity with mucin droplets in a homogeneous granular pattern inside the globules of goblet cells. Mucus-secreting cells from remaining parts of the gastrointestinal tract and other mucus-secreting organs such as respiratory, genitourinary tracts, salivary and mammary glands did not show any reactivity to 7E6A5. These findings indicate that the antigen recognized by 7E6A5 is shared by the goblet cells of both the small and large intestines and is unique to them. The monoclonal antibody may be useful in the study of function of mucus-secreting goblet cells and may represent an important tool in the evaluation of diseases such as ulcerative colitis, colon cancer, and intestinal metaplasia in gastric mucosa that are associated with quantitative changes in goblet cell numbers or with qualitative differences in mucin secretion.

Antibodies, Monoclonal↗
PubMed1986

Survival, organization, and function of microcarrier-attached hepatocytes transplanted in rats.

Hepatocytes harvested by collagenase perfusion of rat liver were attached to collagen-coated microcarriers and injected intraperitoneally into congeneic or allogeneic bilirubin-UDP-glucuronosyltransferase (EC 2.4.1.17)-deficient (Gunn) rats or allogeneic analbuminemic (NAR) rats. Five days later, the microcarriers were observed to have formed conglomerates chiefly on the anterior surface of the pancreas. Scanning electron microscopy showed hepatocytes attached to the granular collagen-coated surface of the microcarriers and newly formed connective tissue. Light microscopy revealed that the microcarriers formed a lattice with the collagen tissue; hepatocytes were seen within this lattice or on the surface of the microcarriers. Hepatocyte plasma membranes were nucleoside-diphosphatase (NDPase)-positive. Newly formed blood islands, blood vessels containing erythrocytes and leukocytes and NDPase-positive endothelium were observed in close proximity to the hepatocytes and fibroblasts. Transmission electron microscopic examination showed hepatocytes with microvilli and nucleoid-containing peroxisomes with catalase activity. Hepatocytes were present for up to 2 months in congeneic recipients, the longest period of observation after transplantation. After normal microcarrier-attached hepatocytes were transplanted into allogeneic Gunn rats, bilirubin glucuronides were present in bile for 6 days. When congeneic Gunn rat recipients were used, bilirubin glucuronides were present in bile throughout the study (28 days); this was accompanied by reduction of serum bilirubin concentrations to nearly normal levels. After injection of normal hepatocytes into allogeneic NAR rats, plasma albumin concentration progressively increased for 6 days and then declined. In NAR recipients which were immunosuppressed with cyclosporin A, peak plasma albumin levels were reached in 14 days and persisted nearly at that level throughout the study (28 days).

Animals↗
PubMed1985

Distribution of UDPglucuronosyltransferase in rat tissue.

UDPglucuronosyltransferase [UDPglucuronate beta-D-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] is a group of enzymes with distinct but partially overlapping substrate specificity. A rabbit antiserum raised against one purified rat liver UDPglycuronosyltransferase isoform was specific for UDPglucuronosyltransferase and recognized all transferase isoforms by immunodiffusion or immunotransblot analysis. The transferase activity toward all substrates was immunoabsorbed from solubilized rat liver microsomes by IgG purified from the antiserum. The purified IgG was used for immunocytochemical localization of UDP-glucuronosyltransferase in rat liver, jejunum, kidney, and adrenal gland. In the liver, UDPglucuronosyltransferase was present exclusively in hepatocytes and was uniformly distributed within all zones of the hepatic lobule. In the jejunum, the transferase was present exclusively in the epithelial cells and showed a progressive increase in concentration from the crypt to the villar tip. In the kidney, the greatest concentration of the transferase was observed in the epithelial cells of the proximal convoluted tubule. Adrenal medullary cells showed intense immunocytochemical staining; the zona glomerulosa and the zona reticularis of the adrenal cortex were more intensely stained than the zona fasciculata. By light microscopy, UDPglucuronosyltransferase was found in the endoplasmic reticulum and nuclear envelope of all the four organs; this was confirmed in the hepatocyte by electron microscopy. The transferase was not observed in mitochondria, Golgi apparatus, lysosomes, peroxisomes, and plasma membrane, even after 3- to 4-fold induction of various substrate-specific UDPglucuronosyltransferase activities.

Adrenal Glands↗
PubMed1985

Effects of swainsonine on rat liver and kidney: biochemical and morphological studies.

Among the reported effects of the plant toxin swainsonine in animals are a decreased level of Golgi mannosidase II activity, an increase in lysosomal alpha-D-mannosidase activity, oligosaccharide accumulation, vacuolization of cells, and neurological changes. We now find that, in the rat, the alkaloid rapidly induces vacuolization of both liver and kidney cells, but oligosaccharides accumulate only in the latter. We demonstrate by enzyme- and immunocytochemistry that the induced pleomorphic vacuoles are lysosomal in nature. The vacuoles do not appear to be derived from the Golgi apparatus, which retains its typical ultrastructural appearance, but are formed by autophagy. In swainsonine-fed rats, the lysosomal system is highly developed in hepatocytes, Kupffer cells, and cells of the proximal convoluted tubules. The relation of this hypertrophy of the lysosomal system to the known effects of swainsonine on glycoprotein biosynthesis and on Golgi and lysosomal alpha-mannosidases is not clear. In addition, in liver there occurs a marked increase in mitotic figures in the hepatocytes. This occurred in the absence of both cell death and increased liver size as estimated by gross morphology.

Alkaloids↗
PubMed1983

Immunocytochemical localization of alpha-D-mannosidase II in the Golgi apparatus of rat liver.

Mannosidase II is involved in the trimming of alpha-1,6-mannosyl residues during the biosynthesis of glycoproteins containing N-linked oligosaccharides of the complex type. A highly specific polyclonal antibody (IgG) was isolated from rabbits immunized with a homogeneous preparation of mannosidase II prepared from rat liver. With this antibody, light and electron microscopic immunocytochemical studies on rat liver reveal that essentially all mannosidase II in hepatocytes is localized in the Golgi apparatus, the only other site with reaction product being the endoplasmic reticulum. The indirect immunocytochemical method used in this study involved three major steps: exposure of aldehyde-fixed tissue to immune and nonimmune IgG, treatment with staphylococcal protein A labeled with horseradish peroxidase, and incubation in diaminobenzidine to reveal sites of peroxidase activity. The procedures described overcome major problems in immunocytochemistry, allowing preservation of antigenic sites and maintaining adequate ultrastructural integrity. The in situ localization of other carbohydrate-processing enzymes, involved in either trimming or attachment of sugar residues, should be possible with this procedure. Because biosynthetic precursors of the processing enzymes may be revealed by an immunocytochemical approach, it is potentially significant that mannosidase II reaction product is present in areas of the endoplasmic reticulum as well as in the Golgi apparatus.

Animals↗
PubMed1983

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.

Animals↗
PubMed1981

Lysosomal compartment of macrophages: extending the definition of GERL.

"Professional phagocytes" such as Kupffer cells show an extensive network of interconnected residual bodies with cytochemically demonstrable acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2]. In such cells and in other (hepatocytes, etc.) this compartment may be considered a part of GERL [a hydrolase-rich region of endoplasmic reticulum (ER) situated at the trans face of the Golgi apparatus from which various Lysosomes appear to arise].

Acid Phosphatase↗
PubMed1980

Organelle relationships in cultured 3T3-L1 preadipocytes.

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha-naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol.

Adipose Tissue↗