Biomedical subjects

T J Raub

Publications and source records attributed to T J Raub.

At least 37 records · Page 2Linked to original sources

PubMed1992

Permeability of bovine brain microvessel endothelial cells in vitro: barrier tightening by a factor released from astroglioma cells.

It has been shown both in vivo and in culture that astrocytes communicate with brain microvessel endothelial cells (BMECs) to induce many of the blood-brain barrier characteristics attributed to these unique cells. However, the results using cultured cells are conflicting as to whether this communication is dependent upon cell-cell contact. In this study we used primary cultures of bovine BMECs grown as monolayers on polycarbonate filters to study the formation of the barrier in vitro and examine its modulation by rat C6 glioma cells. Effects were examined by treating postconfluent BMEC monolayers with medium conditioned continually by C6 cells from the basolateral side to mimic the in vivo orientation. Cell monolayer integrity was assessed using electrical resistance and by measuring diffusion of uncharged molecules. BMEC monolayers form a functionally polarized and leaky barrier, with maximal resistance of 160 omega . cm2 and significant flux of molecules of molecular weight less than 350 Da. Treatment with rat or human astroglioma cells rather than pericytoma cells or transformed fibroblasts results in a concentration-dependent 200-440% increase in electrical resistance and a coincident 50% decrease in permeability to sucrose and dextran (70 kDa). The decrease in passive diffusion is most likely due to a change in tight junctions and not to transcellular vesicular traffic. The findings support that astroglioma cells release one or more signals that are required for cultured BMECs to express a "differentiated" phenotype associated with a tighter barrier, increased gamma-glutamyl transpeptidase activity, and decreased pinocytic activity. The relative ease and quickness of this culture system makes it amenable to studies on cell-cell interaction and regulation of barrier maintenance.

Animals↗

PubMed1992

The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation.

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.

Animals↗

PubMed1991

Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. I. Basic characteristics and effects of D-glucose and insulin.

The basic characteristics of hexose uptake and regulation of the glucose transporter (GLUT1) by D-glucose and insulin were studied in primary cultures of bovine brain microvessel endothelial cells (BMECs). A non-metabolizable glucose analog, 3-O-[3H]methyl-D-glucose [( 3H]3MG), was used as a model substrate, and the uptake was studied using BMECs grown in tissue culture plates. Uptake of [3H]3MG was equilibrative, temperature-dependent, and independent of sodium. The uptake also decreased gradually with culture age from 7 to 13 days. Saturation kinetics were observed for [3H]3MG uptake and the apparent Km and Vmax values were determined to be 13.2 mM and 169 nmol/mg per min, respectively. Pre-incubation with high concentrations of D-glucose and 3MG accelerated [3H]3MG uptake by BMECs by a counter-transport mechanism. D-Glucose, 2-deoxy-D-glucose, D-mannose, D-xylose, D-galactose and D-ribose showed significant competitive inhibition with [3H]3MG, whereas L-glucose, D-fructose, and sucrose did not affect [3H]3MG uptake by BMECs. [3H]3MG uptake was inhibited significantly by cytochalasin B and phloretin but not by phlorizin, 2,4-dinitrophenol, or ouabain. D-Glucose starvation of BMECs by incubation with D-glucose-free media for 24 h resulted in a significant increase (40-70%) in uptake of [3H]3MG compared with control conditions (7.3 mM D-glucose). Low D-glucose treatments (2.43 and 1.83 mM) for 7 days induced a slight but significant increase (20%) in [3H]3MG uptake, while long-term high glucose treatments (25 mM) showed no significant effect on [3H]3MG uptake irrespective of exposure time. The increase in [3H]3MG accumulation following D-glucose starvation was dependent upon starvation time (12 to 48 hr) and protein synthesis. Refeeding of D-glucose (7.3 mM) to D-glucose-starved BMECs resulted in a return of [3H]3MG uptake to control levels in 48 h. The D-glucose-starvation-induced increase in [3H]3MG uptake was shown to result from an increase in Vmax; the Km remained constant. In addition, D-glucose-starved BMECs were shown to have an increased level of GLUT1 using an antibody against human GLUT1 and an enzyme-linked immunosorbent assay (ELISA). The increased uptake following D-glucose starvation was not significantly affected by the presence of L-glucose, was partially impaired by the presence of D-galactose, D-fructose, and D-xylose, and was completely inhibited by the presence of D-mannose and 3MG. Furthermore, preincubation of BMECs with insulin (10 micrograms/ml) for 20 min did not affect the uptake of [3H]3MG or 2-deoxy-D-[3H]glucose ([3H]2DG).(ABSTRACT TRUNCATED AT 400 WORDS)

2,4-Dinitrophenol↗

PubMed1991

Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. II. Effects of conditioned media from astroglial and glioma cells.

Regulation of glucose uptake by an astroglial cell secreted factor(s) was studied in primary cultures of brain microvessel endothelial cells (BMECs). Uptake of a non-metabolizable glucose analog, 3-O-[3H]methyl-D-glucose ([3H]3MG), was measured after the BMECs were treated with media conditioned by primary cultures of rat astrocytes (Astrocyte Conditioned Media: ACM) or rat C6 glioma cells (Glioma Cell Conditioned Media: GCM). Uptake of [3H]3MG was significantly increased by ACM (30-50%) and GCM (60-200%) treatments, whereas conditioned medium from 3T3 fibroblasts (3T3) caused no significant effect. The elevation in [3H]3MG uptake increased with increasing time of exposure of BMECs to these conditioned media (CM), and the effect was shown to be reversible. Glucose depletion of CM was shown not to be a factor. The presence of cycloheximide, a protein synthesis inhibitor, during treatment of the BMECs with ACM and GCM blocked the increase in [3H]3MG uptake by the cells. These results suggested that ACM or GCM treatment elevated de novo synthesis of brain-type glucose transporter (GLUT1). Indeed, enhanced GLUT1 expression by these treatments in BMECs was demonstrated directly by enzyme-linked immunosorbent assay (ELISA) using antibodies against human GLUT1. After trypsinization of ACM and GCM, both conditioned media still induced significant stimulation of [3H]3MG uptake by BMECs. A significant increase in [3H]3MG uptake was also observed when ACM or GCM was exposed to BMECs through a dialysis membrane with a molecular weight cutoff of 1000. To examine whether the effects were specific to brain endothelial cells, [3H]3MG uptake experiments were performed employing aortic endothelial cells (AECs), pulmonary microvessel endothelial cells (PMECs), and 3T3 cells. ACM treatment did not alter 3MG uptake by these cells, suggesting that the ACM effect was specific to BMECs. On the other hand, [3H]3MG uptake by AECs and PMECs treated with GCM was significantly enhanced. The present study demonstrated that some factor(s) of relatively small molecular weight, which was released from astrocytes or glioma cells, stimulated glucose uptake by enhancing GLUT1 synthesis in BMECs.

3T3 Cells↗

PubMed1991

Primaquine blocks transport by inhibiting the formation of functional transport vesicles. Studies in a cell-free assay of protein transport through the Golgi apparatus.

The lysosomotropic amine primaquine has previously been shown to inhibit both secretory and recycling processes of cells in culture. We have used a cell-free assay that reconstitutes glycoprotein transport through the Golgi apparatus to investigate the mechanism of action of primaquine. In this assay, primaquine inhibits protein transport at a half-maximal concentration of 50 microM, similar to the concentration previously reported to disrupt protein secretion in cultured cells. Kinetic analysis of primaquine inhibition indicates that its point of action is at an early step in the vesicular transport mechanism. Primaquine does not inhibit the fusion of vesicles already attached to their target membranes. Primaquine irreversibly inactivates the membranes that form transport vesicles (donor), but not the membranes that are the destination of those vesicles (acceptor). Morphological data indicate that primaquine inhibits the budding of vesicles from the donor membranes. Once formed, the vesicles are refractile to primaquine action, and their attachment to and fusion with acceptor membranes proceeds unimpeded. In addition to illuminating the mechanism of action of primaquine, this study suggests that the selective action of this agent will make it a useful tool in the study of the formation of transport vesicles.

Animals↗

PubMed1991

Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells.

Primary cultures of bovine brain microvessel endothelial cells (BMECs) were used to examine the cycling kinetics of ferrotransferrin (Tf) and to provide evidence for a transcytotic pathway in vitro. Binding of 125I-Tf to BMECs grown on matrix-coated plastic was measured in the presence of saponin to calculate the total number of transferrin receptors (TfRs). Nonlinear regression analysis of the binding isotherm showed that there were 100,000 high-affinity receptors per cell and that expression was maximum at cell confluence. Binding of Tf at 4 degrees C indicated that there was a large intracellular receptor pool comprising 85-90% of the total cellular receptors. Accumulation of Tf at 37 degrees C, inhibited at low temperature and in the presence of metabolic poisons, occurred with an initial rate coefficient of 0.030 min-1 and this decreased by 83% after 60 min. Concomitant accumulation of 59Fe from Tf-59Fe was linear. In the absence of externally added ligand, 80% of the accumulated 125I-Tf was released into the medium with a rate coefficient of 0.017 min-1 and this was inhibited at low temperature. In the presence of the weak base primaquine, the accumulation of Tf and 59Fe and the efflux of Tf were decreased. Moreover, phorbol myristate acetate (PMA) caused a 30% increase in surface TfRs and an 82% increase in Tf accumulation, although the size of the recycling pool remained unchanged. Despite the low numbers of TfR expressed by post-confluent cells, filter-grown BMEC monolayers were used to measure transcytosis of Tf. A small portion of the Tf that was accumulated from the apical side entered a transcytotic pathway. Most of the Tf and all of an accumulated fluid-phase tracer were recycled towards the apical side. These results showed that cultured BMECs cycle Tf-TfR complexes slowly and vectorially and suggested that the large intracellular receptor pool may facilitate steady state accumulation and regulate transcellular transport of iron.

Animals↗

PubMed1990

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network.

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.

Animals↗

PubMed1990

Rapid endocytosis and recycling of wheat germ agglutinin binding sites on CHO cells: evidence for two compartments in a nondegradative pathway.

The internalization and recycling of CHO cell plasma membrane components have been quantified by using iodinated wheat germ agglutinin (WGA) as an adsorptive tracer. Most of these binding sites are thought to be composed of a subpopulation of plasma membrane proteins called high-molecular-weight acidic glycoproteins (HMWAG). Greater than 90% of the WGA bound on the cell surface can be removed by brief treatment with N-acetylglucosamine (GlcNAc). At 37 degrees C, endocytosis of WGA that had been allowed to bind to the surface at 4 degrees C is curvilinear with an initial rapid phase occurring with a t1/2 of 6-8 min. Within 20 min, accumulation has slowed gradually to steady-state with 65% of the cell-associated WGA located intracellularly or resistant to removal by GlcNAc. These portions are unaffected by increasing the extracellular concentration of WGA from 0.003 microM to 2.8 microM. By using pulse-chase experiments, the observed decrease in rate of endocytosis is shown to be due to return of the WGA-HMWAG complexes to the cell surface. More than 60% of the WGA that had been internalized is recycled within 30 min, with a mean t1/2 of 17 min. Recycling involved at least two intracellular populations where a significant fraction (less than 30%) of the WGA-HMWAG complexes are lost gradually from the rapidly recycling pool. Most of the WGA-HMWAG complexes that had internalized are not delivered to the lysosome. These results demonstrate the magnitude of rapid and continuous recycling of WGA binding sites between the cell surface and endosomes in fibroblasts.

Acetylglucosamine↗

PubMed1990

Fluid-phase endocytosis by primary cultures of bovine brain microvessel endothelial cell monolayers.

Blood-brain barrier (BBB) fluid-phase endocytosis was examined in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. By fluorescence spectroscopy. Lucifer yellow (LY, a fluorescent, soluble molecule used as a marker for pinocytosis) accumulation by BMEC was observed to be linear over a concentration range of 0.05 to 1.0 mg/ml. Time-dependent uptake of LY exhibited curvilinear kinetics composed of an initially rapid uptake rate of 1338 ng of LY/mg protein per hour at 0.5 mg/ml LY. Within 20 min, the rate of LY accumulation slowed to a steady-state rate of 23 ng of LY/mg protein per hour. Accumulation of LY was inhibited in the presence of metabolic inhibitors, potassium cyanide or 2-deoxyglucose, and was decreased, but not completely inhibited, at 4 degrees. Pulse-chase experiments revealed that efflux of LY was very rapid with at least 80% of the accumulated LY being lost within 2 min and was not sensitive to low temperature. Only 3-5% of the LY initially accumulated by BMEC remained cell-associated after a 30-min chase. The calculated turnover of the endocytic compartment's total volume (per hour) is 8- to 20-fold less than values for fibroblasts and macrophages, respectively. We have interpreted these data to suggest that the efflux of most of the LY involves loss from a rapidly recycled compartment of finite volume, possibly caveolae, that had sequestered marker during accumulation and suggest that these results are consistent with the present understanding of BBB pinocytosis in vivo.

Animals↗

PubMed1990

Biophysical transport properties of the cuticle of Ascaris suum.

The transport properties of isolated cuticle from Ascaris suum were studied using standard two-chamber diffusion cells and a number of radiolabeled permeants which varied in molecular size, lipophilicity and electrical charge. The permeability coefficient of the collagen matrix (lipid-extracted cuticle) vs. molecular radius relationship showed the interdependence of molecular size and electrical charge of the permeants with respect to the aqueous pores of the negatively charged matrix. The permeability of neutral solutes decreased monotonically with size. Protonated amines permeated the aqueous pores faster than neutral solutes of comparable size, while the permeation of anions was slower. The average pore size was estimated to be 1.5 nm in radius. A biophysical model which accounted for diffusion of molecules within a fixed electrostatic field of force and for molecular sieving by the pore channels was used in the mechanistic interpretation of the data. The effective permeability coefficient of the non-lipid-extracted cuticle was delineated into the permeability coefficients of the water-filled collagen matrix and the lipoidal component of the cuticle to determine which layer was the rate-controlling barrier. While each solute was capable of penetrating the water-filled collagen matrix, the rate-determining step for the majority of compounds was passive diffusion across the lipid component, which controlled 75-99% of transport. The exception was water, for which transport kinetics was 75% matrix-controlled. In general, permeation across the lipid-filled tissue was more favorable for small lipophilic compounds because of molecular restriction not only in the aqueous pores, but also in the lipid-filled pores.

Animals↗

PubMed1990

Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers.

The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75% of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90% of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the t1/2 values for RCAI binding, internalization and efflux were 5, 18 and 13-14 min, respectively. By comparing efflux with and without galactose present, we found that 60% of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4 degrees C became localized within more caveolae within 2.5 min of warming to 37 degrees C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.

Adsorption↗

PubMed1989

Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using an anti-fibronectin receptor beta subunit monoclonal antibody.

Monoclonal antibody (mAb) 7E2.2, which recognizes the beta subunit of the hamster fibronectin receptor (FnR) (Brown, P.J., and Juliano, R. L. (1988) Exp. Cell Res. 177. 303), was used to examine the distribution of and to quantify the internalization of the FnR and possibly related integrins on adherent fibroblasts. Purified 7E2.2 IgG was iodinated and used in binding and internalization studies. Binding to Chinese hamster ovary cells was saturable with a Km of 0.3 nM and an estimated total number of cell surface beta subunits at 2 x 10(5) per cell. The FnR colocalized with fibronectin at cell adhesion contact sites and also was distributed evenly over the dorsal cell surface as discrete clusters. By using a direct immunocolloidal gold approach, the FnR was not associated with coated pits at 4 degrees C until internalization followed warming of the labeled cells to 37 degrees C. A proportion of the FnRs were endocytosed with a half-time of 6.5 min and, consistent with clathrin-mediated uptake, this was sensitive to hypertonic conditions. Receptor-immunocomplexes rapidly became localized within coated pits, small diameter tubules, and peripheral endosomes but the majority remained at the cell surface. At subsaturating concentrations of bound 7E2.2, approximately one-fourth of the total cell receptor population resided intracellularly at any one moment following steady-state; however, appreciable degradation of the iodinated mAb was not detected following accumulation for 4 h at 37 degrees C. These data showed that at least a portion of the FnR are endocytosed via a receptor-mediated pathway and suggested that these receptors do not immediately enter a degradative compartment.

Animals↗

PubMed1989

Demonstration of acid hydrolase activity in primary cultures of bovine brain microvessel endothelium.

The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood-brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and beta-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69-77%). The majority of beta-galactosidase (approximately 48%) and total sulfatase (approximately 58%) activity was associated with the lysosome fraction of the BMECs. In contrast, approximately 52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.

Acid Phosphatase↗

PubMed1989

Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability.

Caco-2 cells develop morphologic characteristics of normal enterocytes when grown on plastic dishes or nitrocellulose filters. The purpose of this study was to determine whether Caco-2 cells undergo similar differentiation when grown on Transwell polycarbonate membranes, and to study the suitability of Caco-2 monolayers as an intestinal epithelial transport model system. Transepithelial electrical resistance values after confluence were 173.5 omega.cm2 and remained unchanged through day 17. Permeabilities to the water-soluble fluid-phase markers that do not permeate the membrane, Lucifer yellow CH, [14C]inulin, [14C]polyethylene glycol, and [3H] dextran were less than 0.25% of the administered amount per hour after day 10. Qualitative evaluation of uptake and permeability to horseradish peroxidase confirmed the similarity in uptake and barrier properties between this cell system and the small intestinal epithelial layer. We conclude that Caco-2 cells grown on collagen-coated polycarbonate membranes should represent a valuable transport model system for the small intestinal epithelium.

Adenocarcinoma↗

PubMed1986

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling.

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 degrees C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t 1/2 approximately 5 min). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

Animals↗

PubMed1986

Cell surface glycoproteins of CHO cells. II. Surface distribution and pathway of internalization.

The surface distribution and pathway for internalization of the major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent trinitrobenzenesulfonate. Molecules, haptenized with trinitrophenol groups, the majority of which are in a group of high molecular weight acidic glycoproteins (HMWAG), were labelled at 4 degrees C with anti-dinitrophenol immunoglobulins coupled to fluorescein isothiocyanate (FITC), horseradish peroxidase, or colloidal gold and either immediately fixed for mapping their distribution or followed intracellularly after warming to allow endocytosis to proceed. The distribution of label on the CHO cell surface was non-random with a large proportion arranged in clusters from 100 to 300 nm in diameter. Antibody label was concentrated heavily on microvilli, and about 10% of the molecules were always associated with clathrin-coated pits. Upon warming the cells to 37 degrees C, HMWAG were internalized immediately into smooth-membraned tubules (less than 80 nm luminal diameter) that appeared to connect with vesicles (less than 300 nm luminal diameter) located in the cortical cytoplasm. By 60 min, labelled antibody was located within larger vesicles (greater than 300 nm luminal diameter) that had a morphology characteristic of multivesicular bodies and not lysosomes. There was no evidence for entry of labelled molecules into either electron-dense, secondary lysosomes or into the Golgi cisternae, suggesting that neither compartment is involved in the major pathway of cell surface endocytosis. Our results are consistent with the view that the majority of plasma membrane protein are internalized as small discrete domains by a pathway very similar to that described by others for adsorptive endocytosis.

Animals↗

PubMed1986

Role of uteroferrin in transplacental iron transport in the pig.

The pig possesses a noninvasive, diffuse type of epitheliochorial placentation in which the blood supply of the mother is well separated from the absorptive surface of the chorion, a feature that must complicate the movement of nutrient molecules across the placenta. Evidence is presented that a protein synthesized and secreted by the glandular epithelial cells of the maternal uterus of the pig is involved in iron transport to the fetus. This protein, uteroferrin, is induced by progesterone; is purple, which results from an unusual iron center; and possesses acid phosphatase activity. Secreted uteroferrin is taken up by specialized chorionic epithelial cells located in domed structures, called areolae, overlying the mouth of each uterine gland. Uteroferrin then enters the placental venous drainage and its iron is efficiently incorporated into fetal hemoglobin. It is taken up by the fetal liver or cleared by the kidney. The liver is the main site of erythropoiesis in the fetus. From the kidney uteroferrin enters the allantoic sac where it exchanges its iron with fetal transferrin. The rate of uteroferrin biosynthesis in the uterus and its rate of metabolism in the fetus can theoretically provide sufficient iron for the needs of pregnancy, at least until around day 70 of the 115-day gestation. Uteroferrin and transferrin, the iron transport protein of plasma, appear to be unrelated proteins.

Acid Phosphatase↗

PubMed1985

The carbohydrate structure of porcine uteroferrin and the role of the high mannose chains in promoting uptake by the reticuloendothelial cells of the fetal liver.

Uteroferrin, the iron-containing, progesterone-induced phosphatase of the porcine uterus, is a glycoprotein carrying a single oligosaccharide chain. Most of the uteroferrin isolated from either uterine secretions or allantoic fluid has endoglycosidase H-sensitive carbohydrate chains with either five or six mannose residues. As determined by 1H-NMR spectroscopy, the Man6 oligosaccharide has the following structure. (Formula: see text) The Man5 species lacks the terminal alpha 1,2-linked residue. Uteroferrin is transported across the pig placenta and has been proposed to be involved in iron transfer to the fetus (see Buhi, W. C., Ducsay, C. A., Bazer, F. W., and Roberts, R. M. (1982) J. Biol. Chem. 257, 1712-1721). Injection of 125I-labeled uteroferrin into the umbilical vein of midpregnant fetuses resulted in incorporation of label into the liver, the major site of fetal erythropoiesis. Light and electron microscope autoradiography revealed that the primary sites of uteroferrin uptake were the reticuloendothelial cells lining the liver sinusoids. Reticuloendothelial cells isolated from either fetal pig or adult rat livers were shown to accumulate uteroferrin when cultured in vitro. Uptake was inhibited by yeast mannan and by glycopeptides isolated from either ovalbumin or uteroferrin. Rat cells did not accumulate uteroferrin whose high mannose chains had been removed using endoglycosidase H. Moreover, the K uptake values (3 X 10(-7) M), specific competition by D-mannose and L-fucose bovine serum albumin, and inhibition by EDTA are consistent with an uptake mechanism involving a receptor for high-mannose oligosaccharides on the liver sinusoidal cells. It is suggested that one function of this receptor in the fetal pig is to remove maternally derived uterine glycoproteins from the fetal circulation. In the case of uteroferrin this process provides iron to the fetal liver.

Acid Phosphatase↗