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Biomedical subjects

R E Fine

Publications and source records attributed to R E Fine.

At least 73 records · Page 4Linked to original sources
PubMed1987

Receptor-mediated transcytosis of transferrin across the blood-brain barrier.

The perfusion of rat brain with 125I-transferrin resulted in a receptor-mediated uptake of transferrin into the endothelium of the blood-brain barrier followed by its detection in the brain. During a pulse-chase experiment, 125I-transferrin accumulated in the endothelial cells during the pulse, with a decrease of this intraendothelial radioactivity during the chase associated with a concomitant increase in the nonvascular elements of the brain. The receptor-mediated movement of transferrin across the blood-brain barrier suggests that the brain may derive its iron through the transcytosis of iron-loaded transferrin across the brain microvasculature. We discuss the likelihood that aluminum and other potentially toxic heavy metals, which also bind tightly to transferrin, may enter the brain by this pathway. We also discuss the possibility that other large molecules including neuroactive peptides and neurotrophic viruses may enter the brain through a similar receptor-mediated, vesicular transcytotic route.

Animals↗
PubMed1987

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.

Animals↗
PubMed1987

Regional variation in the levels of transferrin in the CNS of normal and myelin-deficient rats.

Transferrin (Tf), the iron mobilization protein, is synthesized mainly in the liver. Recently, both Tf and a mRNA for Tf have been demonstrated in oligodendrocytes in the rat brain. The present study used a biochemical assay for determining the levels of Tf in various brain regions of normal rats compared with the level of those obtained from rats with a genetic mutation characterized by an almost complete failure to develop myelin. In myelin-deficient (md) rats, no Tf-positive oligodendrocytes were seen immunohistochemically in the gray or white matter of the CNS. Quantitatively, levels of Tf throughout the CNS of the md rat were decreased to approximately 5% of the normal values despite a normal hepatic synthetic rate. In the normal rat brain, the cerebellum contained the highest concentration of Tf, followed by the pons, the cerebral cortex, and the caudate-putamen, with the latter two sites being similar. Regional variation in the amount of Tf was in general agreement with published reports on the variation of iron and Tf receptor levels in the CNS. Immunohistochemical examination with antiserum to galactocerebroside (a myelin-specific lipid) was used for extending biochemical reports that glycolipid-synthesizing enzymes are deficient in md rats. No immunostaining in the md rat was observed following immunoreaction for galactocerebroside, whereas white matter oligodendrocytes were intensely marked in the normal rat. Robust astrogliosis was present in both the gray and white matter of the md rats. It is not known at present whether the ability to accumulate Tf is necessary for oligodendrocytic survival or if Tf accumulation is more directly related to myelinogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Reversible inactivation of vasopressin and angiotensin II binding to hepatocyte membranes by a calcium-dependent, cytosolic protein.

A cytosolic protein is described which inhibits the binding of vasopressin and angiotensin to their rat liver receptors in the presence of calcium. The binding of insulin and transferrin was unaffected. Inhibition was temperature-dependent; it was maximal in 10 min at 37 degrees C, but required longer incubation times at lower temperatures. The pH optimum was 7.4. Inhibition also required the presence of calcium, with half-maximal inhibition at 6-8 microM calcium, but did not require any other low molecular weight cofactors. Inhibition could be reversed by washing the membranes at pH 5.5, but not by incubation with EGTA. Sephacryl S-300 chromatography showed that activity eluted in two peaks with approximate molecular weights of 70,000 and 150,000. In the presence of calcium, the inhibitory activity eluted at 150,000; in the absence of calcium, most of the inhibitory activity eluted at 70,000. A radiolabeled cytosolic protein with a molecular weight of 70,000 was eluted from inhibited rat liver membranes at pH 5.5 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that vasopressin and angiotensin II, which both mobilize calcium in hepatocytes via phosphatidylinositol turnover, can, by this same mechanism, activate a protein(s) which reduces further binding to their receptors.

Angiotensin II↗
PubMed1986

The distribution of transferrin immunoreactivity in the rat central nervous system.

Recent studies have demonstrated receptors in the nervous system for transferrin, the iron binding and transport protein in the blood. This study using immunohistochemistry at the light and electron microscopic levels demonstrates that transferrin (Tf) is found predominantly in oligodendrocytes in both the gray and white matter of the cerebral cortex, cerebellum and spinal cord. Within the cerebral cortex, layer V has more Tf-labeled cells than the other cortical layers. In the spinal cord, lamina VII has the highest density of Tf-positive cells. Based on location, 3 types of oligodendrocytes can be described: perineuronal, interfascicular and perivascular. In addition to oligodendrocytes, endothelial cells and possibly some neuronal membranes of layer V pyramidal and anterior horn cells label with Tf antiserum. Ultrastructurally, Tf reaction product is homogeneously distributed throughout the perinuclear cytoplasm of both oligodendrocytes and endothelial cells. The importance of iron in motor and behavior function is well established although the mechanism of action of iron in the CNS is not well understood. The presence of Tf in oligodendrocytes implies that these neuroglia are involved in iron mobilization and storage in the CNS. Stored quantities of iron and the ability to mobilize the iron through stored transferrin may be the reason for the extreme dietary restrictions necessary to induce iron-deficient CNS disorders.

Animals↗
PubMed1986

Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique.

Coated vesicles isolated from rat liver perfused with diisopropylfluorophosphate (DFP) to inactivate endogenous cholinesterase contained newly synthesized secretory cholinesterase after a 30 min recovery. The cholinesterase is found in coated vesicles of presumed endocytic origin following DFP treatment and perfusion for 3 min with galactosylated cholinesterase, a ligand for the asialoglycoprotein receptor. Highly enriched populations of endocytic and exocytic coated vesicles can be separated by use of a novel cholinesterase mediated density shift technique. The two coated vesicle classes have very similar polypeptide compositions but differ significantly in the ratio of cholesterol to phospholipid.

Acetylcholinesterase↗
PubMed1986

Chick embryo myotubes contain transferrin receptors and internalize and recycle transferrin.

Embryonic chick skeletal myotubes grown in cell culture require transferrin to provide iron for proliferation and differentiation. We demonstrate here that cultured myotubes contain transferrin receptors as demonstrated by the finding of specific, saturable, and reversible high-affinity binding sites. Scatchard analysis of equilibrium binding data indicates an apparent Kd of 37 nM and one muscle cell equivalent contains 7,500 transferrin receptors. Myotubes exhibit a Kd 100 times higher for apotransferrin than for iron-saturated transferrin. Internalization of specifically bound transferrin is temperature dependent and occurs rapidly at 37 degrees C with a steady state reached after 10 min. Internalization studies using either 125I-ovotransferrin or 55Fe-ovotransferrin suggest that transferrin is internalized, depleted of iron, and recycled intact to the extracellular medium as shown in other cell systems. Autoradiography of muscle cell cultures incubated with 125I-ovotransferrin at 4 degrees C reveals clusters of receptors along the myotubes. The possible mechanisms by which transferrin is supplied to muscle in vivo are discussed in light of the evidence that motor neurons contain transferrin.

Animals↗
PubMed1986

An acetylcholinesterase-mediated density shift technique demonstrates that coated vesicles from chick myotubes may contain both newly synthesized acetylcholinesterase and acetylcholine receptors.

Coated vesicles isolated from 17 d chick embryo skeletal muscle contain acetylcholine receptors (AChRs) as shown by the presence of specific, latent binding sites for 125I-alpha bungarotoxin (125I-alpha-BTX). Since these coated vesicles also contain AChE (Benson et al., 1985), we hypothesized that a coated vesicle could carry both molecules: one an integral membrane protein, the other a secreted protein. An AChE-mediated density shift technique was used to obtain data that indicate that most isolated coated vesicles contain AChE and that some contain AChRs as well. Similar results were obtained with coated vesicles isolated from cultured chick embryo myotubes treated briefly with diisopropylfluorophosphate (DFP) to inactivate all preexisting AChE and allowed to synthesize AChE for 2 1/2 hr. These data are compatible with the hypothesis that both an integral plasma membrane protein, AChR, and a secretory protein, AChE, traverse the identical pathway after synthesis, as proposed by Rotundo and Fambrough (1980a). We suggest that coated vesicles are important intermediates in the exocytotic pathway, and that the large percentage of coated vesicles utilized for exocytotic transport can explain the rapid net increase in surface area achieved during myotube development. We also discuss the potential utility of the AChE-mediated density shift in studying the exocytotic and endocytotic pathways in other cell types, and possible pitfalls associated with its use.

Acetylcholinesterase↗
PubMed1985

Generalized inhibition of cell-free translation by the amino-terminal propeptide of chick type I procollagen.

Fragments of the amino-terminal propeptide of procollagen have been shown to inhibit the synthesis of procollagen in cultured cells and in a reticulocyte lysate cell-free system (for review see Timpl, R. and Glanville, R.W. (1981) Clin. Orth. Rel. Res. 158, 224-242). In this report, we show that the full-length amino-terminal propeptide of chick pro alpha1(I) chains inhibits the translation of chick tendon mRNA and rat brain mRNA in a reticulocyte lysate cell-free system. The synthesis of procollagen and non-collagenous proteins was equally affected. Inhibition was dose-dependent up to 10 microM. A similar pattern of inhibition was observed for the collagenase-resistant fragment, col 1(I).

Animals↗
PubMed1985

Internalization, recycling, and redistribution of vasopressin receptors in rat hepatocytes.

Three hours after isolation, cultured hepatocytes have approximately 150,000 surface vasopressin receptors/cell, and these exhibit a Kd for 125I-vasopressin of 6 nM based on calculation of Koff/Kon, or a Kd of 9.5 nM based on Scatchard plot analysis. After the binding of 125I-vasopressin to its receptor on the hepatocyte surface, this complex is internalized with a t1/2 of 3-6 min. Following this internalization, the number of vasopressin receptors on the cell surface is restored both in vitro and in the isolated perfused liver with a t1/2 of 8-10 min. This restoration is blocked in vitro by incubation of the hepatocytes at 18 degrees C, but not by cycloheximide, suggesting that internalized vasopressin receptors recycle back to the cell surface. Prolonged incubation of hepatocytes with vasopressin results in the loss of greater than 75% of the vasopressin surface binding at concentrations of vasopressin approximately equivalent to its Kd. The binding of vasopressin to cultured hepatocytes 3-5 h after isolation resembles binding to the isolated perfused whole liver with respect to receptor dynamics. During culture for 48 h, however, we observe a progressive loss of hepatocyte surface vasopressin receptors. Concomitant with this reduction in surface receptors with time in culture, there appears to be a marked elevation in intracellular receptors.

Animals↗
PubMed1985

Coated vesicles contain a phosphatidylinositol kinase.

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.

1-Phosphatidylinositol 4-Kinase↗
PubMed1985

Coated vesicles purified from chick tendon fibroblasts contain newly synthesized type I procollagen.

Coated vesicles were purified from embryonic chick tendon fibroblasts pulsed with [3H]proline. They were morphologically and biochemically similar to coated vesicles purified from other sources. Furthermore, they contained newly synthesized Type I procollagen which was protected from bacterial collagenase digestion unless detergent was present. The procollagen remained associated with coated vesicles during immune precipitation and agarose gel electrophoresis. Data from pulse-chase experiments demonstrated that the specific activity of the coated vesicle preparations was approx. 5-fold higher at the 10 min chase point than at either the 0 or 40 min chase points. These data are consistent with the hypothesis that coated vesicles are intermediates in the intracellular transport of newly synthesized Type I procollagen in chick tendon fibroblasts.

Animals↗
PubMed1985

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase.

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219-221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.

Acetylcholinesterase↗
PubMed1984

Identification of a protein kinase as an intrinsic component of rat liver coated vesicles.

Purified rat liver coated vesicles phosphorylate two peptides, Mr 53 000 and Mr 51 000, in the presence of [gamma-32P]ATP. Incorporation of phosphate into these peptides is not stimulated by cAMP, Ca2+, or Ca2+ plus calmodulin and occurs principally on a threonine residue. Mild conditions that result in removal of coat proteins from coated vesicles remove most of the protein kinase activity, suggesting the enzyme(s) is (are) not an integral membrane protein. Photolabeling of coated vesicles with 8-azido-[alpha-32P]ATP results in specific labeling of only the Mr 53 000 and Mr 51 000 peptides. Preincubation with 10 mM N-ethylmaleimide inhibits kinase activity and concomitantly reduces photolabeling of the two peptides. Thus, the data are consistent with the hypothesis that protein kinase activity resides with these two coated vesicle proteins and that they are catalyzing an autophosphorylation reaction.

Adenosine Triphosphate↗
PubMed1983

Chick embryo spinal cord neurons synthesize a transferrin-like myotrophic protein.

Highly enriched cultures of chick embryo spinal cord neurons synthesize and secrete a protein which is immunoprecipitable by anti-ovotransferrin. Ovotransferrin, an iron-binding glycoprotein of Mr 80 000, is also shown to stimulate in vitro myogenesis of cultured chick embryo myotubes as measured by saturable dose-dependent increase in acetylcholine receptors. This effect is probably dependent on ovotransferrin's ability to donate iron to the cells. In many respects ovotransferrin is similar to 'sciatin', a myotrophic protein isolated from chicken sciatic nerves.

Animals↗
PubMed1983

Coated vesicles from rat liver and calf brain contain cryptic mannose 6-phosphate receptors.

Highly purified clathrin-coated vesicles, isolated from rat liver and calf brain, contain mannose 6-phosphate receptors. The coated vesicle receptors appear to have the same subunit molecular weight and similar binding affinity as the receptor previously purified from bovine liver and rat chondrosarcoma microsomes (Sahagian, G. G., Distler, J. J., and Jourdian, G. W. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4289-4293 and Steiner, A. W., and Rome, L. H. (1982) Arch. Biochem. Biophys. 214, 681-687). There is a considerable (greater than 60-fold) enrichment of receptors in liver coated vesicles as compared to liver microsomes. Experiments carried out with intact and detergent-disrupted coated vesicles indicated that the receptors face toward the inside of the coated vesicles. The data suggest that coated vesicles are involved in the intracellular transport of the mannose 6-phosphate receptor.

Animals↗
PubMed1983

Coated vesicles participate in the receptor-mediated endocytosis of insulin.

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.

Animals↗