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R E Fine

Publications and source records attributed to R E Fine.

At least 55 records · Page 3Linked to original sources
PubMed1993

Kinesin is rapidly transported in the optic nerve as a membrane associated protein.

We have investigated the membrane vs. cytosolic distribution of newly synthesized and total kinesin in rabbit retinal ganglion cell axons which comprise the optic nerve. We find that kinesin is rapidly transported into the axon and that this newly synthesized protein is completely membrane-associated while approximately two third of the total kinesin in the optic nerve is membrane associated. Of this membrane associated kinesin about half is resistant to removal by treatment with 100 mM Na2CO3 (pH 11.3) and none can be stripped by 1 M NaCl. The newly synthesized axonal kinesin is completely resistant to removal by Na2CO3 treatment. By these criteria, at least one third of the total and essentially all of the rapidly transported axonal kinesin appears to exist as an integral membrane protein, consistent with it functioning as the anterograde motor for rapid vesicle transport from the cell body through the axon.

Animals↗
PubMed1993

Differences in the subcellular localization of calreticulin and organellar Ca(2+)-ATPase in neurons.

It has become clear that calcium is an important mediator in the transduction of signals due to ligand binding to cell surface receptors. Cytosolic calcium is typically maintained at low levels in both muscle and non-muscle cells and intracellular sequestering of calcium appears to be important in this process. The identification of intracellular calcium pools has been the subject of much recent study, and it has been proposed that such pools would contain three components: a calcium-activated pump or Ca(2+)-ATPase, a calcium channel such as the inositol trisphosphate receptor or ryanodine receptor, and a high-capacity calcium-binding protein such as calsequestrin or calreticulin. We report here on the localization of two components, the organellar Ca(2+)-ATPase (SERCA) and calreticulin, in neuronal tissues. Using immunofluorescence and subcellular fractionation, we have found that for the most part, these two proteins do not co-localize in neuron cell bodies, dendrites, or axons; but may co-localize at the axon terminal.

Animals↗
PubMed1993

Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized.

We have investigated the synthesis, axonal transport, and processing of the beta-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a t1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]-methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is approximately 110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.

Amino Acid Sequence↗
PubMed1993

Receptor-mediated endocytosis of transferrin at the blood-brain barrier.

Rat brains were perfuse with a transferrin-peroxidase conjugate (Tf-HRP) to characterize morphologically the endocytic pathway of transferrin in blood-brain barrier endothelial cells. Electron microscopic evaluation of rat brains perfused with Tf-HRP at 4 degrees C and subsequently warmed to 37 degrees C for brief periods of time (2 minutes) showed sequestration of Tf-HRP in clathrin coated pits and vesicles on the luminal membrane of the brain endothelium. After 5 minutes of warming, diaminobenzidine (DAB) reaction product was present in vesicular structures 250-500 nm in diameter and in associated tubules morphologically identified as large or sorting endosomes. Recycling endosomes were also heavily labelled at this time point. Almost no DAB reaction product remained in the cerebral endothelial cells when the warming period was longer than 15 minutes. Other rat brains were perfused with Tf-HRP at 30 degrees C for 15 minutes prior to fixation and DAB cytochemistry. In these studies, brain endothelial cells contained large amounts of DAB reaction product, mostly localized in 50-100 nm vesicles and tubules, often in the Golgi region of the cells. Coated pits and vesicles and large endosomes were also heavily labelled. Transcytosis of Tf-HRP was not identified in either perfusion protocol. Ultrastructural, indirect immunocytochemical localization of transferrin receptors showed that the transferrin receptor is highly polarized at the blood-brain barrier and is localized only on the apical membrane, in contrast to other polarized epithelial cells, like hepatocytes, in which the receptor is present on the basolateral membrane. The evidence supports an iron transport model in which iron-loaded transferrin is taken up by receptor-mediated endocytosis at the luminal membrane of brain capillaries. The iron then dissociates from transferrin in endosomal compartments and is transcytosed by unknown mechanisms, while the transferrin is retroendocytosed.

Animals↗
PubMed1992

Studies of the mechanism of iron transport across the blood-brain barrier.

The mechanism by which iron enters the central nervous system from the blood is not well understood. Iron in blood plasma is totally bound to transferrin (Tf), a major plasma glycoprotein. Tf receptors are present on the blood-brain barrier (BBB) endothelium. It is not known whether iron separates from Tf during its passage across the endothelial cells and then enters the brain by another mechanism, or whether the two proteins enter the brain together. We characterize here the morphological pathway for endocytosis of a monomeric horseradish peroxidase-transferrin conjugate by the rat BBB endothelium. Our results indicate that this conjugate binds to Tf receptors on the luminal BBB, is internalized via clathrin-coated vesicles, enters early or sorting endosomes, and, subsequently, late or recycling endosomes near the Golgi apparatus. No evidence is found for Tf transcytosis. It is likely that iron separates from Tf in early endosomes, which are assumed to be acidic, as they are in other cells, and enters the brain by an as yet undefined pathway. A clonal line of brain capillary endothelial cells that mimics the BBB when grown on permeabilized membranes can transcytose iron provided as Fe55-Tf. This cell line may provide a useful system to determine the pathway that iron uses to enter the brain. We also present evidence that cultured chick embryo forebrain neurons contain a large number of a unique Tf receptor.

Animals↗
PubMed1992

Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer's disease.

It is well established that iron, which is of considerable importance for normal neurological function, is highly regulated in all organ systems. However, until recently, iron regulation in the nervous system has received little attention. This study quantitatively compares the levels of the major iron-regulatory proteins, transferrin and ferritin, and iron itself in three cerebral cortical regions of the human brain from material collected at autopsy. Three groups were studied: 1) normal adult (under 65 yr of age), 2) aged (greater than 65), and 3) Alzheimer's disease. Normally, transferrin is more abundant in white matter than in gray matter. Ferritin is approximately 10x more abundant than transferrin throughout the brain regions examined and is evenly distributed, as is iron, in the gray and white matter. In Alzheimer's disease transferrin is consistently decreased particularly in the white matter of the various cerebral cortical regions examined whereas the iron and ferritin changes are inconsistent. The observations in this study are consistent with our general hypothesis that iron homeostasis is disrupted in the aging brain and the alterations in iron-regulatory proteins are exacerbated in Alzheimer's disease. The decrease in transferrin levels could indicate a decreased mobility and subsequent utilization of iron in the brain. Such a decrease in iron availability could play a significant role in neuronal degeneration and increased peroxidative damage known to occur in Alzheimer's disease.

Adult↗
PubMed1992

Isolation of a calreticulin-like calcium binding protein from bovine brain.

Intracellular calcium levels are stringently regulated in all cells. The nature of this regulation is incompletely understood, but recent evidence indicates that the endoplasmic reticulum plays an important role in sequestering intracellular calcium. Using methods for isolating both calsequestrin and calreticulin, we have isolated a 58 kDa, high capacity calcium binding protein that exists in microsomes that shift their density in an oxalate-mediated density shift assay. This protein which we call CBP-58 bears similarities to the endoplasmic reticulum protein, calreticulin, in that it has a pI of 4.7 containing approximately 30% glutamate and aspartate, has a high capacity for calcium, and stains blue with the carbocyanine dye, 'Stains-all'. Peptide, amino acid, nucleotide and immunochemical analyses reveal further similarities between CBP-58 and calreticulin, but also some marked differences. Its tissue distribution suggests it is highly enriched in brain versus other tissues. We believe that CBP-58 is a calreticulin-like protein and that differences in the amino acid composition and sequences may reflect species diversity in calreticulin.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of rapid transport vesicle subtypes from rabbit optic nerve.

Subcellular fractionation of rabbit optic nerve resolves three populations of membranes that are rapidly labelled in the axon. The lightest membranes are greater than 200 nm and are relatively immobile. The intermediate density membranes consist of 84 nm vesicles which disappear from the nerve with kinetics identical to those of the rapid component. A third population of membranes, displaying a distinct protein profile, is present in the most dense region of the gradient. Immunological characterization of these membranes suggests the following. (1) The lightest peak contains rapidly transported glucose transporter and most of the total glucose transporters present in the nerve; this peak is therefore enriched in axolemma. (2) The intermediate peak contains rapidly transported glucose transporters and synaptophysin, an integral synaptic vesicle protein, and about half of the total synaptophysin; this peak therefore contains transport vesicles bound for both the axolemma and the nerve terminal, and these subpopulations can be separated by immunoadsorption with specific antibodies against the aforementioned proteins. (3) The heaviest peak contains rapidly transported synaptophysin and tachykinin neuromodulators and about half of the total synaptophysin, and 80% of the total tachykinins present in the nerve; this peak appears to represent a class of synaptic vesicle precursor bound for the nerve terminal exclusively. (4) Synaptophysin is present in the membranes of vesicles carrying tachykinins. (5) Both the intermediate and the heaviest peaks are enriched in kinesin heavy chain, suggesting that both vesicle classes may be transported by the same mechanism.

Adenosine Triphosphatases↗
PubMed1989

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis.

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.

Animals↗
PubMed1989

Newly synthesized calsequestrin, destined for the sarcoplasmic reticulum, is contained in early/intermediate Golgi-derived clathrin-coated vesicles.

We have examined the possible role of clathrin-coated vesicles (CVs) in the genesis of the sarcoplasmic reticulum (SR) in developing chick skeletal myotubes. Calsequestrin (CSQ) a luminal Ca2+ binding protein of the terminal SR cisternae, is contained within the vesicle lumen of skeletal muscle CVs in substantial amounts, approximately four molecules/CV. Employing 3-day cultures of chick skeletal myotubes we demonstrate that after a 30-min labeling with [35S]methionine and cysteine, radioactivity in CSQ remains high in the CVs 45 min later and then declines, while labeled CSQ in the SR continues to rise. No CSQ appears to be secreted. All of the CSQ in both the CVs and SR is sensitive to the activity of endoglycosidase H, and a significant fraction also binds to wheat germ agglutinin. Based on these results, we discuss the hypothesis that a selective CV-mediated pathway exists in developing skeletal muscle cells for the transport of CSQ from the early/intermediate Golgi apparatus to the SR.

Acetylcholinesterase↗
PubMed1989

Intracellular transport, sorting, and turnover of acetylcholinesterase. Evidence for an endoglycosidase H-sensitive form in Golgi apparatus, sarcoplasmic reticulum, and clathrin-coated vesicles and its rapid degradation by a non-lysosomal mechanism.

Tissue-cultured muscle cells synthesize several oligomeric forms of acetylcholinesterase (AChE) destined for the cell surface or secretion. Previous studies on the biogenesis of AChE polypeptide chains have shown that only a small fraction become assembled into catalytically active oligomers which transit the Golgi apparatus and acquire endoglycosidase H (endo H) resistance. Most of the AChE polypeptides remain endo H-sensitive and are rapidly degraded intracellularly. We now show that all newly synthesized AChE polypeptides are transported from the rough endoplasmic reticulum to the Golgi apparatus where they acquire N-acetylglucosamine. However, approximately 80% of these AChE polypeptides remain endo H-sensitive and are degraded intracellularly with a half-life of about 1.5 h by a mechanism which is insensitive to lysosomotropic agents. These endo H-sensitive AChE molecules can be chased into clathrin-coated vesicles and/or the sarcoplasmic reticulum prior to degradation. Pulse-chase studies of isotopically labeled or catalytically active AChE molecules suggest that there are at least two discreet populations of clathrin-coated vesicles which leave the Golgi, one whose origin is cis/medial and one whose origin is trans. These studies indicate the existence of a post-rough endoplasmic reticulum, non-lysosomal degradative pathway for intra-luminal proteins and suggest that post-translational events at the levels of protein sorting and degradation may play a role in regulating the abundance of exportable proteins.

Acetylcholinesterase↗
PubMed1988

Specific trophic factor-receptor interactions. Key selective elements in brain development and "regeneration".

An hypothesis is presented which emphasizes the key role of specific trophic factor-receptor interactions in the development of the brain. We postulate that very early in development neurons become dependent on external factors (mainly neuropeptides) for guidance and survival. These requirements are the key to the selection process which results in the creation of a functional nervous system. These specific localized trophic factor requirements are postulated to persist throughout life. Disruptions in specific trophic factor-receptor systems are postulated to be responsible for a variety of age-related neurodegenerative diseases. The implications of recent animal and human transplant experiments in the context of the theoretical framework discussed above are profound. It would appear that the mature mammalian brain possesses an exquisite ability to regenerate specific connections to replace those lost due to death or injury to nerve cells. Unfortunately, it does not contain a population of undifferentiated stem cells to supply the necessary healthy neurons. The reason for this appears obvious based on the theoretical considerations given above, that the specific trophic factor-receptor interactions needed to produce a functional brain circuitry are necessarily stringently selective. Therefore, a significant stem cell population does not survive. However, if an appropriate stem cell population, ie, a fetal transplant, is provided, the brain will "heal itself" according to the program outlined above. In the future it may be technically feasible to perform genetic testing of newborns to determine to which genetic neurological diseases they are susceptible and at an appropriate time provide the appropriate fetal transplant. Obviously, society will have to deal with the profound ethical questions this technology will raise.

Animals↗
PubMed1987

Purification and characterization of the rat liver vasopressin (V1) receptor.

Utilizing a proteoliposome reconstitution system, we have purified the rat liver V1 vasopressin receptor to near homogeneity. The receptor was purified approximately 21,000-fold from rat liver membranes, using differential detergent solubilization, size exclusion gel filtration, lectin affinity, and ion-exchange chromatography. The purified receptor exhibits a Kd of 6 nM, when, prior to solubilization, the membranes were exposed to 1 microM vasopressin. This resulted in the association of a pertussis toxin-insensitive guanine nucleotide-binding protein with the receptor during most of the purification procedure. In the absence of this association, the receptor had a Kd of approximately 30 nM. Association of the receptor with a G-protein was confirmed by the ability of vasopressin to stimulate the hydrolysis of [gamma-32P]GTP. The specific activity of the vasopressin-stimulated hydrolysis was 25 nmol/min/mg, approximately 8,000-fold higher than values obtained with crude reconstituted receptor preparations. Cross-linking of 125I-vasopressin to a partially purified preparation of receptor demonstrated that the receptor had a molecular weight of approximately 68,000 under reducing conditions, and 58,000 under nonreducing conditions. The purification procedure may prove useful in purifying a number of small peptide hormone receptors (e.g. bradykinin, angiotensin II) and perhaps their associated G-proteins as well.

Animals↗
PubMed1987

Reconstitution of the rat liver vasopressin receptor coupled to guanine nucleotide-binding proteins.

The V1 vasopressin receptor has been solubilized from rat liver membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate (CHAPS) and reconstituted into phospholipid vesicles. There is essentially complete solubilization of the receptor by 3% CHAPS at a protein concentration of 15 mg/ml. Reconstitution into soybean phospholipid vesicles is readily achieved either by gel filtration chromatography or by membrane dialysis. The binding of [3H]vasopressin to proteoliposomes is specific, saturable, reversible, and magnesium-dependent. In contrast, the detergent-soluble vasopressin receptor does not display specific binding. The apparent affinity of the reconstituted receptor for [3H]vasopressin is approximately 4-fold lower than that of the receptor in native membranes. In addition, the binding of [3H]vasopressin to reconstituted vesicles is not sensitive to 100 microM guanosine 5'-O-thiotriphosphate (GTP gamma S) as it is in native membranes. However, the apparent affinity of the reconstituted receptor for ligand approximates that of native membranes when membranes are prebound with vasopressin prior to solubilization and reconstitution into vesicles. Furthermore, vesicles reconstituted from membranes prebound with vasopressin show GTP gamma S sensitivity of [3H] vasopressin binding. This finding strongly suggests that vasopressin stabilizes a receptor-G-protein complex during solubilization. The rat liver vasopressin receptor is a glycoprotein, as shown by its specific binding to the lectin "wheat germ agglutinin." The vasopressin receptor can be reconstituted from the N-acetylglucosamine-eluted peak of a wheat germ agglutinin-Sepharose column, and [3H] vasopressin binding activity is purified 5-6-fold from membranes by this chromatographic procedure. The functionality of the partially purified receptor is indicated by its ability to bind ligand with high affinity and by its ability to functionally interact with a G-protein when vasopressin is bound prior to solubilization.

Animals↗
PubMed1987

A trans Golgi-derived exocytic coated vesicle can contain both newly synthesized cholinesterase and internalized transferrin.

We used a cholinesterase-mediated density shift protocol to investigate the movement of internalized transferrin (Tf) through endo- and exocytic coated vesicles (CVs) in the perfused rat liver. Upon internalization, exogenous 125I-Tf was found in endocytic CVs but not in cholinesterase-containing (i.e., exocytic) CVs (0-40 min). Between 1 and 2 hr, 125I-Tf began to appear in exocytic CVs. The origin of the exocytic CV was further investigated. After perfusion of the liver with asialotransferrin, the exocytic CVs were shown to contain resialylated Tf, indicating that the trans Golgi was the origin of this class of CVs. The resialylated Tf accumulated in the extracellular medium with kinetics very similar to the time course for appearance of Tf in cholinesterase-containing, exocytic CVs, suggesting that these CVs are directly involved in the transfer of material from the trans Golgi to the cell surface.

Animals↗
PubMed1987

Development of transferrin-positive oligodendrocytes in the rat central nervous system.

Transferrin is the second most abundant plasma protein and functions to transport iron. It is an essential constituent in culture media for virtually all cells. In a recent study, we reported that transferrin (Tf) is specifically located in oligodendrocytes in the rat nervous system. This investigation examines immunohistochemically the development of Tf in the cerebral cortex, corpus striatum, and spinal cord. Tf is first seen in oligodendrocytes in the spinal cord white matter at 5 days of age. The immunoreactivity is confined to the white matter in the periphery of the spinal cord between 5 and 8 days of age. By 10-12 days of age, the number of immunoreactive oligodendrocytes in the spinal cord white matter increases considerably, corresponding to the onset of myelination. Tf-positive oligodendrocytes are first found in the gray matter at 15 days of age. By 30 days of age, the number and distribution of Tf-positive oligodendrocytes in both the brain and spinal cord have reached the adult pattern. The results of this study demonstrate a spatial and temporal association between Tf development and myelinogenesis. This suggests that part of the process of differentiation of oligodendrocytes includes the accumulation of Tf, perhaps in order to support the metabolic demands associated with the production and maintenance of myelin.

Animals↗