Judy Garner - Academia.edu (original) (raw)

Papers by Judy Garner

Research paper thumbnail of Biogenesis of Presynaptic Terminal Proteins

Journal of Neurochemistry, 1987

The delivery of proteins to the presynaptic terminals of guinea pig retinal ganglion cells by two... more The delivery of proteins to the presynaptic terminals of guinea pig retinal ganglion cells by two of the major components of axonal transport, and the subsequent persistence and turnover of those proteins were examined in this study. Ganglion cell proteins were radiolabeled by intravitreal injection of radiolabeled amino acids and radioactive axonally transported proteins were analyzed in synaptosomes prepared from the superior colliculi. This procedure allowed examination of presynaptic components of ganglion cell synapses without having to compensate for postsynaptic or other unidentified contaminants. Each of the two major axonal transport components supplies a large number of proteins to the presynaptic terminal, in relative quantities similar although not identical to those seen in the axon. Proteins conveyed by the fast component of axonal transport reached the terminals by 3 h after intraocular injection, peaked by 24 h, and were largely undetectable by I5 days. Slow component b proteins reached the terminals by 12 days, peaked around 2 I days. and persisted up to 63 days in the terminals. Proteins in both components demonstrated differential turnover relative to cotransported proteins once they reached the terminals. Differential turnover may account for change in relative concentration of a particular protein required to meet new functional demands on that protein once it enters the terminal.

Research paper thumbnail of Selective Alterations in Presynaptic Cytomatrix Protein Organization Induced by Calcium and Other Divalent Cations That Modulate Exocytosis

Journal of Neurochemistry, 1990

Rises in intracellular calcium cause several events of physiological significance, including the ... more Rises in intracellular calcium cause several events of physiological significance, including the regulated release of neuronal transmitters. In this study, the effects of divalent cations on the structural organization of cytomatrix in presynaptic terminals was examined. [35S]Methionine-radiolabeled guinea pig retinal ganglion cell cytomatrix proteins were axonally transported [in slow component b (SCb) of axonal transport] to the neuron terminals in the superior colliculus. When the peak of radiolabeled cytomatrix proteins reached the terminals, synaptosomes containing the radiolabeled cytomatrix proteins were prepared. Approximately 40% of each SCb protein was soluble after hypoosmotic lysis of the radiolabeled synaptosomes in the presence of divalent cation chelators. Lysis of synaptosomes in the presence of calcium ions over a range of concentrations, however, caused a dramatic decrease in solubility of the presynaptic SCb proteins. The cytoplasmic effects may result from a calcium-dependent condensation of cytoplasm around presynaptic terminal membrane systems. There are two major presynaptic SCb proteins (at 60 and 35 kDa), that exhibited exceptional behavior: they remained as soluble in the presence of calcium as under control conditions, suggesting that they were relatively unaffected by the mechanism causing the decrease in SCb protein solubility. Also examined were the effects of other alkaline earth and transition metal divalent cations on the presynaptic SCb proteins. Key Words: Presynaptic terminal-Axonal transport-Cytomatnx-Calcium-Divalent cations-Transmitter release. Garner J. A. Selective alterations in presynaptic cytomatrix protein organization induced by calcium and other divalent cations that modulate exocytosis.

Research paper thumbnail of Cytoplasmic matrix proteins in central nervous system presynaptic terminals: turnover and effects of osmotic lysis

Brain Research, 1990

Cytomatrix proteins, of primary functional importance in central nervous system neuron terminals,... more Cytomatrix proteins, of primary functional importance in central nervous system neuron terminals, are provided to their site of action in the terminal by axonal transport. Slow component b (SCb) of axonal transport has been proposed to be the biochemical counterpart of the moving cytoplasmic matrix, or cytomatrix, in axons. In the current study, axonally transported SCb proteins destined for neuron terminals were pulse-radiolabeled with [35S]methionine in guinea pig retinal ganglion cells. After SCb proteins reached the terminals in the superior collioili, synaptosomes were prepared to distinguish between SCb proteins in the preterminal axons and those of the presynaptic terminals, Study of the initial entry and turnover of individual SCb proteins in presynaptic terminals revealed different residence times of certain SCb proteins in comparison with their cohorts. Preliminary information about the structural relationships of the proteins comprising the presynaptic cytomatrix was obtained by examining the solubility of individual SCb proteins relative to other SCb proteins, or membranes from osmotically lysed terminals. Last, treatment of those radiolabeled synaptosomes with varying concentrations of salts was performed to determine possible effects on observed structural relationships.

Research paper thumbnail of Differential turnover of tubulin and neurofilament proteins in central nervous system neuron terminals

Brain Research, 1988

The transport of tubulin and neurofilament protein subunits from the preterminal axons of guinea ... more The transport of tubulin and neurofilament protein subunits from the preterminal axons of guinea pig retinal ganglion cells into their presynaptic terminals in the superior colliculus was examined. Newly synthesized tubulin and neurofilament proteins were radiolabeled with tritiated amino acids in the cell bodies and were allowed to be axonally transported through the optic axons and into the terminals in the superior colliculi. Superior colliculi were harvested at appropriate times, synaptosomes were prepared, and radiolabeled proteins were examined by gel electrophoresis and fluorography. Proteins in the radiolabeled synaptosomes were compared with those in the portion of the optic tract immediately proximal to the superior colliculus. Tubulin subunits entered the terminals by 100 days after intraocular labeling, and at least one isoform of tubulin appeared to persist as long as 400 days. Neurofilament proteins, despite the fact that they are axonally transported and delivered to the terminals in concert with the tubulin subunits, disappear rapidly upon entry into the terminals themselves.

Research paper thumbnail of Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals

Journal of Neurochemistry, Aug 1, 1996

Research paper thumbnail of Cohesive axonal transport of the slow component b complex of polypeptides

The Journal of Neuroscience the Official Journal of the Society For Neuroscience, 1983

The axonal transport of the diverse group of polypeptides characteristic of slow component b (SCb... more The axonal transport of the diverse group of polypeptides characteristic of slow component b (SCb), or Group IV, may be accomplished either by a bulk flow mechanism acting on each of the individual polypeptides or by their movement through the axoplasm as part of a physical complex or structural assembly. In order to determine if the SCb polypeptides travel as a cohesive unit (which would be consistent with the latter alternative above), we examined in detail the quantitative distribution of the individual SCb polypeptides in the guinea pig optic system at three different times after pulse radiolabeling the neuron cell bodies. Eighteen SCb polypeptides were selected as appropriate for analysis. Individual optic systems at the three select times after labeling were harvested, cut into small segments, and subjected to SDS-PAGE. Individual radioactive polypeptide bands were identified and located by fluorography, excised, and then quantified by scintillation counting.

Research paper thumbnail of Rapid Axonal Transport of the Neural Cell Adhesion Molecule

The neural cell adhesion molecule (NCAM) is a cell-surface glycoprotein that mediates cell-cell i... more The neural cell adhesion molecule (NCAM) is a cell-surface glycoprotein that mediates cell-cell interactions in the nervous system during development. In the present study, we demon- strate that NCAM is axonally transported in 3-d-old chick ret- inal ganglion cells and that it travels within the fast component of axonal transport (FC). Proteins were radiolabeled in retinal ganglion cell bodies after

Research paper thumbnail of Clathrin is axonally transported as part of slow component b: the microfilament complex

The Journal of Cell Biology, 1981

During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal el... more During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal elements travel in either of two slow components, SCa (with tubulin and neurofilament protein) and SCb (with actin) . Clathrin, the highly ordered, structural coat protein of coated vesicles, has recently been shown to be able to interact in vitro with cytoskeletal proteins in addition to membranes. The present study examines whether clathrin travels preferentially with the membrane elements or the cytoskeletal elements when it is axonally transported. Guinea pig visual system was labeled with tritiated amino acids. Radioactive SDS-polyacrylamide gel electrophoresis profiles from the major components of transport were coelectrophoresed with clathrin . Only SCb had a band comigrating with clathrin . In addition, radioactive clathrin was purified from guinea pig brain containing only radioactive SCb polypeptides . Kinetic analysis of the putative clathrin band in SCb revealed that it travels entirely within the SCb wave . Thus we conclude that clathrin travels preferentially with the cytoskeletal proteins making up SCb, rather than with the membranes and membrane-associated proteins in the fast component.

Research paper thumbnail of Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes

Science, 1981

The proteins of the three major rate components of axonal transport in guinea pig retinal ganglio... more The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.

Research paper thumbnail of Factors that contribute to the transneuronal spread of herpes simplex virus

Journal of Neuroscience Research, 1997

In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread b... more In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread between neurons in the central nervous system (CNS) by distinctly different routes. The steps of viral infection and spread in a single neuron type and nearby glial cells in vivo have been determined for three different strains of HSV (F, H129, and McIntyre-B). The corneas of mice were inoculated with equivalent titers of the strains. Two to 5 days later, the animals were killed. The spread of viral proteins within trigeminal cells was examined using immuno- and electron microscopy and Western blots with anti-HSV polyclonal antiserum. McIntyre-B virus infection resulted in fewer labeled ganglion cells, possibly as a result of reduced viral production in the corneal epithelium or trigeminal ganglion cells. Although the McIntyre-B strain was at least as, if not more efficient, at retrograde transport than the other strains, the amount of McIntyre-B virus that was transported in the trigeminal roots in an anterograde direction was significantly less than the other strains. Uptake by ganglionic satellite cells was qualitatively similar for the three strains, but maturation and release of virus from satellite cells to other neurons were reduced in the McIntyre-B strain. These characteristics may account for the preferential retrograde transneuronal spread of McIntyre-B strain.

Research paper thumbnail of Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals

Journal of Neurochemistry, 2002

Research paper thumbnail of Protein changes during anterograde-to-retrograde conversion of axonally transported vesicles

Brain Research, 1989

In the axon tip, cell biological mechanisms convert anterogradely transported membranous elements... more In the axon tip, cell biological mechanisms convert anterogradely transported membranous elements. To study the effects of these anterograde-to-retrograde (A-R) converting mechanisms on the electrophoretic behaviour of vesicle proteins, we compared the proteins of anterograde vesicles (before A-R conversion at the axon tip) with those of retrograde vesicles (after A-R conversion at the axon tip). The proteins in transported vesicles were pulse-labeled with [35S]methionine, and the radiolabeled vesicles were concentrated by ligating the axons-anterograde vesicles accumulate selectively on the proximal side of the ligature and retrograde vesicles accumulate on the distal side of the ligature. Analyses of vesicle proteins by polyacrylamide gel electrophoresis (SDS-PAGE) show that most of the anterograde proteins were also present in the retrograde vesicles. In addition to the conservation of these anterograde proteins in the retrograde vesicles, there were also many differences: some anterograde proteins were diminished in the retrograde vesicles, other anterograde proteins were absent from the retrograde vesicles, and the retrograde vesicles contained some new protein bands that were not present in the anterograde vesicles. These results indicate that A-R converting mechanisms modify membranous vesicle proteins in the axon tip. We propose that some of these post-translational protein modifications change the directional code on the vesicle surfaces, thereby converting anterograde membranous elements into retrograde membranous elements.

Research paper thumbnail of Characterization of spherical amyloid protein from a prolactin-producing pituitary adenoma

Acta Neuropathologica, 1997

Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abun... more Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abundant spherical amyloid; however, the origin of the amyloid has not been established. In this report, a PRL-producing pituitary adenoma composed almost entirely of spherical amyloid was analyzed biochemically. The tumor was removed surgically from a 56-year-old man. Immunohistochemical analysis revealed that residual tumor cells were strongly positive for PRL, while the spherical amyloid was not. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of approximately 4 kDa associated with the amyloid, which was not present in a nonamyloid producing prolactinoma. The 4-kDa band is similar in size to other known amyloidogenic peptides. Immunoblot analysis of the tumor material using polyclonal anti-human PRL antibodies revealed a small amount of normal-sized PRL; however, the abundant 4-kDa band was nonimmunoreactive. Amino acid sequencing showed that this peptide represents the first 34 amino acids of the intact PRL protein with a predicted size of 4313 Da. The presence of a small amount of normal-sized PRL in this tumor, as well as elevated circulating levels of PRL implies that intact PRL is being abnormally processed in the formation of spherical amyloid.

Research paper thumbnail of Axonal transport of neuronal antigens characteristic of subpopulations of central nervous system (CNS) neurons

Metabolic Brain Disease, 1989

Monoclonal antibodies (MAbs) are useful for the identification of nervous system antigens localiz... more Monoclonal antibodies (MAbs) are useful for the identification of nervous system antigens localized to neuronal subpopulations. We have examined the transport of the corresponding antigens of four such MAbs in guinea pig retinal ganglion-cell axons. Determination of the axonal transport rate of radiolabeled antigens allowed their assignment to one of the three major anterograde axonal transport rate components, each of which is through to convey a subcellular structural system in the axon. Antigens identified by three of the MAbs were found to be transported in slow component b of axonal transport, the component thought to convey the cytoplasmic matrix, and an antigen identified by the fourth MAb was found in slow component a, similarly thought to contain the linear cytoskeletal elements. Assignment of these antigens to the different rate components suggests that they may be associated with a particular structural system in neurons. Additionally, in cases where more than one nervous system cell type may express a particular antigen, the identity of the neuronal form of the antigen has been confirmed by its axonal transport. The roles that these antigens may play in the nervous system during normal axonal function and during neuropathogenesis can now be further examined.

Research paper thumbnail of Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in Lacrimal Acini

Journal of Virology, 2006

The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves f... more The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to ␣ v integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.

Research paper thumbnail of Factors that contribute to the transneuronal spread of herpes simplex virus

Journal of Neuroscience Research, 1997

In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread b... more In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread between neurons in the central nervous system (CNS) by distinctly different routes. The steps of viral infection and spread in a single neuron type and nearby glial cells in vivo have been determined for three different strains of HSV (F, H129, and McIntyre-B). The corneas of mice were inoculated with equivalent titers of the strains. Two to 5 days later, the animals were killed. The spread of viral proteins within trigeminal cells was examined using immuno- and electron microscopy and Western blots with anti-HSV polyclonal antiserum. McIntyre-B virus infection resulted in fewer labeled ganglion cells, possibly as a result of reduced viral production in the corneal epithelium or trigeminal ganglion cells. Although the McIntyre-B strain was at least as, if not more efficient, at retrograde transport than the other strains, the amount of McIntyre-B virus that was transported in the trigeminal roots in an anterograde direction was significantly less than the other strains. Uptake by ganglionic satellite cells was qualitatively similar for the three strains, but maturation and release of virus from satellite cells to other neurons were reduced in the McIntyre-B strain. These characteristics may account for the preferential retrograde transneuronal spread of McIntyre-B strain.

Research paper thumbnail of Retinal Pigment Epithelial Cells Secrete and Respond to Hepatocyte Growth Factor

Biochemical and Biophysical Research Communications, 1998

gen (10). The receptor for HGF is the membrane-span-Hepatocyte growth factor (HGF) is normally ex... more gen (10). The receptor for HGF is the membrane-span-Hepatocyte growth factor (HGF) is normally exning tyrosine kinase c-Met (11,. Through its activapressed by mesenchymal cells while its receptor, ction of c-Met, HGF has been found to be critical for Met, is expressed in epithelial cells. Since HGF is critiepithelial-mesenchymal interactions with widespread cally involved in epithelial-mesenchyme interactions effects in embryogenesis and regeneration (reviewed in and the retinal pigment epithelium (RPE) is present .

Research paper thumbnail of Differential anterograde transport of HSV type 1 viral strains in the murine optic pathway

Active anterograde transport of herpes simplex virus Type 1 (HSV-1) in neurons is often assumed b... more Active anterograde transport of herpes simplex virus Type 1 (HSV-1) in neurons is often assumed based on early appearance of infection in postsynaptic target cells of a primary infected cell, and is further logically inferred by good evidence of microtubule-motor based mechanisms of retrograde transport. However, direct evidence of mechanisms of anterograde movement of newly synthesized virus in CNS neurons actually has yet to be obtained. In efforts to investigate the latter, we will be greatly aided by viral strains that exhibit differences in their ability to move in an anterograde direction. We compared the anterograde axonal transport of three HSV strains (F strain, H129, and MacIntyre B) in the murine visual system. Equivalent titers of virus were injected intraocularly in BALB/c mice. From 2 ± 6 days after inoculation, segments of the infected optic pathway were harvested and Western blots using an anti-HSV polyclonal antibody performed. H129 traveled very rapidly towards the terminals (3 days post-inoculation). F strain spread more slowly than H129, but also reached terminal regions by 4 days. MacIntyre B accumulated only in the most proximal optic nerve, and was seen only very faintly in distal optic pathway after 5 days. Coincidentally, a single viral protein appeared to be greatly reduced in expression in MacIntyre B. Our results suggest that different viral strains display variability in their capacity to spread anterogradely, and that further comparison of these strains may reveal how virus engages the host cell transport machinery.

Research paper thumbnail of Biogenesis of Presynaptic Terminal Proteins

Journal of Neurochemistry, 1987

The delivery of proteins to the presynaptic terminals of guinea pig retinal ganglion cells by two... more The delivery of proteins to the presynaptic terminals of guinea pig retinal ganglion cells by two of the major components of axonal transport, and the subsequent persistence and turnover of those proteins were examined in this study. Ganglion cell proteins were radiolabeled by intravitreal injection of radiolabeled amino acids and radioactive axonally transported proteins were analyzed in synaptosomes prepared from the superior colliculi. This procedure allowed examination of presynaptic components of ganglion cell synapses without having to compensate for postsynaptic or other unidentified contaminants. Each of the two major axonal transport components supplies a large number of proteins to the presynaptic terminal, in relative quantities similar although not identical to those seen in the axon. Proteins conveyed by the fast component of axonal transport reached the terminals by 3 h after intraocular injection, peaked by 24 h, and were largely undetectable by I5 days. Slow component b proteins reached the terminals by 12 days, peaked around 2 I days. and persisted up to 63 days in the terminals. Proteins in both components demonstrated differential turnover relative to cotransported proteins once they reached the terminals. Differential turnover may account for change in relative concentration of a particular protein required to meet new functional demands on that protein once it enters the terminal.

Research paper thumbnail of Selective Alterations in Presynaptic Cytomatrix Protein Organization Induced by Calcium and Other Divalent Cations That Modulate Exocytosis

Journal of Neurochemistry, 1990

Rises in intracellular calcium cause several events of physiological significance, including the ... more Rises in intracellular calcium cause several events of physiological significance, including the regulated release of neuronal transmitters. In this study, the effects of divalent cations on the structural organization of cytomatrix in presynaptic terminals was examined. [35S]Methionine-radiolabeled guinea pig retinal ganglion cell cytomatrix proteins were axonally transported [in slow component b (SCb) of axonal transport] to the neuron terminals in the superior colliculus. When the peak of radiolabeled cytomatrix proteins reached the terminals, synaptosomes containing the radiolabeled cytomatrix proteins were prepared. Approximately 40% of each SCb protein was soluble after hypoosmotic lysis of the radiolabeled synaptosomes in the presence of divalent cation chelators. Lysis of synaptosomes in the presence of calcium ions over a range of concentrations, however, caused a dramatic decrease in solubility of the presynaptic SCb proteins. The cytoplasmic effects may result from a calcium-dependent condensation of cytoplasm around presynaptic terminal membrane systems. There are two major presynaptic SCb proteins (at 60 and 35 kDa), that exhibited exceptional behavior: they remained as soluble in the presence of calcium as under control conditions, suggesting that they were relatively unaffected by the mechanism causing the decrease in SCb protein solubility. Also examined were the effects of other alkaline earth and transition metal divalent cations on the presynaptic SCb proteins. Key Words: Presynaptic terminal-Axonal transport-Cytomatnx-Calcium-Divalent cations-Transmitter release. Garner J. A. Selective alterations in presynaptic cytomatrix protein organization induced by calcium and other divalent cations that modulate exocytosis.

Research paper thumbnail of Cytoplasmic matrix proteins in central nervous system presynaptic terminals: turnover and effects of osmotic lysis

Brain Research, 1990

Cytomatrix proteins, of primary functional importance in central nervous system neuron terminals,... more Cytomatrix proteins, of primary functional importance in central nervous system neuron terminals, are provided to their site of action in the terminal by axonal transport. Slow component b (SCb) of axonal transport has been proposed to be the biochemical counterpart of the moving cytoplasmic matrix, or cytomatrix, in axons. In the current study, axonally transported SCb proteins destined for neuron terminals were pulse-radiolabeled with [35S]methionine in guinea pig retinal ganglion cells. After SCb proteins reached the terminals in the superior collioili, synaptosomes were prepared to distinguish between SCb proteins in the preterminal axons and those of the presynaptic terminals, Study of the initial entry and turnover of individual SCb proteins in presynaptic terminals revealed different residence times of certain SCb proteins in comparison with their cohorts. Preliminary information about the structural relationships of the proteins comprising the presynaptic cytomatrix was obtained by examining the solubility of individual SCb proteins relative to other SCb proteins, or membranes from osmotically lysed terminals. Last, treatment of those radiolabeled synaptosomes with varying concentrations of salts was performed to determine possible effects on observed structural relationships.

Research paper thumbnail of Differential turnover of tubulin and neurofilament proteins in central nervous system neuron terminals

Brain Research, 1988

The transport of tubulin and neurofilament protein subunits from the preterminal axons of guinea ... more The transport of tubulin and neurofilament protein subunits from the preterminal axons of guinea pig retinal ganglion cells into their presynaptic terminals in the superior colliculus was examined. Newly synthesized tubulin and neurofilament proteins were radiolabeled with tritiated amino acids in the cell bodies and were allowed to be axonally transported through the optic axons and into the terminals in the superior colliculi. Superior colliculi were harvested at appropriate times, synaptosomes were prepared, and radiolabeled proteins were examined by gel electrophoresis and fluorography. Proteins in the radiolabeled synaptosomes were compared with those in the portion of the optic tract immediately proximal to the superior colliculus. Tubulin subunits entered the terminals by 100 days after intraocular labeling, and at least one isoform of tubulin appeared to persist as long as 400 days. Neurofilament proteins, despite the fact that they are axonally transported and delivered to the terminals in concert with the tubulin subunits, disappear rapidly upon entry into the terminals themselves.

Research paper thumbnail of Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals

Journal of Neurochemistry, Aug 1, 1996

Research paper thumbnail of Cohesive axonal transport of the slow component b complex of polypeptides

The Journal of Neuroscience the Official Journal of the Society For Neuroscience, 1983

The axonal transport of the diverse group of polypeptides characteristic of slow component b (SCb... more The axonal transport of the diverse group of polypeptides characteristic of slow component b (SCb), or Group IV, may be accomplished either by a bulk flow mechanism acting on each of the individual polypeptides or by their movement through the axoplasm as part of a physical complex or structural assembly. In order to determine if the SCb polypeptides travel as a cohesive unit (which would be consistent with the latter alternative above), we examined in detail the quantitative distribution of the individual SCb polypeptides in the guinea pig optic system at three different times after pulse radiolabeling the neuron cell bodies. Eighteen SCb polypeptides were selected as appropriate for analysis. Individual optic systems at the three select times after labeling were harvested, cut into small segments, and subjected to SDS-PAGE. Individual radioactive polypeptide bands were identified and located by fluorography, excised, and then quantified by scintillation counting.

Research paper thumbnail of Rapid Axonal Transport of the Neural Cell Adhesion Molecule

The neural cell adhesion molecule (NCAM) is a cell-surface glycoprotein that mediates cell-cell i... more The neural cell adhesion molecule (NCAM) is a cell-surface glycoprotein that mediates cell-cell interactions in the nervous system during development. In the present study, we demon- strate that NCAM is axonally transported in 3-d-old chick ret- inal ganglion cells and that it travels within the fast component of axonal transport (FC). Proteins were radiolabeled in retinal ganglion cell bodies after

Research paper thumbnail of Clathrin is axonally transported as part of slow component b: the microfilament complex

The Journal of Cell Biology, 1981

During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal el... more During axonal transport, membranes travel down axons at a rapid rate, whereas the cytoskeletal elements travel in either of two slow components, SCa (with tubulin and neurofilament protein) and SCb (with actin) . Clathrin, the highly ordered, structural coat protein of coated vesicles, has recently been shown to be able to interact in vitro with cytoskeletal proteins in addition to membranes. The present study examines whether clathrin travels preferentially with the membrane elements or the cytoskeletal elements when it is axonally transported. Guinea pig visual system was labeled with tritiated amino acids. Radioactive SDS-polyacrylamide gel electrophoresis profiles from the major components of transport were coelectrophoresed with clathrin . Only SCb had a band comigrating with clathrin . In addition, radioactive clathrin was purified from guinea pig brain containing only radioactive SCb polypeptides . Kinetic analysis of the putative clathrin band in SCb revealed that it travels entirely within the SCb wave . Thus we conclude that clathrin travels preferentially with the cytoskeletal proteins making up SCb, rather than with the membranes and membrane-associated proteins in the fast component.

Research paper thumbnail of Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes

Science, 1981

The proteins of the three major rate components of axonal transport in guinea pig retinal ganglio... more The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.

Research paper thumbnail of Factors that contribute to the transneuronal spread of herpes simplex virus

Journal of Neuroscience Research, 1997

In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread b... more In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread between neurons in the central nervous system (CNS) by distinctly different routes. The steps of viral infection and spread in a single neuron type and nearby glial cells in vivo have been determined for three different strains of HSV (F, H129, and McIntyre-B). The corneas of mice were inoculated with equivalent titers of the strains. Two to 5 days later, the animals were killed. The spread of viral proteins within trigeminal cells was examined using immuno- and electron microscopy and Western blots with anti-HSV polyclonal antiserum. McIntyre-B virus infection resulted in fewer labeled ganglion cells, possibly as a result of reduced viral production in the corneal epithelium or trigeminal ganglion cells. Although the McIntyre-B strain was at least as, if not more efficient, at retrograde transport than the other strains, the amount of McIntyre-B virus that was transported in the trigeminal roots in an anterograde direction was significantly less than the other strains. Uptake by ganglionic satellite cells was qualitatively similar for the three strains, but maturation and release of virus from satellite cells to other neurons were reduced in the McIntyre-B strain. These characteristics may account for the preferential retrograde transneuronal spread of McIntyre-B strain.

Research paper thumbnail of Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals

Journal of Neurochemistry, 2002

Research paper thumbnail of Protein changes during anterograde-to-retrograde conversion of axonally transported vesicles

Brain Research, 1989

In the axon tip, cell biological mechanisms convert anterogradely transported membranous elements... more In the axon tip, cell biological mechanisms convert anterogradely transported membranous elements. To study the effects of these anterograde-to-retrograde (A-R) converting mechanisms on the electrophoretic behaviour of vesicle proteins, we compared the proteins of anterograde vesicles (before A-R conversion at the axon tip) with those of retrograde vesicles (after A-R conversion at the axon tip). The proteins in transported vesicles were pulse-labeled with [35S]methionine, and the radiolabeled vesicles were concentrated by ligating the axons-anterograde vesicles accumulate selectively on the proximal side of the ligature and retrograde vesicles accumulate on the distal side of the ligature. Analyses of vesicle proteins by polyacrylamide gel electrophoresis (SDS-PAGE) show that most of the anterograde proteins were also present in the retrograde vesicles. In addition to the conservation of these anterograde proteins in the retrograde vesicles, there were also many differences: some anterograde proteins were diminished in the retrograde vesicles, other anterograde proteins were absent from the retrograde vesicles, and the retrograde vesicles contained some new protein bands that were not present in the anterograde vesicles. These results indicate that A-R converting mechanisms modify membranous vesicle proteins in the axon tip. We propose that some of these post-translational protein modifications change the directional code on the vesicle surfaces, thereby converting anterograde membranous elements into retrograde membranous elements.

Research paper thumbnail of Characterization of spherical amyloid protein from a prolactin-producing pituitary adenoma

Acta Neuropathologica, 1997

Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abun... more Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abundant spherical amyloid; however, the origin of the amyloid has not been established. In this report, a PRL-producing pituitary adenoma composed almost entirely of spherical amyloid was analyzed biochemically. The tumor was removed surgically from a 56-year-old man. Immunohistochemical analysis revealed that residual tumor cells were strongly positive for PRL, while the spherical amyloid was not. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of approximately 4 kDa associated with the amyloid, which was not present in a nonamyloid producing prolactinoma. The 4-kDa band is similar in size to other known amyloidogenic peptides. Immunoblot analysis of the tumor material using polyclonal anti-human PRL antibodies revealed a small amount of normal-sized PRL; however, the abundant 4-kDa band was nonimmunoreactive. Amino acid sequencing showed that this peptide represents the first 34 amino acids of the intact PRL protein with a predicted size of 4313 Da. The presence of a small amount of normal-sized PRL in this tumor, as well as elevated circulating levels of PRL implies that intact PRL is being abnormally processed in the formation of spherical amyloid.

Research paper thumbnail of Axonal transport of neuronal antigens characteristic of subpopulations of central nervous system (CNS) neurons

Metabolic Brain Disease, 1989

Monoclonal antibodies (MAbs) are useful for the identification of nervous system antigens localiz... more Monoclonal antibodies (MAbs) are useful for the identification of nervous system antigens localized to neuronal subpopulations. We have examined the transport of the corresponding antigens of four such MAbs in guinea pig retinal ganglion-cell axons. Determination of the axonal transport rate of radiolabeled antigens allowed their assignment to one of the three major anterograde axonal transport rate components, each of which is through to convey a subcellular structural system in the axon. Antigens identified by three of the MAbs were found to be transported in slow component b of axonal transport, the component thought to convey the cytoplasmic matrix, and an antigen identified by the fourth MAb was found in slow component a, similarly thought to contain the linear cytoskeletal elements. Assignment of these antigens to the different rate components suggests that they may be associated with a particular structural system in neurons. Additionally, in cases where more than one nervous system cell type may express a particular antigen, the identity of the neuronal form of the antigen has been confirmed by its axonal transport. The roles that these antigens may play in the nervous system during normal axonal function and during neuropathogenesis can now be further examined.

Research paper thumbnail of Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in Lacrimal Acini

Journal of Virology, 2006

The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves f... more The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to ␣ v integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.

Research paper thumbnail of Factors that contribute to the transneuronal spread of herpes simplex virus

Journal of Neuroscience Research, 1997

In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread b... more In viral encephalitis and retinal necrosis, different herpes simplex virus (HSV) strains spread between neurons in the central nervous system (CNS) by distinctly different routes. The steps of viral infection and spread in a single neuron type and nearby glial cells in vivo have been determined for three different strains of HSV (F, H129, and McIntyre-B). The corneas of mice were inoculated with equivalent titers of the strains. Two to 5 days later, the animals were killed. The spread of viral proteins within trigeminal cells was examined using immuno- and electron microscopy and Western blots with anti-HSV polyclonal antiserum. McIntyre-B virus infection resulted in fewer labeled ganglion cells, possibly as a result of reduced viral production in the corneal epithelium or trigeminal ganglion cells. Although the McIntyre-B strain was at least as, if not more efficient, at retrograde transport than the other strains, the amount of McIntyre-B virus that was transported in the trigeminal roots in an anterograde direction was significantly less than the other strains. Uptake by ganglionic satellite cells was qualitatively similar for the three strains, but maturation and release of virus from satellite cells to other neurons were reduced in the McIntyre-B strain. These characteristics may account for the preferential retrograde transneuronal spread of McIntyre-B strain.

Research paper thumbnail of Retinal Pigment Epithelial Cells Secrete and Respond to Hepatocyte Growth Factor

Biochemical and Biophysical Research Communications, 1998

gen (10). The receptor for HGF is the membrane-span-Hepatocyte growth factor (HGF) is normally ex... more gen (10). The receptor for HGF is the membrane-span-Hepatocyte growth factor (HGF) is normally exning tyrosine kinase c-Met (11,. Through its activapressed by mesenchymal cells while its receptor, ction of c-Met, HGF has been found to be critical for Met, is expressed in epithelial cells. Since HGF is critiepithelial-mesenchymal interactions with widespread cally involved in epithelial-mesenchyme interactions effects in embryogenesis and regeneration (reviewed in and the retinal pigment epithelium (RPE) is present .

Research paper thumbnail of Differential anterograde transport of HSV type 1 viral strains in the murine optic pathway

Active anterograde transport of herpes simplex virus Type 1 (HSV-1) in neurons is often assumed b... more Active anterograde transport of herpes simplex virus Type 1 (HSV-1) in neurons is often assumed based on early appearance of infection in postsynaptic target cells of a primary infected cell, and is further logically inferred by good evidence of microtubule-motor based mechanisms of retrograde transport. However, direct evidence of mechanisms of anterograde movement of newly synthesized virus in CNS neurons actually has yet to be obtained. In efforts to investigate the latter, we will be greatly aided by viral strains that exhibit differences in their ability to move in an anterograde direction. We compared the anterograde axonal transport of three HSV strains (F strain, H129, and MacIntyre B) in the murine visual system. Equivalent titers of virus were injected intraocularly in BALB/c mice. From 2 ± 6 days after inoculation, segments of the infected optic pathway were harvested and Western blots using an anti-HSV polyclonal antibody performed. H129 traveled very rapidly towards the terminals (3 days post-inoculation). F strain spread more slowly than H129, but also reached terminal regions by 4 days. MacIntyre B accumulated only in the most proximal optic nerve, and was seen only very faintly in distal optic pathway after 5 days. Coincidentally, a single viral protein appeared to be greatly reduced in expression in MacIntyre B. Our results suggest that different viral strains display variability in their capacity to spread anterogradely, and that further comparison of these strains may reveal how virus engages the host cell transport machinery.