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 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.

Retinal pigment epithelial cells secrete and respond to hepatocyte growth factor.

Hepatocyte growth factor (HGF) is normally expressed by mesenchymal cells while its receptor, c-Met, is expressed in epithelial cells. Since HGF is critically involved in epithelial-mesenchyme interactions and the retinal pigment epithelium (RPE) is present at the interface between the retina and choroid, this study was initiated to determine whether the RPE expresses or responds to HGF in vitro. Cultured adult and fetal human RPE expressed mRNA for HGF and c-Met by RT-PCR. ELISA assay demonstrated the secretion of HGF into RPE culture supernatants. Tyrosine phosphorylation of c-Met was constitutively found in 72 hour RPE cultures and could be rapidly induced in serum-starved cells by concentrated RPE supernatants. HGF was mitogenic for cultured RPE (100 ng/ml.) and stimulated their chemotaxis (maximal response at 50 ng/ml). RPE are one of only a very limited number of epithelia that express both HGF and its receptor, suggesting the possibility of an autocrine action for this growth factor.

Factors that contribute to the transneuronal spread of herpes simplex virus.

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.

Characterization of spherical amyloid protein from a prolactin-producing pituitary adenoma.

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.

Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells.

Many Helicobacter pylori strains produce a cytotoxin (VacA) that induces vacuolation in epithelial cells. In this study, binding and internalization of the cytotoxin by HeLa or AGS (human gastric adenocarcinoma) cells were characterized by indirect fluorescence microscopy. Cells incubated with the cytotoxin at 4 degrees C displayed a uniform fluorescent plasma membrane signal. Preincubation of the cytotoxin with either rabbit antiserum to approximately 90-kDa H. pylori VacA or sera from H. pylori-infected persons inhibited its binding to cells and blocked its capacity to induce cytoplasmic vacuolation. Recombinant VacA fragments (approximately 34 and approximately 58 kDa), corresponding to two proteolytic cleavage products of approximately 90-kDa VacA, each bound to the plasma membrane of HeLa cells. Antiserum reactive with the approximately 58-kDa VacA fragment inhibited the binding of native H. pylori cytotoxin to cells and inhibited cytotoxin activity, whereas antiserum to the approximately 34-kDa fragment had no effect. When incubated with cells at 37 degrees C for > or = 3 h, the H. pylori cytotoxin localized intracellularly in a perinuclear location but did not localize within cytotoxin-induced vacuoles. When cells with previously bound cytotoxin were incubated with anticytotoxin serum at 4 degrees C and then shifted to 37 degrees C, vacuolation was completely inhibited. Bound cytotoxin became inaccessible to the neutralizing effects of antiserum after 60 to 120 min of incubation with cells at 37 degrees C. These data suggest a model in which (i) VacA binds to cells primarily via amino acid sequences in its 58-kDa fragment, (ii) VacA internalization occurs slowly in a temperature-dependent process, and (iii) VacA interacts with an intracellular target.

Type I brain hexokinase: axonal transport and membrane associations within central nervous system presynaptic terminals.

While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.

Analysis of genetic diversity in cytotoxin-producing and non-cytotoxin-producing Helicobacter pylori strains.

Analysis of 32 Helicobacter pylori strains indicated a strong association between the presence of the cagA gene and a specific type of vacA allele found predominantly in cytotoxin-producing strains (P < .001). To determine whether tox+/CagA+ and tox-/CagA- strains constituted two separate noncombining lineages, sequences of the H. pylori ureC gene, cysS homologue, and the intergenic region between cysS and vacA were determined for multiple strains. The mean levels of nucleotide identity in the three regions were 96.7% +/- 0.5%, 95.0% +/- 1.0%, and 89.0% +/- 2.9%, respectively. Multiple sequence alignments and dendrograms based on these three regions failed to identify two clonal populations of organisms for which cagA and vacA genotypes were markers. The presence of a 63- to 64-bp insertion in the cysS-vacA intergenic region was unrelated to the vacA genotype of the strains. These data suggest that recombination between Helicobacter genomes may occur in vivo.

High radiocesium levels in granite outcrop vegetation and reductions through time.

Levels of fallout radiocesium in vegetation were examined on three granite outcrops and a forested area in the Georgia piedmont during 1976-1980. Mean values averaged 4.3 times higher in three species collected on an outcrop than in the same species collected on clay soils in a nearby pine-hardwood forest. Levels in reindeer moss (Cladonia spp.) were significantly (p less than 0.01) lower in species that formed deep, entangled tufts with abundant, slender branches. Cesium levels decreased by as much as 90% from the mid-1960s but were virtually unchanged in the late 1970s. Dry-weight levels in mushrooms reached 18,470 Bq kg-1 (499.2 pCi g-1). Radiation levels in outcrop vegetation were higher than values found anywhere in the piedmont and were comparable to levels reported in plants from the sterile sandy soils of the temperate region coastal plains. These data fit well with earlier reported values and correlate well with the availability of atmospheric fallout. Outcrops can be used as sensitive environmental barometers for some contaminants.

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

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 colliculi, 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.

R56865, a potent new antiarrhythmic agent, effective during ischemia and reperfusion in the rat heart.

The antiarrhythmic effects of R56865 were characterized both in vivo and in vitro. Four groups (n = 12 per group) of anesthetized rats, subjected to 5- or 30-min coronary artery ligation and reperfusion, were studied: saline, dimethyl sulfoxide (DMSO) carrier, and R56865 (0.5 or 2 mg/kg) were administered as an intravenous (i.v.) bolus before ligation. After 5 min of ischemia, the incidences of reperfusion-induced ventricular tachycardia (VT) and fibrillation (VF), which were high in the saline (100 and 75%, respectively) and DMSO (100 and 82%, respectively) control groups, were abolished with both doses of R56865. With 30 min of ischemia, R56865 (2 mg/kg) significantly reduced the incidences of ischemia-induced VT and VF (from 100 and greater than 50% to 25 and 8%, respectively). For in vitro studies, five groups (n = 12 per group) of isolated rat hearts subjected to 10- or 30-min coronary ligation and reperfusion were studied: unmodified buffer and buffer containing DMSO or R56865 (10(-7), 10(-8), 10(-9) M). After 10 min of ischemia, R56865 (10(-7) M) decreased reperfusion-induced VT and VF (from 100 and 75% in buffer controls to 42 and 8%, respectively) when administered throughout the experiment. With 30 min of ischemia, R5685 (10(-7) M) reduced the incidences of ischemia-induced VT and VF (from 75 and 67% in the buffer controls to 25 and 25%, respectively). Although reperfusion after 30 min of ischemia did not induce VF in any of the groups studied, VT and other arrhythmias did occur and their incidences were reduced significantly by R56865. To investigate whether calcium overload might mediate the effects of R56865, hearts were perfused aerobically with a high-calcium/low-sodium medium. VT and VF occurred in 80% of control hearts; R56865 (10(-7) M) did not prevent these arrhythmias. In conclusion, R56865 exerts a potent effect against ischemia- and reperfusion-induced arrhythmias through a mechanism which appears to operate during ischemia.

Selective alterations in presynaptic cytomatrix protein organization induced by calcium and other divalent cations that modulate exocytosis.

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.

Axonal transport of neuronal antigens characteristic of subpopulations of central nervous system (CNS) neurons.

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.

Differential turnover of tubulin and neurofilament proteins in central nervous system neuron terminals.

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.

Biogenesis of presynaptic terminal proteins.

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 15 days. Slow component b proteins reached the terminals by 12 days, peaked around 21 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.

Rapid axonal transport of the neural cell adhesion molecule.

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 demonstrate that NCAM is axonally transported in 3-d-old chick retinal ganglion cells and that it travels within the fast component of axonal transport (FC). Proteins were radiolabeled in retinal ganglion cell bodies after intraocular injection of 35S-methionine. The presence of radiolabeled NCAM in the optic nerves and contralateral tecta was detected by specific immunoadsorption to a monoclonal antibody. Major radioactive polypeptide bands at relative mobilities of approximately 200,000, 150,000, and 120,000 Mr (after SDS-PAGE) were recognized by the anti-NCAM antibody. These bands comigrated in 1-dimensional gels with components of purified NCAM from chick brain. The 2 largest NCAM polypeptides (at 200,000 and 150,000 Mr) were found to be transported in this system, while the 120,000 Mr form was apparently not transported. The ratio and electrophoretic profiles of the 2 transported forms of NCAM remained similar in the retina, optic nerve, chiasm, tract, and tectum, suggesting that there is no interconversion of the 2 major polypeptides. The fraction of NCAM in the 35S-labeled FC proteins appears to be at least an order of magnitude less than in the plasma membrane, suggesting that the turnover rate of NCAM at this age is slower than for other membrane proteins of the CNS.

Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes.

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.