The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role

Protostellar Disk Evolution Over Million-Year Timescales with a Prescription for Magnetized Turbulence

Magnetorotational instability (MRI) is the most promising mechanism behind accretion in low-mass protostellar disks. Here we present the first analysis of the global structure and evolution of non-ideal MRI-driven T-Tauri disks on million-year timescales. We accomplish this in a 1+1D simulation by calculating magnetic diffusivities and utilizing turbulence activity criteria to determine thermal structure and accretion rate without resorting to a 3-D magnetohydrodynamical (MHD) simulation. Our major findings are as follows. First, even for modest surface densities of just a few times the minimum-mass solar nebula, the dead zone encompasses the giant planet-forming region, preserving any compositional gradients. Second, the surface density of the active layer is nearly constant in time at roughly 10 g/cm2, which we use to derive a simple prescription for viscous heating in MRI-active disks for those who wish to avoid detailed MHD computations. Furthermore, unlike a standard disk with constant-alpha viscosity, the disk midplane does not cool off over time, though the surface cools as the star evolves along the Hayashi track. The ice line is firmly in the terrestrial planet-forming region throughout disk evolution and can move either inward or outward with time, depending on whether pileups form near the star. Finally, steady-state mass transport is a poor description of flow through an MRI-active disk. We caution that MRI activity is sensitive to many parameters, including stellar X-ray flux, grain size, gas/small grain mass ratio and magnetic field strength, and we have not performed an exhaustive parameter study here.Comment: Accepted for publication in Astrophysical Journal. 19 pages, including 8 figure

The Effects of Nerve Growth Factor on Adult and Aged Dorsal Root Ganglion Neurons Maintained in Primary Culture

Nerve growth factor (NGF) is, a prototype neurotrophic factor, well documented for its biochemical character, distribution and functions in embryonic and neonatal animals. However, only very tentative evidence exists for its effects on mature neurons, and results are controversial. Studying NGF by using mature neurons should be a significant and attractive field, not only for its clinical value in nerve regeneration by adults, but also for understanding the mechanisms of aging. Studies in vitro present a comparatively simple environment for analysis of the effective factors. Dorsal root ganglia (DRGs), containing an enormous heterogeneity of sensory neuronal types, are a useful model in understanding the nervous system. Hence, in the present study, mouse DRG neurons were maintained in vitro supplemented with NGF V5 cultures without NGF or with anti-NGF present, to examine any NGF effects on survival, morphological phenotype, neuropeptide expressions and neurite outgrowth (equivalent to regeneration in vitro). Comparison of adult (6 months) and aged (2 years) animals was made throughout the present study. In the survival study, a range of NGF (25-200ng/ml) was added to adult neurons in co-cultures, and neuronal survival monitored for 14 days in vitro (div). 100ng/ml NGF effectively maintained survival, compared with cultures without exogenous NGF. Aged as well as adult neurons were cultured [with non-neuronal cells (NNCs) present] without exogenous NGF or with 100ng/ml NGF added for up to 29div. NGF enhanced the survival of both adult and aged neurons (P<0.005 by ANOVA). To exclude possible endogenous NGF from NNCs, or any mediation of an NGF effect in co-culture by NNCs , a neuron- enriched culture system was also used. In these neuronal loss was avoided during cell preparation and well dispersed neurons were obtained. 100ng/ml NGF and/or 1:100 anti-NGF were re-examined in the modified neuron-enriched cultures for 9div and an enhanced survival of both adult and aged neuron persisted, although this effect was lower than in co-cultures. In general, about 20% of DRG neurons were NGF-dependent for their survival in adult and aged cultures. By size analysis of over 6500 neurons, intermediate-sized neurons (24-33mum in diameter) were predominant for both adult and aged in co-culture in the presence of NGF. The immunocytochemistiy (ICC) study using the avidin-biotin- peroxidase complex (ABC) method revealed that higher proportions of SP- immunoreactive (ir), CGRP-ir and NPY-ir aged, as well as adult, neurons were present in enriched cultures supplemented with 10 or 100ng/ml NGF than in cultures without NGF added (P<0.05-0.01 by ANOVA and t-tests), whereas SOM-ir adult and aged neurons showed little difference in cultures without or with NGF added. By counting at the peak day (9div) of SP expression, enhancement of the SP-ir subset in the presence of vs absence of NGF was similar for adult or aged neurons; the proportion of the SP-ir subset was not reduced following aging. Hence the SP-ir subset, together with the subsets of CGRP-ir and NPY-ir, were considered as NGF-dependent survival neurons in adult, and even aged mouse DRGs. Enhancement of the SP-ir proportion was more rapid in aged neuronal cultures supplemented with NGF than in cultures without exogenous NGF. Also, the staining intensities for CGRP and NPY were greater in both adult and aged cultures with NGF added than those in cultures without NGF. In addition to maintaining survival, NGF affected the mature neuronal phenotypes. Scanning election microscopy demonstrated that in the presence of NGF, abundant microvilli projections were distributed on the neuronal surface; in contrast, neurons were deformed and lost their smooth appearance, if 1:100 anti-NGF was present in enriched cultures. In the study of neurite geometry in enriched culture, major neurite length (maximal extension of a neurite), entire neurite length (lengths of major neurite plus all its branches), total neurite length (lengths of all neurite outgrowth from each neuron), soma size, neurite number (for each neuron) and branch number (per neurite) were selected as parameters. Tracing was carried out blindly at 1, 3, 6 and 9div by computer image analysis and a digitizing tablet. Neurites and neurons from at least 5 mice for each adult or aged cultures were measured directly from culture or from photomicrograph montages and data were statistically tested by ANOVA. The results demonstrated that NGF enhanced neurite outgrowth (P<0.0005) at 6 and 9div for both adult and aged neurons Total neurite lengths of adult and aged neurons were distinct in the presence of NGF; neurite length was predominantly enhanced by adult neurons, whereas increased branch lengths were evident for aged neurons. Neurites predominantly extended from intermediate- and large-sized neurons. In summary, aged neurons showed comparatively lower abilities for survival and functional peptide expression compared to adult neurons in vitro, but continued to be responsive to exogenous NGF. In addition, reconstruction of neurites by aged and adult neurons in vitro was demonstrated in the present study

The role of neurotrophin-3 in primary sensory neurons

Neurotrophin-3 (NT-3) is a member of the nerve growth factor family of neurotrophins. These molecules regulate aspects of sensory neuron survival, proliferation, phenotype, regeneration, and nociception. NT-3 presumably acts upon binding to its preferred receptors, trkC and p75, but much of the literature concerning its role involves embryological studies; little is known regarding its role in the adult, aside from its part in proprioception. Here, the effect of a 7 day intrathecal infusion of NT-3 on the phenotype of intact or axotomized adult rat dorsal root ganglion (DRG) neurons was examined. Serial sections were processed for in situ hybridization and computer-assisted image analysis was undertaken to characterize the NT-3 responsive subpopulation and to compare relative levels of mRNA for neurotrophin receptors, neuropeptides, cytoskeletal elements, injury- and regeneration-associated molecules, and other markers in individual neurons. 125I-NGF was utilized to determine the influence of NT-3 on high-affinity NGF binding site densities. Data show that ~ 40% of neurons coexpress trkC and p75 mRNAs; some of these cells also exhibit transcripts for trkA, NFM, Ta1 a-tubulin, a-CGRP, SP, galanin, NPY, GAP-43, cjun, and SNAP-25 - establishing the presence of potentially functionally significant micropopulations within the trkC-positive subset. Following injury, levels of many biochemical markers are altered in a positive or negative fashion. In all cases described here, if the marker colocalizes with trkC, post-trauma treatment with NT-3 allows for a return towards normal message levels, suggesting a role for NT-3 in the maintenance of normal adult phenotype in these cells. In the intact state, NT-3 effects a reduction in trkA, high-affinity NGF binding sites, and SP levels, within non-trkC neurons. The importance of these markers in nociception suggests a role for NT-3 in analgesia. But, in addition, NT3 also reduces SNAP-25 mRNA levels in otherwise normal trkC-expressing cells, which might negatively alter their functioning. Together, these data indicate that multiple subsets of mature DRG neurons are responsive to NT-3, not all of which express trkC, and not all of which respond to the neurotrophin in the same manner: these factors must be taken into account when considering therapeutic applications for NT-3

Studies on Herpes Simplex Virus Type 1 Latency in Tissue Culture Cells

After primary infection, herpes simplex virus type 1 (HSV-1) remains latent in neurons of the host. To facilitate studies on HSV-1 latency, models of latency have been developed in tissue culture. The HSV-1 protein Vmw65 is an essential structural component of the virus tegument. Following fusion of the virus envelope with the cell membrane Vmw65 is released into the cell and forms a complex with cellular factors which bind to TAATGARAT (where R is a purine) elements upstream of the viral immediate early (IE) genes. Once the complex has bound to DNA, the acidic carboxy-terminal domain of Vmw65 stimulates transcription by recruiting components of the RNA polymerase II transcription initiation complex to the promoter. The mutant ml814 has a4 amino acid insertion in Vmw65 which disrupts its ability to form the transactivation complex, as a consequence levels of IE transcription are reduced. During infection of cells with in1814 at low multiplicity of infection (MOI), only a minor proportion of infecting viruses undergo replication, whereas the majority of viral genomes enter a quiescent state. These quiescent genomes can be reactivated by expression within the cell of the HSV-1 encoded protein Vmwl 10 and probably by other herpesvirus proteins functionally equivalent to Vmw110. At high MOI in1814 replicates as efficiently as wild type virus. To perform structural and functional studies on the quiescent ml814 genomes, it is necessary to permit infection of cultured cells at high MOI. An in vitro latency system has been developed by D.R.S. Jamieson and C.M. Preston using two modifications which further reduce the expression of viral IE proteins, thereby enabling infection of human foetal lung (HFL) cells with in1814 at high MOI without extensive cell destruction. Firstly, the regulatory region controlling transcription of the gene encoding the IE protein Vmwl 10 has been replaced by the Moloney murine leukaemia virus (MMLV) enhancer. Since the MMLV enhancer is a relatively inefficient promoter in HFL cells the resulting mutant, named ml820, exhibits a phenotype equivalent to that of a Vmwl 10 deletion mutant of in1814 when titrated on HFL cells. Another modification is pretreatment of the cells with human interferon alpha (IFNalpha). Treatment of cells with IFNalpha results in an antiviral state against HSV-1 which has been attributed to reduced viral IE gene expression. Previous studies demonstrated that infection of IFNalpha-treated HFL cells with ml820 at high MOI results in high efficiency retention of viral genomes in a transcriptionally repressed state. The purpose of the study reported here was to extend previous analysis by D.R.S. Jamieson on the structure of the ml820 genome during latency in vitro, and to characterise early events in the establishment of latency. Initial experiments confirmed previous data showing that the ml820 genome is nonlinear in the in vitro latency system, as was reported to occur in neurons in vivo. Quantification of Southern blots revealed that after an initial input MOI of 120 particles of in820 per cell, approximately 84% of the total nuclear viral DNA was nonlinear and that on average each cell retained approximately 12 viral genomes. Upon reactivation of latent in820 by superinfection of latently infected HFL cells in the presence of an inhibitor of DNA replication, the reactivated genomes remained nonlinear, demonstrating that a change from a nonlinear to a linear form is not a requirement for reactivation. The configuration of the linear genomes also remained unchanged upon reactivation. During in vitro latency, the nonlinear viral genomes were more sensitive to nucleases than the linear genomes. Linearity and resistance to nucleases is indicative of genomes which are not uncoated and are thus unable to circularise. Comparison of rates of uncoating after infection of BHK, HeLa, CV-1 and HFL cells revealed that uncoating appeared significantly slower in HFL cells than in the other cell types tested. Latent HSV-1 DNA in the brainstems of mice is associated with nucleosomes in a chromatin structure. Previous studies examining the structure of in820 in the in vitro latency system showed that the thymidine kinaase (TK) gene is not bound by nucleosomes with regular spacing. The study reported here also showed the absence of a regular chromatin structure on the TK gene and furthermore that the nature of the inhibitor of DNA replication used to prevent the spread of nonlatent virus was not a significant variable. Fascinatingly, the LAT/Vmw110 encoding region, the only region transcriptionally active during latency in vivo, was in a regular chromatin structure in the in vitro latency system. However, when the LAT / Vmwl 10 encoding region and the TK gene were examined in a parallel experiment, both regions exhibited a possible regular nucleosomal structure suggesting that a regular nucleosomal structure exists on the entire genome and is not confined to the LAT/Vmw110 region. Thus the previous inability to detect chromatin on the TK gene may have been caused by differences in the arrangement of nucleosome between experiments or experimental differences in the ability to detect nucleosome-associated DNA. (Abstract shortened by ProQuest.)