Biomedical subjects

Richard W Burry

Publications and source records attributed to Richard W Burry.

8 recordsLinked to original sources

PubMed2006

Role of the human T-cell leukemia virus type 1 PTAP motif in Gag targeting and particle release.

Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent.

Amino Acid Motifs↗

PubMed2005

Ultrasound-mediated gene transfer into neuronal cells.

A new field of gene transfer is emerging as a simple, effective means to drive the expression foreign genes in cells: ultrasound-mediated gene transfer or sonoporation. We report here that sonoporation is an effective means of gene transfer for cultured neurons, a cell type that has been difficult to transfect. Neuronal cell types that are effectively sonoporated include chick retinal neurons, chick dorsal forebrain, chick optic tectum, PC12 cells, rat cerebellar neurons and mouse hippocampal neurons. Depending on the type of cell and conditions of sonoporation the transfection efficacy was as high as 20%. Sonoporation of plasmid DNA was effective for cells adherent to a substrate and for free-floating cells that were freshly dissociated. In the free-floating preparations, between 60 and 95% of the cells that were transfected were neuronal, as much as 90% higher than that observed for other methods of gene transfer including adenovirus and lipid-based transfection methods. We conclude that sonoporation is a simple, effective and inexpensive means by which to preferentially transfect DNA into neuronal cells.

Animals↗

PubMed2005

Open access.

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Online Systems↗

PubMed2004

Corticotropin releasing factor enhances survival of cultured GABAergic cerebellar neurons after exposure to a neurotoxin.

Corticotropin-releasing factor (CRF), in addition to its role as a hormone in the stress response, functions as a neuromodulator in the cerebellum, where it enhances both the spontaneous and amino acid induced firing rate of Purkinje cells. In the cerebellum, CRF and its two types of receptors (CRF-R(1) and CRF-R(2)) are present during cerebellar development at ages that precede the onset of afferent ingrowth and synaptogenesis, suggesting a distinct role during early cerebellar development. The present study was undertaken to determine whether CRF enhances the survival of cerebellar neurons, in particular GABAergic neurons. Primary cultures of cerebellar neurons obtained from embryonic day 18 mice were composed primarily, but not exclusively, of GABAergic neurons. Although CRF-R(1) is present in most neurons in this culture system, when CRF was added to the medium, no significant change in neuronal survival was observed when compared to control cultures. It is possible that a role for CRF is not seen in growth-promoting culture medium at the plating density chosen for this study and may only be evident when the cells have been exposed to conditions that reduce the likelihood of survival, such as exposure to neurotoxins such as AraC. We propose that, because AraC increases the number of cleaved caspase-3 positive cells, indicating apoptosis, it is possible that a CRF effect involves an inhibition of the apoptotic pathway. Cultures treated with AraC had a decrease in the total number of GABAergic neurons and an increase in apoptotic cells as measured with the apoptotic marker cleaved caspase-3. Co-treatment with CRF rescued many GABAergic neurons. It is interesting to note that apoptotic cells do not exhibit GABA or c-fos positive immunolabeling. Thus, these data support the concept that CRF plays a neuroprotective role in the survival of GABAergic cerebellar neurons in culture after exposure to a neurotoxin.

Animals↗

PubMed2004

GAP-43 mRNA in growth cones is associated with HuD and ribosomes.

The neuron-specific ELAV/Hu family member, HuD, interacts with and stabilizes GAP-43 mRNA in developing neurons, and leads to increased levels of GAP-43 protein. As GAP-43 protein is enriched in growth cones, it is of interest to determine if HuD and GAP-43 mRNA are associated in developing growth cones. HuD granules in growth cones are found in the central domain that is rich in microtubules and ribosomes, in the peripheral domain with its actin network, and in filopodia. This distribution of HuD granules in growth cones is dependent on actin filaments but not on microtubules. GAP-43 mRNA is localized in granules found in both the central and peripheral domains, but not in filopodia. Ribosomes were extensively colocalized with HuD and GAP-43 mRNA granules in the central domain, consistent with a role in the control of GAP-43 mRNA stability in the growth cone. Together, these results demonstrate that many of the components necessary for GAP-43 mRNA translation/stabilization are present within growth cones.

Animals↗

PubMed2003

Domain-dependent modulation of PDGFRbeta by ganglioside GM1.

The regulation of receptor tyrosine kinases (RTKs) is important in several cellular events, including proliferation, differentiation, and apoptosis. Gangliosides are sialic acid-containing glycosphingolipids that can regulate RTK activity. The addition of ganglioside GM1 to the medium of Swiss 3T3 fibroblasts inhibits both platelet-derived growth factor (PDGF)-mediated tyrosine phosphorylation of PDGF receptor beta (PDGFRbeta) and receptor-mediated endocytosis. However, GM1 did not affect PDGF-mediated receptor phosphorylation, neuritogenesis, or endocytosis in PC12 cells stably transfected with the gene for PDGFRbeta. The ability of GM1 to modulate PDGFRbeta in 3T3 cells but not in transfected PC12 cells indicates a cell context-dependent response. We hypothesized that this inhibition of PDGFRbeta by GM1 must map to one or more domains of the receptor. Thus, a chimeric receptor was created that possessed the extracellular and transmembrane domains of the nerve growth factor (NGF) receptor TrkA and the cytoplasmic domain of PDGFRbeta (TTbeta). In 3T3 cells transfected with the TTbeta construct, GM1 did not inhibit NGF-induced tyrosine phosphorylation of the chimeric receptor or of Erk1/2 in this cell line. GM1 still inhibited PDGF-mediated tyrosine phosphorylation of endogenous PDGFRbeta and of Erk1/2 in Swiss TTbeta cells. Thus, the cytoplasmic domain of PDGFRbeta is not required for GM1-dependent inhibition of PDGFRbeta in 3T3 cells. This suggests that the inhibition of PDGFRbeta by GM1 in Swiss 3T3 fibroblasts maps to either the extracellular and/or transmembrane domain of PDGFRbeta.

3T3 Cells↗

PubMed2002

Upregulation of FGF-2 in reactive spinal cord astrocytes following unilateral lumbar spinal nerve ligation.

Spinal nerve ligation results in dramatic changes in spinal cord primary C-afferent fibers, which include atrophy with an accompanied decrease in calcitonin-gene-related peptide (CGRP). These changes parallel the activation of astrocytes, which have been implicated in the ensuing neuropathic pain states. As part of an effort to elucidate the role of the downstream effectors of astrocyte reactivity in the context of allodynia, the expression of fibroblast growth factor-2 (FGF-2) was examined following tight ligation of L5 and L6 spinal nerves. FGF-2 is a pleiotropic cytokine that is synthesized and secreted by neurons and astrocytes. FGF-2 immunoreactivity was increased in ipsilateral dorsal horn reactive astrocytes at 1 and 3 weeks following nerve ligation. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) of laser-captured dorsal spinal cord sections revealed an increase in FGF-2 mRNA in the dorsal horn ipsilateral to nerve injury compared to contralateral and SHAM. Furthermore, an increase in FGF-2 mRNA in ispilateral dorsal root ganglia (DRG) was seen by in situ hybridization. These results demonstrate that, in response to ligation-induced injury of sensory neurons, FGF-2 is upregulated in both DRG neurons and in spinal cord astrocytes, suggesting neurotrophic functions of this growth factor following peripheral nerve lesion and possibly in astrocyte-related maintenance of pain states.

Animals↗

PubMed2002

Kainate-induced excitotoxicity is dependent upon extracellular potassium concentrations that regulate the activity of AMPA/KA type glutamate receptors.

In addition to well-known N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, recent studies suggest that non-NMDA type ionotropic glutamate receptors are also important mediators of excitotoxic neuronal death, and that their functional expression can be regulated by the cellular environment. In this study, we used cerebellar granule cells (CGCs) in culture to investigate kainate (KA)-induced excitotoxicity. Although previous reports indicated that KA induces apoptosis of CGCs in culture, no KA-induced excitotoxic cell death was observed in CGCs treated with KA when cells were maintained in high potassium media (24 mm K+). In contrast, when mature CGCs were shifted into low potassium media (3 mm K+), KA produced significant excitotoxicity. In electrophysiological studies, the KA-induced inward current density was significantly elevated in CGCs shifted into low K+ media compared with those maintained in high K+ media. Non-desensitizing aspects of KA currents observed in this study suggest that these responses were mediated by AMPA rather than KA receptors. In immunofluorescence studies, the surface expression of GluR1 subunits increased when mature CGCs were shifted into a low K+ environment. This study suggests that KA-induced excitotoxicity in mature CGCs is dependent upon the extracellular potassium concentration, which modulates functional expression and excitability of AMPA/KA receptors.

Animals↗