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Biomedical subjects

N Nash

Publications and source records attributed to N Nash.

11 recordsLinked to original sources
PubMed2000

Dopamine D(5) receptor immunolocalization in rat and monkey brain.

Dopamine D(5) receptor localization has been difficult because even the most specific ligands cannot distinguish between molecular subtypes of the D(1)-like receptor subfamily. Antifusion protein rabbit polyclonal antibodies directed against the C-terminus of human D(5) receptor were therefore developed for immunolocalization of the D(5) receptor protein in brain. The antibodies were characterized by immunoblot analysis and immunoprecipitation and used for light microscopic immunocytochemistry in rat and monkey brain. Affinity purified D(5) antibodies were specific for D(5) fusion protein as well as cloned and native D(5) receptor on Western blots, and D(5) antisera specifically immunoprecipitated solubilized, cloned D(5) receptor. Regional distribution of D(5) receptor immunoreactivity was consistent across species and correlated well with D(5) mRNA distribution previously reported in monkey brain. Immunoreactivity was widespread and tended to label perikarya and proximal dendrites of neurons in cerebral cortex, basal ganglia, basal forebrain, hippocampus, diencephalon, brainstem, and cerebellum. Neuropil was immunoreactive in olfactory bulb, islands of Calleja, cerebral cortex, superior colliculus, and molecular layer of cerebellum. The distribution of D(5) in brain was clearly different from that of other dopamine receptor subtypes, including D(1), the other member of the D(1)-like receptor subfamily. This unique distribution corroborates the idea that the D(5) receptor subtype has a distinct role in dopamine neurotransmission.

Animals↗
PubMed1999

Overexpression and functional characterization of kinin receptors reveal subtype-specific phosphorylation.

G protein-coupled receptors such as the receptors for bradykinin are present in low copy numbers in most natural cells. To overcome the problems associated with the analysis of these receptors at the protein level, we used highly efficient expression systems such as the baculovirus/insect cell system. However, the structural and functional statuses of recombinant receptors have often remained elusive. We have expressed the two types of human kinin receptors, B1 and B2, in Sf9 cells. Both receptors are found on the surface of infected cells where they display the same pharmacological profiles as their cognate receptors of native cells. The functional analysis of kinin receptors coupled to the intracellular signaling pathways of Sf9 cells revealed differential patterns of ligand-induced phosphorylation for the two kinin receptors. The B1 receptor failed to undergo ligand-induced phosphorylation. However the B2 receptor showed selective phosphorylation of a minor 38 kDa band and lack of phosphorylation of a dominant 33 kDa band, indicating that only a fraction of the receptor protein is functionally linked to the kinase pathway. A striking discrepancy between the number of binding sites and the amount of receptor protein per cell (molar ratio of 1:20 to 1:1000) indicated that a significant portion of kinin receptors is associated with the intracellular compartments of Sf9 cells. Pulse-chase and immunoprecipitation experiments demonstrated that the heterogeneity of recombinant receptors is not due to proteolytic processing but likely reflects incomplete or lacking N-glycosylation. We conclude that the baculovirus/Sf9 system is suitable for the recombinant expression and functional analysis of kinin receptors though limitations of the system have to be considered.

Animals↗
PubMed1998

Cloning of m-calpain 80 kD subunit from the axonal degeneration-resistant WLD(S) mouse mutant.

Calpains are calcium-activated cysteine proteases that are involved in cellular degradation in models of neurodegeneration. Calpains are the effectors of cytoskeletal disruption during axonal degeneration, a pathological feature of many neurological disorders. The WLD(S) mouse mutant is resistant to axonal degeneration and demonstrates prolonged survival of the cytoskeleton after nerve injury. To investigate the possibility that a mutation in calpain or abnormalities in calpain protein expression is responsible for the resistance to axonal degeneration seen in the WLD(S) mouse mutant, we 1) cloned and sequenced the large subunit of the high calcium-requiring form of calpain (m-calpain) from nervous system tissues of WLD(S) and from wild-type C57BL/6 mice, and 2) generated polyclonal m-calpain antibodies for comparison of relative protein levels by Western blot. We found our sequence for mouse m-calpain to be almost identical to another recently published mouse sequence, and the wild-type and WLD(S) sequences to be identical. Our fusion protein and peptide polyclonal antibodies were specific for the 80 kD subunit and recognized appropriate protein bands from pure m-calpain, fusion protein, and in tissue. There was no apparent difference in m-calpain expression in nerve or spinal cord in noninjured adult animals. These data suggest that a defect in m-calpain 80 kD subunit does not likely underlie the WLD(S) phenotype, but raise questions about other subunits of calpain and possibly other proteases.

Amino Acid Sequence↗
PubMed1995

Cloning and transient expression of genes encoding the human alpha 4 and beta 2 neuronal nicotinic acetylcholine receptor (nAChR) subunits.

Partial cDNA clones generated by RT-PCR were used as probes to clone the cDNAs encoding the human alpha 4 and beta 2 neuronal nicotinic acetylcholine receptor (nAChR) subunits. The 2.1-kb alpha 4 cDNA shows 84 and 76% identity to the rat and chicken cDNA sequences, respectively. The deduced amino-acid sequence shares 89 and 84% similarity, respectively, with the corresponding rat and chicken proteins, with most of the divergence occurring in the cytoplasmic domain. The 1721-nucleotide beta 2 sequence was identical to the human beta 2 sequence previously reported. Transfection of the alpha 4 and beta 2 clones into HEK293 cells resulted in the formation of binding sites that display high affinity towards [3H] cytisine, a characteristic of the alpha 4 beta 2 subtype produced in vivo.

Amino Acid Sequence↗
PubMed1994

Localization of neuronal and glial glutamate transporters.

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.

Amino Acid Sequence↗
PubMed1989

Vectors permitting visual monitoring of simple transposition events.

The construction and use of two novel transposon(Tn)-delivery vectors is described. These vectors carry Inc.W or Inc.N broad-host-range transfer functions cloned next to the narrow-host-range replicon of pBR329. The host specificities of pSLX10 and pSLX23 both complement and extend the host specificities of existing Tn delivery vectors. Plasmids pSLX10 and pSLX23 were shown to transfer at high frequency in intergeneric matings. The lux genes which are present on each vector permit the visual monitoring of transconjugants which have retained a Tn element, but are devoid of plasmid molecules. pSLX10 and pLSX23 were efficiently used to generate a range of auxotrophic mutants in various strains of Pseudomonas as well as to clone genes from Serratia liquefaciens. These vectors may have general applicability to identify and clone genes in a wide range of Gram-negative bacteria.

Bacterial Proteins↗
PubMed1988

A novel vector allowing the expression of genes in a wide range of gram-negative bacteria.

The construction and use of a novel vector allowing the expression of genes in a wide range of Gram-negative bacteria is described. The vector utilizes the regulatory region from IS50. The 70-bp promoter region was isolated from one of the terminal inverted repeats of Tn5 by creating EcoRI and Sa/I or PstI restriction sites by in vitro mutagenesis. This 70-bp region was shown to direct the expression of cat and lacZ genes in different bacterial genera including Alcaligenes, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens. Different strains containing the cat gene behind the regulatory elements of IS50 were able to tolerate high concentrations (300 micrograms/ml) of chloramphenicol in the medium. The 70-bp promoter region was cloned into a broad-host-range plasmid behind multiple cloning sites to create pAV10, which has unique restriction sites for BamHI, KpnI, SstI, and XbaI. Genes cloned into pAV10 can be expressed in a variety of Gram-negative bacteria.

DNA Transposable Elements↗
PubMed1988

Toxic shock syndrome due to an infected human bite.

We report a case of nonmenstrual toxic shock syndrome. A 21-year-old man presented with severe pain in the right hand after an altercation in which he sustained a bite from a person. Physical examination revealed a small abscess on the right proximal interphalangeal joint with edema in the fourth and fifth digits and tenderness of the flexor sheath. The patient was hypotensive with diffuse macular erythematous skin. He responded to surgical drainage and antibiotics.

Adult↗