Identification of glyoxalase-I as a protein marker in a mouse model of extremes in trait anxiety.

For >15 generations, CD1 mice have been selectively and bidirectionally bred for either high-anxiety-related behavior (HAB-M) or low-anxiety-related behavior (LAB-M) on the elevated plus-maze. Independent of gender, HAB-M were more anxious than LAB-M animals in a variety of additional tests, including those reflecting risk assessment behaviors and ultrasound vocalization, with unselected CD1 "normal" control (NAB-M) and cross-mated (CM-M) mice displaying intermediate behavioral scores in most cases. Furthermore, in both the forced-swim and tail-suspension tests, LAB-M animals showed lower scores of immobility than did HAB-M and NAB-M animals, indicative of a reduced depression-like behavior.

Mining the human cerebrospinal fluid proteome by immunodepletion and shotgun mass spectrometry.

We have analyzed the proteome of human cerebrospinal fluid with the help of shotgun mass spectrometry. In order to identify low-abundant proteins in these fluids, we have found it necessary to remove the abundant protein components from the mixture. Immunodepletion of the abundant proteins has allowed us to identify more than 100 proteins in cerebrospinal fluids from a patient suffering from normal pressure hydrocephalus.

Binding of NCK to SOS and activation of ras-dependent gene expression.

NCK, an SH2- and SH3 domain-containing protein, becomes phosphorylated and associated with tyrosine kinase receptors upon growth factor stimulation. The sequence of NCK suggests that NCK functions as a linker between receptors and a downstream signaling molecule. To determine if NCK can mediate growth factor-stimulated responses, we measured the ability of NCK to activate the fos promoter.

cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor.

Using immobilized PDGF receptor as an affinity reagent, we purified an 85 kd protein (p85) from cell lysates and we cloned its cDNA. The protein contains an SH3 domain and two SH2 domains that are homologous to domains found in several receptor-associated enzymes. Recombinant p85 overexpressed in mammalian cells inhibited the binding of endogenous p85 and a 110 kd protein to the receptor and also blocked the association of PI3-kinase activity with the receptor.

Agents that increase cAMP accumulation block endothelial c-sis induction by thrombin and transforming growth factor-beta.

Endothelial cells express the product of the c-sis gene, which encodes the B-chain of platelet-derived growth factor (PDGF). Through local production of growth factors such as PDGF in vascular sites, endothelial cells may stimulate proliferation of adjacent cells through a paracrine mechanism. Previously, we have shown that the expression of c-sis mRNA and release of growth factor activity by human renal endothelial cells is induced by thrombin.

Biosynthetic and glycosylation studies of cell surface platelet-derived growth factor receptors.

The cell surface pool of metabolically labeled platelet-derived growth factor (PDGF) receptors in BALB/c3T3 fibroblasts was studied using an antiphosphotyrosine antibody. Exposure of intact cells to PDGF stimulates autophosphorylation of surface PDGF receptors and allowed immunoaffinity purification of only PDGF-activated receptors. Pulse-chase experiments demonstrated appearance of newly synthesized receptors in a surface activatable pool within 30-45 min of synthesis.

Thrombin stimulates c-sis gene expression in microvascular endothelial cells.

We have determined whether expression of the c-sis gene product, platelet-derived growth factor (PDGF), is regulated in cultured renal microvascular endothelial cells by factors to which vascular endothelial cells may be exposed at sites of perivascular cellular proliferation. Thrombin exposure increased endothelial cell levels of c-sis message by 3-5-fold over a time course that peaked at 4 h after exposure. Similarly, thrombin-exposed microvascular endothelial cells released increased amounts of PDGF activity into their media.

nu 1, a Mr 43,000 component of postsynaptic membranes, is a protein kinase.

Acetylcholine receptor-enriched membranes from the electric organ of Torpedo californica show a major band at Mr 43,000 on NaDodSO4/polyacrylamide gels. This band is composed of three polypeptides: nu 1, nu 2, and nu 3. Polypeptide nu 1 has been found to be localized exclusively at the innervated face of the electrocyte and at the neuromuscular junction in rat muscle.

Identification of a molecular weight 43,000 protein kinase in acetylcholine receptor-enriched membranes.

A photoaffinity ATP ligand is used to identify the protein kinase present in acetylcholine receptor-enriched membranes from Torpedo californica. Incubation of these membranes with 8-azido-[alpha-32P]ATP and subsequent irradiation with UV light resulted in covalent labeling of a major band of Mr 43,000. Alkali-stripped membranes that show a selective reduction in the Mr 43,000 polypeptide also show a corresponding reduction in incorporation of photoaffinity label.

Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes.

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor.

Phosphorylation of the membrane-bound acetylcholine receptor: inhibition by diphenylhydantoin.

Purified postsynaptic membranes can be used as a model system to study the regulation of synaptic membrane proteins. These membranes contain protein kinase activity that phosphorylates the acetylcholine receptor (AChR). We find that diphenylhydantoin (DPH) interacts with these membranes to inhibit phosphorylation of the membrane-bound AChR.

The relationship of choline acetyltransferase activity at the neuromuscular junction to changes in muscle mass and function.

1. The role of muscle mass and function in the regulation of choline acetyltransferase activity at the neuromuscular junction has been investigated in the rat.2.