Patients with detectable cocaethylene are more likely to require intensive care unit admission after trauma.

Cocaethylene (CE) is a toxic metabolite that is formed after simultaneous consumption of cocaine and ethanol. This potent stimulant is more toxic than cocaine and has a longer half-life. The deleterious hemodynamic and cardiovascular effects of CE have been proven in animal models.

Histoplasma capsulatum secreted gamma-glutamyltransferase reduces iron by generating an efficient ferric reductant.

The intracellular fungal pathogen Histoplasma capsulatum (Hc) resides in mammalian macrophages and causes respiratory and systemic disease. Iron limitation is an important host antimicrobial defence, and iron acquisition is critical for microbial pathogenesis. Hc displays several iron acquisition mechanisms, including secreted glutathione-dependent ferric reductase activity (GSH-FeR).

Cpc2/RACK1 is a ribosome-associated protein that promotes efficient translation in Schizosaccharomyces pombe.

Cpc2/RACK1 is a highly conserved WD domain protein found in all eucaryotes. Cpc2/RACK1 functions on mammalian signal transduction pathways most notably as an adaptor protein for the betaII protein kinase C isozyme. In single cell eucaryotes, Cpc2/RACK1 regulates growth, differentiation, and entry into G0 stationary phase.

Activation of JNK3 alpha 1 requires both MKK4 and MKK7: kinetic characterization of in vitro phosphorylated JNK3 alpha 1.

JNK3 alpha 1 is predominantly a neuronal specific MAP kinase that is believed to require, like all MAP kinases, both threonine and tyrosine phosphorylation for maximal enzyme activity. In this study we investigated the in vitro activation of JNK3 alpha 1 by MAP kinase kinase 4 (MKK4), MAP kinase kinase 7 (MKK7), and the combination of MKK4 + MKK7. Mass spectral analysis showed that MKK7 was capable of monophosphorylating JNK3 alpha 1 in vitro, whereas both MKK4 and MKK7 were required for bisphosphorylation and maximal enzyme activity.

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity.

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function.

Assay for IkappaB kinases using an in vivo biotinylated IkappaB protein substrate.

IkappaB kinases (IKK)-1 and -2 are related kinases that are induced by stimuli such as TNF or IL-1 to phosphorylate serines 32 and 36 of IkappaBalpha, the regulatory subunit of the transcription factor NF-kappaB. A procedure for an IKK protein kinase assay is described that uses an in vivo biotinylated IkappaB protein substrate, [gamma-(33)P]ATP, and capture onto a streptavidin membrane. Residues 1-54 of the IkappaBalpha substrate were expressed as a fusion with glutathione S-transferase (GST) and a short (22 amino acid) biotinylation sequence that allowed modification during bacterial expression.

Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR.

Discovery of a small molecule insulin mimetic with antidiabetic activity in mice.

Insulin elicits a spectrum of biological responses by binding to its cell surface receptor. In a screen for small molecules that activate the human insulin receptor tyrosine kinase, a nonpeptidyl fungal metabolite (L-783,281) was identified that acted as an insulin mimetic in several biochemical and cellular assays. The compound was selective for insulin receptor versus insulin-like growth factor I (IGFI) receptor and other receptor tyrosine kinases.

Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine.

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.

Active-site structure analysis of recombinant human inducible nitric oxide synthase using imidazole.

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of L-arginine to nitric oxide and L-citrulline. It is a specialized cytochrome P450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitive and reversible inhibitors versus L-arginine.

Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.

The high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS;NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage.

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

Objective: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo.

Methods: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence.

Inhibition of calcineurin by a novel FK-506-binding protein.

FK-506, a potent immunosuppressive drug, acts during the commitment phase of T-lymphocyte activation to block a subset of calcium-associated events necessary for transcription of certain early lymphokine genes. The drug binds to an abundant, cytosolic 11.8-kDa protein termed the FK-506-binding protein (FKBP12).

Electrospray ionization mass spectrometry as a mechanistic tool: mass of human leucocyte elastase and a beta-lactam-derived E-I complex.

We have utilized liquid chromatography electrospray ionization mass spectrometry (ESI-MS) to probe the nature of the covalent E-I complex of human leucocyte elastase (HLE) and a beta-lactam. The mass spectrum of HLE isozyme 4 displayed one major and two minor components with masses of 25,202, 25,043, and 24,522 Da, respectively. Isozyme 3 displayed three components, with masses of 25,180, 24,030, and 24,523 Da.

Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits.

Human leukotriene C4 (LTC4) synthase was purified > 25,000-fold to homogeneity from the monocytic leukemia cell line THP-1. Beginning with taurocholate-solubilized microsomal membranes, LTC4 synthase was chromatographically resolved by (i) anion exchange, (ii) affinity chromatography (through a resin of biotinylated LTC2 immobilized on streptavidin-agarose), and then (iii) gel filtration. The final preparation contained only an 18-kDa polypeptide.

Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme.

Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM).

Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex.

The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506.

Calmodulin is a subunit of nitric oxide synthase from macrophages.

A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist-induced elevation of Ca2+ and resultant binding of calmodulin (CaM).

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis.

Cloning and characterization of inducible nitric oxide synthase from mouse macrophages.

Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin.

Human plasma fibronectin. Demonstration of structural differences between the A- and B-chains in the III CS region.

Fibronectins are a class of cell-adhesion proteins produced from a single gene. The soluble plasma form is synthesized by hepatocytes and the insoluble cellular form by fibroblasts and other cell types. The proteins possess multiple binding domains for macromolecules including collagen, fibrin and heparin along with at least one cell-binding domain.

Microsequence analysis of peptides and proteins. IX. An improved, compact, automated instrument.

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent.

Brain capillary 46,000 dalton protein is cytoplasmic actin and is localized to endothelial plasma membrane.

The most abundant protein of the brain capillary, which makes up the blood-brain barrier (BBB) in vivo, is a protein that migrates at a molecular weight of approximately 46 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bovine brain capillary 46 kDa protein was purified by SDS-PAGE and Sephadex G-25 gel filtration. The purified protein migrated as a single band of molecular weight of approximately 42,000 Da on subsequent SDS-PAGE followed by silver staining.

Predominant low-molecular-weight proteins in isolated brain capillaries are histones.

Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins.