Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system.

Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C-terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1.

N5-(1-Imino-3-butenyl)-L-ornithine. A neuronal isoform selective mechanism-based inactivator of nitric oxide synthase.

Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion of L-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest.

Characterization of 5-oxo-L-prolinase in normal and tumor tissues of humans and rats: a potential new target for biochemical modulation of glutathione.

5-Oxo-L-prolinase (5-OPase) is an enzyme of the gamma-glutamyl cycle involved in the synthesis and metabolism of glutathione (GSH), which is known to protect cells from the cytotoxic effects of chemotherapy and radiation. Previous studies on rats have shown that administration of the cysteine prodrug L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-proline analogue that is metabolized by 5-OPase, preferentially increases the GSH content of normal tissues while paradoxically decreasing it in the tumor and results in an enhanced in vivo tumor response to the anticancer drug melphalan. These observations initiated the present study of 5-OPase in experimental models and clinical specimens to investigate the potential role of this enzyme in the selective modulation of GSH in normal and tumor tissues.

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis.

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M.

Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis.

Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C.

A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GP) anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker.

Measurements of total plasma nitrite and nitrate in pediatric patients with the systemic inflammatory response syndrome.

Objectives: The systemic inflammatory response syndrome (SIRS) is typified by the presence of fever, hemodynamic changes, and end organ dysfunction. Endothelial cell activation leads to overproduction of nitric oxide, which results in sustained vasodilation and hypotension. This study was undertaken to determine the sensitivity, specificity, and positive and negative predictive values of plasma nitrite/nitrate measurements in identifying patients with clinical characteristics of SIRS, as defined by criteria based on physician diagnosis.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system.

Design of nitric oxide synthase inhibitors and their use to reverse hypotension associated with cancer immunotherapy.

It is now just 10 years since it was first appreciated that NO is endogenously synthesized in mammals. In this period, two constitutive and one inducible isoform of NOS have been isolated, sequenced, and characterized with respect to their protein chemistry and catalytic mechanism. A wide variety of NOS inhibitors, most targeted to the arginine binding site in the oxygenase domain, have been synthesized and used to elucidate the physiological and pathophysiological roles of NO.

Characterization of the mechanisms of busulfan resistance in a human glioblastoma multiforme xenograft.

Busulfan is an alkylating agent commonly used in the treatment of chronic myelogenous leukemia and in combination with cyclophosphamide in preparation for allogeneic bone marrow transplantation. Serial treatment of a childhood high-grade glioma xenograft (D-456 MG) with busulfan resulted in a busulfan-resistant xenograft, D-456 MG(BR). Cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea was seen but not resistance to cyclophosphamide or CPT-11.

L-amino acid oxidase (LOX) modulation of melphalan activity against intracranial glioma.

These studies evaluated the efficacy of sequential pretreatment with L-amino acid oxidase (LOX) and LOX antiserum in the modulation of melphalan activity against intracranial glioma in athymic nude mice. LOX produced statistically significant (P < 0.01) depletion of the large neutral amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine in murine plasma at doses of 100 and 200 micrograms administered intravenously.

Synthesis, structure-activity relationships, and the effect of polyethylene glycol on inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus.

Substrate analog inhibitors of Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) were synthesized and screened for their suitability to map the active site region of the enzyme by protein crystallography. Analogs of the natural substrate phosphatidylinositol (PI) were designed to examine the importance of the lipid portion and the inositol phosphate head group for binding to the enzyme. The synthetic compounds contained pentyl, hexyl, or hexanoyl and octyl lipid chains at the sn-1 and sn-2 positions of the glycerol backbone and phosphonoinositol, phosphonic acid, methyl phosphonate, phosphatidic acid, or methyl phosphate at the sn-3 position.

Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl(alpha 1-->6)-D-myo-inositol, an essential fragment of GPI anchors.

Numerous proteins on the external surface of the plasma membrane are anchored by glycosylated derivatives of phosphatidylinositol (GPI), rather than by hydrophobic amino acids embedded in the phospholipid bilayer. These GPI anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) to release a water-soluble protein with an exposed glycosylinositol moiety and diacylglycerol, which remains in the membrane. We have previously determined the crystal structure of Bacillus cereus PI-PLC, the enzyme which is widely used to release GPI-anchored proteins from membranes, as free enzyme and also in complex with myo-inositol [Heinz, D.

Technetium-99m sestamibi uptake in human breast carcinoma cell lines displaying glutathione-associated drug-resistance.

An in vitro study was designed to evaluate the uptake of sestamibi (MIBI) in P-glycoprotein (Pgp) and glutathione-associated (GSH) multidrug-resistant (MDR) cell lines. MIBI uptake was studied in various human breast carcinoma cell lines, i.e.

Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform.

Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc.

Characterization by electron paramagnetic resonance of the interactions of L-arginine and L-thiocitrulline with the heme cofactor region of nitric oxide synthase.

Nitric oxide synthase (NOS) catalyzes sequential NADPH- and O2-dependent mono-oxygenase reactions converting L-arginine to N omega-hydroxy-L-arginine and N omega-hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer, and that cofactor is thought to mediate both partial reactions. Here we show by electron paramagnetic resonance spectroscopy that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin.

Pulmonary oxygen toxicity in mice is characterized by alterations in ascorbate redox status.

Pulmonary oxygen toxicity results from disruption of the usual antioxidant defenses of the body. We therefore investigated whether mice that suffer from oxygen toxicity show significant alterations in the redox status of ascorbate, an important antioxidant, as reflected by changes in the relative amounts of its oxidized and reduced forms. Mice were exposed to air or hyperoxia (> 97% O2, 760 mmHg).

Photoelectron imaging of cells: photoconductivity extends the range of applicability.

Photoelectron imaging is a sensitive surface technique in which photons are used to excite electron emission. This novel method has been applied successfully in studies of relatively flat cultured cells, viruses, and protein-DNA complexes. However, rounded-up cell types such as tumor cells frequently are more difficult to image.

Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI-specific phospholipase C (PI-PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI-PLC from Bacillus cereus (EC 3.

An extracellular inositol phospholipid-specific phospholipase C is released by cultured Swiss 3T3 cells.

A phosphatidylinositol-cleaving phospholipase C (PI-PLC) activity is released into the extracellular environment by intact Swiss 3T3 cell cultures. The activity is found in both serum-containing and serum-free defined culture medium. The cells remain attached and intact by Trypan Blue exclusion and lactate dehydrogenase assays.

NG-methyl-L-arginine, an inhibitor of nitric oxide synthase, reverses interleukin-2-induced hypotension.

Objective: To evaluate the role of NG-methyl-L-arginine as a modulator of the hyperdynamic shock induced by the administration of interleukin-2 (IL-2).

Design: A prospective, pilot clinical study.

Setting: Intensive care unit of a tertiary care center.

Characterization of the effects of two new arginine/citrulline analogues on constitutive and inducible nitric oxide synthases in rat aorta.

1. New potent inhibitors of nitric oxide synthase (NOS) may be useful in the treatment of septic shock, a disorder characterized by a vascular hyporeactivity to catecholamines caused by an overproduction of nitric oxide (NO-). We examined the effects of L-thiocitrulline (L-TC) and S-methyl-L-thiocitrulline (L-SMTC), novel NOS inhibitors, on the constitutive and inducible NOS in rat aorta and compared those effects with inhibition due to NG-methyl-L-arginine (L-NMA).