Adverse environmental conditions in the respiratory and medical ICU settings

Sleep deprivation and fragmentation occurring in the hospital setting may have a negative impact on the respiratory system by decreasing respiratory muscle function and ventilatory response to CO2. Sleep deprivation in a patient with respiratory failure may, therefore, impair recovery and weaning from mechanical ventilation. We postulate that light, sound, and interruption levels in a weaning unit are major factors resulting in sleep disorders and possibly circadian rhythm disruption. As an initial test of this hypothesis, we sampled interruption levels and continuously monitored light and sound levels for a minimum of seven consecutive days in a medical ICU, a multiple bed respiratory care unit (RCU) room, a single-bed RCU room, and a private room. Light levels in all areas maintained a day-night rhythm, with peak levels dependent on window orientation and shading. Peak sound levels were extremely high in all areas representing values significantly higher than those recommended by the Environmental Protection Agency as acceptable for a hospital environment. The number of sound peaks greater than 80 decibels, which may result in sleep arousals, was especially high in the intensive and respiratory care areas, but did show a day-night rhythm in all settings. Patient interruptions tended to be erratic, leaving little time for condensed sleep. We conclude that the potential for environmentally induced sleep disruption is high in all areas, but especially high in the intensive and respiratory care areas where the negative consequences may be the most severe.     

Role of maintenance treatment in opioid dependence

Methadone maintenance treatment (MMT) involves the daily administration of the oral opioid agonist methadone as a treatment for opioid dependence-a persistent disorder with a substantial risk of premature death. MMT improves health and reduces illicit heroin use, infectious-disease transmission, and overdose death. However, its effectiveness is compromised if low maintenance doses of methadone (<60 mg) are used and patients are pressured to become prematurely abstinent from methadone. Pregnancy and psychiatric comorbidity are not contraindications for MMT. As an alternative to MMT, other oral opioid agents (eg, naltrexone, buprenorphine) may increase patient choice and avoid some of the more unpleasant aspects of MMT. The public-health challenge for the future is to develop and continue to deliver safe and effective forms of opioid maintenance treatment to as many opioid-dependent individuals as can benefit from them.     

Characterization of the elastin binding domain in the cell-surface 25-kDa elastin-binding protein of staphylococcus aureus (EbpS)

Our previous studies have established that a cell-surface 25-kDa elastin-binding protein of Staphylococcus aureus (EbpS) mediates binding of this pathogen to the extracellular matrix protein elastin. Results from binding assays examining the activity of various EbpS fragments suggested that the elastin recognition domain is contained within the first 59 amino acids. In this report, we have used functional analyses with synthetic peptides and recombinant truncated forms of EbpS to localize the elastin binding domain to a 21-amino acid region contained within residues 14-34 of EbpS. Further evidence for the importance of this domain was obtained by demonstrating that the inhibitory activity of anti-EbpS antibodies on staphylococcal elastin binding was neutralized when these antibodies were pre-absorbed with a truncated recombinant EbpS construct containing residues 1-34. Overlapping synthetic peptides corresponding to EbpS residues 14-36 were then generated and tested for elastin binding activity to define further the elastin binding domain, and results from these studies showed that sequences spanning amino acids Gln14-Asp23, Asp17-Asp23, and Thr18-Glu34 inhibit binding of Staphylococcus aureus to elastin. Our analyses indicate that the hexameric sequence Thr18-Asn-Ser-His-Gln-Asp23 is the minimal sequence common to all active synthetic peptides, proteolytic fragments, and recombinant constructs of EbpS. Furthermore, substitution of Asp23 with Asn abrogated the blocking activity of the synthetic peptides, demonstrating the requirement for a charged amino acid at this location. The composite data indicate that staphylococcal elastin binding is mediated by a discrete domain defined by short peptide sequences in the amino-terminal extracellular region of EbpS.     

Lovastatin maintains nitric oxide--but not EDHF-mediated endothelium-dependent relaxation in the hypercholesterolemic rabbit carotid artery

The endothelium contributes to the regulation of vascular tone by producing nitric oxide (NO) and the endothelium-derived hyperpolarising factor (EDHF). In hypercholesterolemia, endothelium-dependent relaxation is impaired but can be restored by treatment with lovastatin (LOVAS). We investigated the effects of LOVAS on NO and EDHF-mediated relaxation. Rabbits were fed 1% cholesterol diet for 4 weeks and 0.5%) cholesterol for the following 12 weeks (CHOL-group). The LOVAS group additionally received 10 mg of lovastatin over the last 12-week period. Experiments were performed in carotid artery rings. Relaxant responses to acetylcholine (ACh) were recorded in the presence of indomethacin. Nitro-L-arginine (NOARG, 100 microM) and potassium chloride (KCl, 35 mM) were used to differentiate between NO- and EDHF-mediated relaxations. Cholesterol impaired ACh-induced relaxations and this effect was prevented by LOVAS (control 100+/-1%, CHOL 81+/-6%, LOVAS 98+/-1%). In the presence of NOARG, relaxations to ACh were not different between the LOVAS and CHOL groups (control 78+/-4%, CHOL 64+/-6%, LOVAS 64+/-5%). When KCl was used, ACh-induced relaxations were similar in the LOVAS and control group (control 75+/-5%, CHOL 49+/-6%, LOVAS 76+/-2%). In arteries treated with NOARG and KCl together, no relaxations were observed. Relaxations of arteries from the control group were not affected by 18 h preincubation with lovastatin (10 microM). Lovastatin selectively maintains nitric oxide-mediated endothelium-dependent relaxation in hypercholesterolemic rabbit carotid arteries.     

Structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the cholecystokinin receptor. Rapid analysis using fluorescent photoaffinity labeling probes and capillary electrophoresis

The molecular basis of ligand binding to receptors provides important insights for drug development. Here, we explore domains of the cholecystokinin (CCK) receptor that are critical for ligand binding, using a novel series of fluorescent photolabile probes, receptor proteolysis, and rapid high resolution separation of peptide fragments by capillary electrophoresis. Each probe incorporated the same fluorophore and a photolabile p-benzoylphenylalanine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-33) of CCK analogues representing full agonist, partial agonist, and antagonist of this receptor. Each was used to label the CCK receptor expressed on Chinese hamster ovary-CCKR cells, with the labeled domain then released by cyanogen bromide cleavage. Capillary electrophoresis with laser-induced fluorescence detection achieved an on-capillary mass sensitivity of 1.6 attomoles (10(-18) mol), with an excellent signal-to-noise ratio. Each of the biologically divergent, but structurally similar probes saturably and specifically labeled the same receptor domain, consistent with conservation of "docking" determinants. This had an apparent mass of 2.9 kDa, most consistent with the first extracellular loop domain. An additional probe having its site of covalent attachment in a different region of the probe (residue 29 of CCK-33) labeled a distinct receptor fragment with differential migration on capillary electrophoresis (third extracellular loop). Identification of the specific receptor residue(s) covalently linked to the amino-terminal probes must await further fragmentation and sequence analysis.     

Conditions for copackaging rous sarcoma virus and murine leukemia virus Gag proteins during retroviral budding

Rous sarcoma virus (RSV) and murine leukemia virus (MLV) are examples of distantly related retroviruses that normally do not encounter one another in nature. Their Gag proteins direct particle assembly at the plasma membrane but possess very little sequence similarity. As expected, coexpression of these two Gag proteins did not result in particles that contain both. However, when the N-terminal membrane-binding domain of each molecule was replaced with that of the Src oncoprotein, which is also targeted to the cytoplasmic face of the plasma membrane, efficient copackaging was observed in genetic complementation and coimmunoprecipitation assays. We hypothesize that the RSV and MLV Gag proteins normally use distinct locations on the plasma membrane for particle assembly but otherwise have assembly domains that are sufficiently similar in function (but not sequence) to allow heterologous interactions when these proteins are redirected to a common membrane location.     

Reaction of cytochrome P450 with cumene hydroperoxide: ESR spin-trapping evidence for the homolytic scission of the peroxide O-O bond by ferric cytochrome P450 1A2

ESR spin trapping was used to investigate the reaction of rabbit cytochrome P450 (P450) 1A2 with cumene hydroperoxide. Cumene hydroperoxide-derived peroxyl, alkoxyl, and carbon-centered radicals were formed and trapped during the reaction. The relative contributions of each radical adduct to the composite ESR spectrum were influenced by the concentration of the spin trap. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide (DMPO) concentrations was used to quantitate the contributions of each radical adduct to the composite ESR spectrum. The alkoxyl radical was the initial radical produced during the reaction. Experiments with 2-methyl-2-nitrosopropane identified the carbon-centered adducts as those of the methyl radical, hydroxymethyl radical, and a secondary carbon-centered radical. The reaction did not require NADPH-cytochrome P450 reductase or NADPH. It is concluded that the reaction involves the initial homolytic scission of the peroxide O-O bond to produce the cumoxyl radical. Methyl radicals were produced from the beta-scission of the cumoxyl radical. The peroxyl adduct was not observed in the absence of molecular oxygen. We conclude that the DMPO peroxyl radical adduct detected in the presence of oxygen was due to the methylperoxyl radical formed by the reaction of the methyl radical with oxygen. At a higher P450 concentration, a protein-derived radical adduct was also detected.     

Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro

The pharmacological profile of a novel dual inhibitor, tepoxalin and of its carboxylic acid metabolite on cyclooxygenase and lipoxygenase pathways was evaluated by in vitro incubation with synovial tissue. Tissue specimens obtained at surgery in rheumatoid arthritis (RA, n = 10) or osteoarthritis (OA, n = 11) patients were incubated. Tepoxalin (10(-7), 10(-6), 10(-5) M) decreased eicosanoid release calculated in % of tyrode control for OA: LTC4 to 71-33%, 6-keto-PGF1a to 37-20%, PGE2 to 29-6%. For RA: LTC4 to 56-22%, 6-keto-PGF1a to 43-22%, PGE2 to 57-32%. Similarly, its metabolite (10(-7), 10(-5)M) decreased release in OA: LTC4 to 99 and 60%, PGE2 to 42 and 20%, 6-keto-PGF1a to 54 and 25%. In RA:LTC4 to 81 and 45%, PGE2 to 61 and 30%, 6-keto-PGF1a to 46 and 18%. Significance (P < 0.05) was achieved for all but 1 group (LTC4 metabolite at 10(-7)M vs tyrode). In summary a marked and dose dependent decrease of LT and PG release was obtained when incubating the dual inhibitor tepoxalin and its active carboxylic acid metabolite with synovial tissue at doses expected to be reached in the joint during therapy.