Oxidized phospholipids derived from ozone-treated lung surfactant extract reduce macrophage and epithelial cell viability

Ozone is known to be a highly toxic gas present in the urban air which exerts its effect on pulmonary tissue through its facile chemical reactions with target molecules in the airway. One of the first barriers encountered by ozone is epithelial lining fluid which contains pulmonary surfactant rich in glycerophosphocholine lipids. The reaction of ozone with calf lung surfactant extract was found to result in the production of 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho) as an expected product of the ozonolysis of abundant unsaturated phospholipids containing unsaturated fatty acyl groups with a double bond at carbon-9. This oxidized phospholipid was identified as a biologically active product in that it reduced elicited macrophage viability by necrosis with an ED(50) of 6 microM. Further studies of the biological activity of 16:0a/9-al-GPCho revealed that concentrations from 100 to 200 nM initiated apoptosis in pulmonary epithelial-like A549 cells as assessed by TUNEL staining, nuclear size, and caspase-3 activation with loss of viability indicated by reduction of mitochondrial dehydrogenase activity. The release of IL-8, a neutrophil chemokine, from A549 cells was also stimulated by 50-100 nM 16:0a/9-al-GPCho. Exposure of calf lung surfactant to low levels of ozone (62.5, 125, and 250 ppb) for various time periods from 2 to 48 h in a feedback-regulated ozone exposure chamber resulted in a dose- and time-dependent increase in the formation of 16:0a/9-al-GPCho as measured by a specific and sensitive LC/MS/MS assay. The quantity of this biologically active chain-shortened glycerophosphocholine lipid generated even at 125 ppb ozone for 2-4 h (50-100 nM) was consistent with this product mediating the toxic effects of ozone on cells in contact with surfactant.     

Applicability of a modified Edman procedure for measurement of protein adducts: mechanisms of formation and degradation of phenylthiohydantoins

Adducts to N-terminal valine residues in hemoglobin (Hb) are used for monitoring in vivo doses of electrophiles and are quantitated by means of a modified Edman procedure, the "N-alkyl Edman procedure". In the reaction with pentafluorophenyl isothiocyanate, N-alkylated valines cyclize and detach from the protein as pentafluorophenylthiohydantoins (PFPTHs) much more efficiently than do unsubstituted N-terminal valine residues. The mechanisms of this reaction, and of possible degradation reactions, have been studied with model compounds using phenyl- and pentafluorophenyl isothiocyanate. The rapid cyclization to N-alkylvaline-PTHs occurs as a consequence of the influence of substituents on ring formation. This facilitated cyclization favors a direct attack by the thiocarbamoyl nitrogen atom on valine-C-1, and is also observed to occur slowly at unsubstituted N-terminal valines. Such cyclization is favored in protic solvents. Under alkaline conditions and in the presence of air, hydrolytic and oxidative processes give rise to degradation products. The PTH derivatives of N-alkylvaline are less apt to undergo such reactions than are the corresponding derivatives of unsubstituted valine. We conclude that the presence of an N-substituent exerts a greater influence on the cyclization process than the structure of the amino acid or of the Edman reagent. For adducts of different structures, the method has broad applicability, for which the limits, however, are not yet explored. The knowledge from the studies is valid not only for the N-alkyl Edman procedure, but also, to some extent, for the classical Edman degradation reaction. The oxidative side reaction gave rise to the invention of a novel synthesis route for insertion of nucleophiles at carbon-5 in thiohydantoins. The present investigation provides a basis for the N-alkyl Edman procedure, facilitating new toxicological applications.     

Limited and optimal sampling strategies for etoposide and etoposide catechol in children with leukemia

Etoposide is used to treat childhood malignancies, and its plasma pharmacokinetics have been related to pharmacodynamic endpoints. Limiting the number of samples should facilitate the assessment of etoposide pharmacokinetics in children. We compared limited sampling strategies using multiple linear regression of plasma concentrations and clearance with Bayesian methods of estimating clearance using compartmental pharmacokinetic models. Optimal sampling times were estimated in the multiple linear regression method by determining the combination of two samples which maximized the correlation coefficient, and in the Bayesian estimation approach by minimizing the variance in estimates of clearance. Clearance estimates were compared to the actual clearances from Monte Carlo-simulated data and predicted clearances estimated using all available plasma concentrations in clinical data from children with acute lymphoblastic leukemia. Multiple linear regression poorly predicted clearance (mean bias 8.3%, precision 17.5%), but improved if plasma concentrations were logarithmically transformed (mean bias 1.4%, precision 12.5%). Bayesian estimation methods with optimal samples gave the best overall prediction (mean bias 2.5%, precision 6.8%) and also performed better than regression methods for abnormally high or low clearances. We conclude that Bayesian estimation with limited sampling gives the best estimates of etoposide clearance.     

The utility of mandatory depression screening of dementia patients in nursing homes

OBJECTIVE: Current methods for enhancing the recognition and treatment of depression in nursing home patients have been unsuccessful. This study examines the process, outcome, and impact of instituting a mandatory depression screening program for depressed dementia patients in nursing homes. METHOD: The experimental and comparison groups each consisted of two nursing homes of 519 and 363 patients, respectively. Two of the experimental group and one of the comparison group homes were more traditionally staffed facilities; one of the comparison group homes had an enriched staff of psychologists. The Cornell Scale for Depression in Dementia was administered to the residents with dementia. In the experimental group, the patients who scored > or =5 were referred for psychiatric assessment. RESULTS: In the experimental group, 100% of the referred dementia patients who met screening criteria for depression were seen by a psychiatrist. This resulted in a significant increase in the percentage of individuals given antidepressants. This was greater than the percentage of patients receiving antidepressants in the "typical" comparison group home but not the "staff-enriched" comparison group home. White patients were significantly more likely to receive antidepressants; however, screening significantly increased the proportion of depressed nonwhites receiving antidepressants. At the 12-week follow-up, there was a significant difference in scores between patients receiving antidepressants in each group. CONCLUSIONS: Mandatory depression screening can significantly increase the proportion of depressed dementia patients receiving antidepressants, lead to dose adjustments, diminish potential ethnic biases in treatment, and affect the depressive symptoms of treated individuals.     

Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C

Protein kinase C (PKC)-dependent regulation of voltage-gated Ca (Ca(v); with alpha(1)beta1Balpha2/delta subunits) channel 2.3 was investigated using phorbol 12-myristate 13-acetate (PMA), or by M(1) muscarinic receptor activation in Xenopus oocytes. The inward Ca(2+)-current with Ba(2+) (I(Ba)) as the charge carrier was potentiated by PMA or acetyl-beta-methylcholine (MCh). The inactivating [I(inact)] and non-inactivating [I(noninact)] components of I(Ba) and the time constant of inactivation tau(inact) were all increased by MCh or PMA. This may be a PKC-dependent action since the effect of MCh and PMA was blocked by Ro-31-8425 or beta-pseudosubstrate. MCh effect was blocked by atropine, guanosine-5'-O-(2-thiodiphosphate) trilithium (GDPbetaS) or U-73122. The effect of MCh but not PMA was blocked by the inhibition of inositol-1,4,5-trisphosphate (IP3) receptors, intracellular Ca(2+) ([Ca(2+)](i)) or the translocation of conventional PKC (cPKC) with heparin, BAPTA and betaC2.4, respectively. While a lower concentration (25 nM) of Ro-31-8425 blocked MCh, a higher concentration (500 nM) of Ro-31-8425 was required to block PMA action. This differential susceptibility of MCh and PMA to heparin, BAPTA, betaC2.4 or Ro-31-8425 is suggestive of the involvement of Ca(2+)-dependent cPKC in MCh action, whereas cPKC and Ca(2+)-independent novel PKC (nPKC) in PMA action. PMA led to additional increase in I(Ba) that was already potentiated by preadministered MCh (1 or 10 microM), leading to the suggestion that differential phosphorylation sites for cPKC and nPKC may be present in the alpha(1)2.3 subunit of Ca(v) 2.3 channels.     

Stimulation of spinal 5-HT(2A/2C) receptors potentiates the capsaicin-induced in vivo release of substance P-like immunoreactivity in the rat dorsal horn

Stimulation of spinal serotonin (5-HT)_2A/2C receptors has previously been reported to lead to either a pro-nociceptive or an anti-nociceptive response. Behavioral data have indicated that the pro-nociceptive effect is related to the release of substance P (SP). The aim of this in vivo microdialysis study was to investigate if stimulation of spinal 5-HT_2A/2C receptors by the selective agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) induces spontaneous or capsaicin-evoked increase in the release of SP-like immunoreactivity (SP-LI) in the rat dorsal horn. A dose of capsaicin (25 μM in the perfusion medium administered for 30 min), which did not lead to a significant release of SP-LI on its own, induced a significant increase of greater than 4-fold of the SP-LI level following spinal application of 50 nmol DOI. Higher (500 nmol) or lower (5 nmol) doses of DOI failed to induce a similar effect. In rats with a peripheral inflammation, induced by carrageenan, capsaicin (25 μM) induced a non-significant increase of SP-LI. A significant 8-fold increase of the SP-LI level was detected following administration of 50 nmol DOI in combination with capsaicin. The effect of DOI, which was completely prevented by co-administration of the 5-HT_2A receptor antagonist ketanserin in control animals without peripheral inflammation, was only partly blocked in animals with carrageenan induced peripheral inflammation. In conclusion, stimulation of 5-HT_2A/2C receptors facilitates the capsaicin-evoked release of SP-LI in the dorsal horn in both animals with and without carrageenan-induced unilateral inflammation. The observation that the highest dose of DOI failed to induce SP-LI release may be due to an inhibitory postsynaptic action at this dose.