Femtosecond time-resolved guanine oxidation in acridine modified alanyl peptide nucleic acids.

Alanyl peptide nucleic acids have been designed to generate linear and rigid pairing complexes. Femtosecond time resolved electron transfer dynamics studies of alanyl-PNA double strands where both strands contain an intercalated 9-amino-6-chloro-2-methoxy-acridine in its protonated state reveal a strong similarity to nearest neighbor interstrand/intrastrand guanine oxidation in the corresponding B-DNA fragment. This observation implies that the combined influence of electronic couplings and energetic parameters, driving force and reorganization energy, on electron transfer dynamics is similar in both structures. With respect to the alanyl-PNA structure, this result is consistent with the notion of stacking distances in the nucleobase staple similar to the one in B-DNA and thus provides additional structural evidence for nucleobase stacking in alanyl-PNA double strands.

On the apparently anomalous distance dependence of charge-transfer rates in 9-amino-6-chloro-2-methoxyacridine-modified DNA.

From previous thermal and photoinduced charge-transfer reactions in duplex DNA there is accumulative evidence for an attenuation parameter beta of the distance dependence in the range 0.6-0.8 A(-1), with the exception of one specific system exhibiting beta = 1.5 A(-1) which is reinvestigated in this paper. Femtosecond to nanosecond time-resolved pump-probe spectroscopy has been used to follow photoinduced charge-shift dynamics in DNA duplexes containing a covalently appended, protonated 9-alkylamino-6-chloro-2-methoxyacridine chromophore. This acridine derivative (X+) resides in the DNA duplex at a specific abasic site, which is highly defined as reflected in the monoexponentiality of the kinetics. In the presence of only neighboring A:T base pairs, no charge transfer occurs within the excited-state lifetime (18 ns) of the chromophore. However, the presence of a guanine nucleobase as either a nearest neighbor or with one interspersed A:T base pair does result in fluorescence quenching. In the case of nearest neighbors, the intermediate radical state X* is formed within 4 ps and decays on the 30 ps time scale. Placing one A:T base pair between the X+ and guanine slows down the forward transfer rate by 3 orders of magnitude, corresponding to an apparent beta value of >2.0 A(-1). This dramatic decrease in the rate is due to a change in charge-transfer mechanism from a (nearly) activationless to a thermally activated regime in which the forward transfer is slower than the back transfer and the X* state is no longer observed. These observations indicate that the distance dependence of charge injection in the X+-labeled DNA duplex is not solely caused by a decrease in electronic couplings but also by a concomitant increase of the activation energy with increasing distance. This increase in activation energy may result from the loss of driving force due to excited-state relaxation competing with charge transfer, or reflect distance-dependent changes in the energetics, predominantly of the low-frequency reorganization energy in this charge-shift reaction, on purely electrostatic grounds. To test the hypothesis of distance-dependent activation energy, guanine has been replaced by 7-deazaguanine, its easier-to-oxidize purine analogue. In these duplexes, a similar change of charge-transfer mechanism is found. However, consistent with an a priori larger driving force this change occurs at a larger donor-acceptor separation than in the X+-guanine systems. Independent of the detailed contributions to the distance-dependent activation energy, this phenomenon illustrates the complex nature of experimental beta values.

Conformational gating of long distance electron transfer through wire-like bridges in donor-bridge-acceptor molecules.

A series of five donor-bridge-acceptor (DBA) molecules in which the donor is tetracene, the acceptor is pyromellitimide, and the bridge molecules are oligo-p-phenylenevinylenes (OPV) of increasing length has been shown to undergo electron transfer (ET) by means of two mechanisms. When the bridge is short, strongly distance dependent superexchange dynamics dominates, whereas when the bridge is longer, bridge-assisted hopping dynamics prevails. The latter mechanism results in relatively soft distance dependence for ET in which the OPV oligomers act effectively as molecular wires. We now report studies on the critical influence that bridge dynamics have on electron transfer through these oligomers. The temperature dependence of the charge separation (CS) rates in all five molecules does not appear to obey the predictions of standard ET theories based upon the Condon approximation. All five molecules show behavior consistent with CS being "gated" by torsional motion between the tetracene donor and the first bridge phenyl ring. This is based on the near equivalence of the CS activation energies measured for all five molecules with the frequency of a known vibrational mode in 5-phenyltetracene. In the molecule containing a trans-stilbene bridge, a competition occurs between the tetracene-phenyl torsional motion and one that occurs between the vinyl group and the phenyls linked to it. This results in complex temperature-dependent CS that exhibits both activated and negatively activated regimes. The charge recombination (CR) reactions within the molecules which have the two shortest bridges, namely phenyl and trans-stilbene, show a weaker dependence on these molecular motions. The three molecules with the longest bridges all display complex temperature dependencies in both their rates of CS and CR, most likely because of the complex torsional motions, which arise from the multiple phenyl-vinyl linkages. The data show that long-distance electron transfer and therefore wire-like behavior within conjugated bridge molecules depend critically on these low-frequency torsional motions. Molecular device designs that utilize such bridges will need to address these issues.

Bronchoalveolar lavage fluid granulomas in a case of severe sarcoidosis.

A case of pulmonary sarcoidosis is presented in which cytologic analysis of bronchoalveolar (BAL) fluid showed intact granulomas. The patient had severe alveolar inflammation and probable endobronchial sarcoidosis. Thus the granulomas in the BAL fluid probably reflect a high burden of alveolar wall granulomas and/or the removal of granulomas from proximal inflamed airways. This is the first reported case of granulomas in BAL fluid in sarcoidosis. Although an unusual finding, the recovery of BAL granulomas is not diagnostic for sarcoidosis and cannot substitute for the demonstration of granulomatous inflammation in lung tissue.

BAL fluid contains detectable superoxide dismutase 1 activity.

OBJECTIVE: This study determined which, if any, of the three superoxide dismutase (SOD) enzyme activities were detectable in BAL fluid (BALF). BACKGROUND: The identity and concentrations of antioxidant molecules in BALF have not been fully characterized. One important class of antioxidants is that of the SOD enzymes. METHODS: BALF from control nonsmokers (n=9), smokers (n=7), and asthmatic subjects (n=12) were examined for SOD activity by a modified pyrogallol auto-oxidation method. The particular SOD enzyme or enzymes responsible for any activity were identified based on activity inhibition data and gel filtration column chromatography patterns. RESULTS: SOD activity was detected in all samples. Unlike serum or some other fluids, in which the enzyme extracellular SOD accounts for virtually all SOD activity, the enzyme SOD 1 accounted for virtually all SOD activity. BALF SOD activities were lower for smokers than for control or asthmatic subjects (p<0.05). CONCLUSION: BALF SOD 1 activities can be measured as part of lung antioxidant studies. Data from a limited number of subjects suggested that smokers can have low BALF SOD values.

Analysis of the risks associated with calcium channel blockade: implications for the obstetrician-gynecologist.

Calcium channel antagonists are widely prescribed in obstetrics and gynecology for blood pressure control and tocolysis. Concerns have recently arisen regarding the safety of these agents. Several studies found that short-acting forms of calcium channel blockers were associated with increased cardiovascular mortality, malignancy, and gastrointestinal bleeding. A recent meta-analysis found a significant increase in the risk of mortality in patients treated with a short-acting form of nifedipine. Another subgroup analysis of an observational study of older hypertensive patients found a significantly increased risk of cancer and gastrointestinal hemorrhage in patients prescribed calcium channel blockers. Both in vitro and small human in vitro series have reported a potential for cardiac toxicity in pregnant women treated concomitantly with calcium channel blockers and magnesium sulfate. Until additional data are available, we suggest that when calcium channel blockers are used in obstetrics and gynecology, the long-acting variety be prescribed. Concurrent use of calcium channel blockers and magnesium sulfate should be undertaken cautiously because of the potential for synergistic depression of cardiac function.

Aminobenzoic acid compounds as HOCl traps for activated neutrophils.

This study was designed to develop traps for hypochlorous acid (HOCl) which could be used to detect HOCl in the microenvironment of activated neutrophils. Reagent HOCl was found to react with para-aminobenzoic acid (PABA) in aqueous solution to produce a predominant metabolite detectable by high performance liquid chromatography (HPLC). Mass spectroscopy and nuclear magnetic resonance identified this metabolite as the ring addition product 3-chloro PABA. The related compound para-aminosalicylic acid (PAS) was also metabolized by HOCl to 3-chloro PAS. The formation of the 3-chloro metabolite was specific for reactions involving HOCl, since several other oxidants in chloride buffer failed to produce the metabolite. Human blood neutrophils activated by phorbol myristate acetate or zymosan in the presence of PABA (or PAS) used their HOCl to produce large amounts of the 3-chloro metabolite. The formation of 3-chloro PABA was inhibited by azide, catalase, and taurine, which is consistent with the production of the metabolite by the neutrophil myeloperoxidase (MPO) pathway. The reaction of HOCl with PABA and PAS was relatively slow as shown by competitive reactions with endogenous antioxidants like taurine, methionine, and glutathione. This was confirmed in reactions involving PABA/PAS and reagent HOCl or HOCl generated by the MPO enzyme system. In these in vitro systems, glutathione and serum completely inhibited the formation of the 3-chloro metabolite. In contrast, activated neutrophils metabolized PABA/PAS to the 3-chloro metabolite even in the presence of 1% serum. These findings demonstrate that PABA and PAS are specific trapping agents for HOCl produced by neutrophils in complex biological conditions.

Acute eosinophilic pneumonia. A summary of 15 cases and review of the literature.

Idiopathic acute eosinophilic pneumonia (AEP) is an acute febrile illness that may be mistaken for an infectious pneumonia. Patients are often young and otherwise healthy. Clues to considering this disorder in a differential diagnosis include the acuity and severity of the clinical presentation and an initial chest X-ray with diffuse infiltrates, often interstitial, and the presence of Kerley B lines and/or evidence of pleural fluid. The diagnosis can be made through examination of bronchoalveolar lavage fluid in most cases, with careful exclusion of other similar eosinophilic lung disease. Although it can lead to life-threatening respiratory failure, AEP is easily treatable with corticosteroids. This disease has not been reported to recur in any patients to this point.

Eosinophilic pleural effusion: is it always nondiagnostic?

The presence of eosinophils in the pleural effusion is generally considered nondiagnostic. It usually indicates that the patient has had a previous thoracentesis and that air or blood has come in contact with the effusion. Idiopathic acute eosinophilic pneumonia is characterized by acute onset of pulmonary symptoms with hypoxemia, pulmonary infiltrates, eosinophils in bronchoalveolar lavage fluid, and prompt response to steroid therapy. We report a patient who presented with symptoms of acute pneumonia in which the presence of increased eosinophils in the pleural effusion indicated eosinophilic pneumonia.

Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils.

ontivation of neutrophils by phorbol-12-myristate-13-acetate (PMA) causes rapid production of superoxide radical (O2-), leading to the formation of additional reactive oxygen species, including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and possibly hydroxyl radical (.OH). These reactive oxygen species have been associated with the oxidation of some drugs. We investigated the metabolism of phenytoin (5,5-diphenylhydantoin) and the covalent binding of reactive intermediates to cellular macromolecules in activated neutrophils. In incubations with 100 microM phenytoin, PMA-stimulated neutrophils from six human subjects produced p-, m-, and o-isomers of 5-(hydroxyphenyl)-5-phenylhydantoin (HPPH) in a ratio of 1.0:2.1:2.8, respectively, as well as unidentified polar products. Analysis of cell pellets demonstrated that phenytoin was bioactivated to reactive intermediates that bound irreversibly to macromolecules in neutrophils. Glutathione, catalase, superoxide dismutase, azide, and indomethacin all diminished the metabolism of phenytoin and the covalent binding of its reactive intermediates. The iron-inactivating chelators desferrioxamine and diethylenetriaminepentaacetic acid had little or no effect on the metabolism of phenytoin by neutrophils, demonstrating that adventitious iron was not contributing via Fenton chemistry. In an .OH-generating system containing H2O2 and Fe2+ chelated with ADP, phenytoin was oxidized rapidly to unidentified polar products and to p-, m-, and o-HPPH (ratio 1.0:1.7:1.5, respectively). Reagent HOCl and human myeloperoxidase (MPO), in the presence of Cl- and H2O2, both formed the reactive dichlorophenytoin but no HPPH. However, no chlorinated phenytoin was detected in activated neutrophils, possibly because of its high reactivity. These findings, which demonstrated that activated neutrophils biotransform phenytoin in vitro to hydroxylated products and reactive intermediates that bind irreversibly to tissue macromolecules, are consistent with phenytoin hydroxylation by .OH generated by a transition metal-independent process, chlorination by HOCl generated by MPO, and possibly cooxidation by neutrophil hydroperoxidases. Neutrophils activated in vivo may similarly convert phenytoin to reactive intermediates, which could contribute to some of the previously unexplained adverse effects of the drug.

Paraaminosalicylate blocks that ileal injury induced by phorbol myristate acetate.

Activation of neutrophils with the release of oxidant radicals has been implicated in the pathogenesis of gut injury in inflammatory bowel disease (IBD). The pathogenesis of gut injury in the multiple organ dysfunction syndrome associated with acute lung injury, although less focal, appears to be similar. Paraaminosalicylate (PAS) has been shown to be effective in treating IBD, most likely because of its ability to scavenge oxidant radicals. The present study was therefore designed to test the hypothesis that PAS attenuates the gut injury typically seen during systemic neutrophil activation by phorbol myristate acetate (PMA). We assessed gut injury by measuring the concentration ratio of lymph to plasma protein (CL/CP) at steady-state lymph flows in autoperfused cat ileum preparations. As expected, the CL/CP increased in animals given PMA (15 micrograms/kb; n = 6) compared with control animals (n = 5) (0.205 +/- 0.033 versus 0.118 +/- 0.004; p = 0.04) 0.04) and were accompanied by morphologic alterations. In contrast, the intravenous administration of PAS (100 mg/kg) to animals prior to PMA infusion (n = 5) yielded a CL/CP value indistinguishable from that in control animals (0.113 +/- 0.017 versus 0.118 +/- 0.004). Additional in vitro studies suggested that the protective effects of PAS were not the result of altered neutrophil margination, chemotaxis, or oxidant burst. Although PAS appeared to protect the ileum from PMA-induced microvascular injury, it had no protective effects on the lungs.

Tumor necrosis factor increases the elastolytic potential of adherent neutrophils: a role for hypochlorous acid.

Neutrophils adhered to biologic surfaces exhibit proteolytic cleavage of surface proteins even in the presence of proteinase inhibitors. Such proteolysis is restricted to the pericellular space and appears to require the dual action of proteinases and reactive oxygen species. The present study was designed to investigate the mechanism by which tumor necrosis factor-alpha (TNF) stimulates neutrophil proteolysis. Tissue culture wells were coated with insoluble 3H-labeled elastin substrate. Human blood neutrophils (0.5 to 2.0 x 10(6) cells/ml/well) were incubated in the coated wells for 4 to 18 h at 37 degrees C in the presence of varying concentrations of serum or purified alpha 1-antitrypsin (A1AT). TNF (0 to 1,000 U/ml) was also present in the incubations. Elastin degradation was determined as soluble 3H-elastin fragments released into the supernatants. As previously reported, cells (no TNF) exhibited spontaneous elastolysis even in the presence of 1% serum or 4 microM AlAT. Compared with cells incubated alone (no TNF), TNF increased elastolysis 3-fold in the 4-h incubations and 83% in 18-h incubations. TNF also significantly increased proteolysis when neutrophils were concurrently treated with phorbol myristate acetate or N-formylmethionylleucylphenylalanine. Since TNF is known to prime neutrophils for hypochlorous acid (HOCl) release, the present study hypothesized that the enhancement of proteolysis by TNF was related to increased release of HOCl. First, TNF caused a 4-fold increase in HOCl release by neutrophils adhered to elastin surfaces. Second, the effect of methionine on elastolysis by adherent neutrophils was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydroxylation of salicylate by the in vitro diaphragm: evidence for hydroxyl radical production during fatigue.

There is increasing evidence that oxygen-derived free radicals produced during strenuous work by the diaphragm may contribute to diaphragm fatigue and/or injury. However, the precise identity of these oxygen radicals remains unknown, inasmuch as oxygen free radicals are extremely short lived and their detection in biologic systems is quite difficult. There is recent evidence that the salicylate-trapping method may be a useful means of monitoring tissue production of hydroxyl radical (.OH). This method is predicated on the fact that salicylate's phenolic ring can be attacked by .OH at the 3 or 5 position to yield 2,3- or 2,5-dihydroxybenzoic acid (DHB). These metabolites are stable and can be identified by high-performance liquid chromatography (HPLC) coupled with electrochemical or ultraviolet detection. To test the hypothesis that hydroxylated salicylates are produced during diaphragm fatigue, we exposed in vitro rat diaphragm strips to a physiological saline solution containing 2.0 mM sodium salicylate for approximately 15 min. The solution was then removed, and the strips were fatigued (20 Hz, 200-ms train duration, 1 train/s) via phrenic nerve stimulation for 30 s-10 min. The diaphragm strips were subsequently homogenized, and the homogenate was analyzed by HPLC coupled with ultraviolet detection. Levels of 2,3-DHB were significantly higher in fatigued than in control nonfatigued strips. There was also a significant correlation between the amount of 2,3-DHB in the fatigued muscle and the accumulated tension-time product developed during fatigue. 2,5-DHB was not consistently identified in control or experimental strips.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between the monocyte/macrophage and the vascular smooth muscle cell. Stimulation of mitogenesis by a soluble factor and of prostanoid synthesis by cell-cell contact.

The effect of soluble factors from the monocyte/macrophage (M phi) on cell proliferation and the functional effects of cell-cell contact on the arachidonic acid (AA) cascade were studied with vascular smooth muscle cells (SMCs). Peripheral blood M phi s were isolated by adherence or in a Percoll gradient, and alveolar M phi s were obtained by lavage. Conditioned medium (CM) was prepared by preincubating M phi s with medium alone or by separating SMC and M phi cocultures by a membrane insert. Cell proliferation (image analysis) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha, radioimmunoassay) were measured in SMCs. Labeled prostanoids and other eicosanoid metabolites were isolated by high-performance liquid chromatography from SMCs prelabeled with 14C-AA. M phi s did not synthesize 6-keto-PGF1 alpha. The CM enhanced proliferation but did not stimulate 6-keto-PGF1 alpha synthesis in SMCs. However, cell-cell contact in cocultures of SMCs with the same concentration of M phi s used to generate CM resulted in increased 6-keto-PGF1 alpha synthesis by SMCs. Since the stimulatory effect of cell contact was not blocked by butylated hydroxytoluene, it could not be attributed to an oxidative burst from M phi s. Functional studies showed that the stimulatory effect of cell contact was enhanced by exogenous free AA and by endogenous AA release through A23187. Release of total radioactivity from prelabeled SMCs was enhanced by cell contact, and this effect was blocked by indomethacin (IM). Cell contact did not increase the release of free AA from prelabeled SMCs, even in the presence of IM. Finally, cell contact only stimulated the formation of prostanoids (IM-sensitive eicosanoid metabolites) from prelabeled SMCs. Lipoxygenase and other products of AA were not formed through cell-cell contact. These data showed that M phi s express a soluble factor that enhances SMC proliferation without affecting prostanoid synthesis. Subsequent cell contact between SMCs and M phi s stimulates prostanoid synthesis, which may possibly serve as a local and focal homeostatic mechanism for the regulation of uncontrolled SMC proliferation in atherogenesis.

Biotransformation of para-aminobenzoic acid and salicylic acid by PMN.

Para-aminobenzoic acid (PABA) is an essential cofactor for the production of folic acid in bacteria and has mild anti-inflammatory activity. We have recently reported that salicylic acid and benzoic acid are oxidized by stimulated granulocytes Polymorphonuclear Neutrophils (PMN). The oxidation of salicylate appears mediated by a potent oxygen metabolite generated during the respiratory burst which is dependent primarily on superoxide (O2-) for its production. These background studies with the salicylate group of drugs suggested that PABA might be similarly metabolized by PMN. In these studies, we demonstrate that PABA is metabolized by stimulated PMN. However, in contrast to the biochemical mechanism involved in the metabolism of salicylate, our scavenger studies indicate that PABA is metabolized primarily by the myeloperoxidase pathway. Our results may explain the mild anti-inflammatory actions of the drug and suggest that the degradation of PABA by PMN at an inflammatory site may limit the availability of PABA for bacterial growth.

Educational status and blood pressure: the Second National Health and Nutrition Examination Survey, 1976-1980, and the Hispanic Health and Nutrition Examination Survey, 1982-1984.

An inverse association between socioeconomic status, as measured by years of education, and blood pressure has been reported in a number of studies. However, two secular trends may have changed the nature of this relation: a higher mean level of education in the population and intervention for high blood pressure in the community. Given that education is the most commonly used indicator of social class and measurement is a critical issue in epidemiologic research, a study was initiated to examine the validity of education for predicting blood pressure among 11,554 examined persons aged 25-74 years from the Second National Health and Nutrition Examination Survey (1976-1980) or the Hispanic Health and Nutrition Examination Survey (1982-1984). In univariate analysis, a consistent, inverse association between education and blood pressure was found for whites and blacks, but not for Mexican Americans. After adjustment for age and body mass, the effect persisted only for systolic blood pressure in whites. The association of education and blood pressure was positive in Mexican-American females. Education was inversely related to hypertensive status in whites and in black females. These findings suggest that information on education may be of little value for identifying populations at risk of high blood pressure, particularly if age and body mass are known. However, information on education may be of considerable value in the design and implementation of appropriate interventions.

Antioxidant activity of bronchoalveolar lavage fluid in the adult respiratory distress syndrome.

Bronchoalveolar lavage (BAL) fluid from normal subjects is a potent inhibitor of lipid peroxidation. This antioxidant activity (AOA) of BAL fluid is primarily due to the serum proteins transferrin and ceruloplasmin. In the adult respiratory distress syndrome (ARDS), there is an influx of protein-rich edema fluid into the alveolar space that may increase antioxidant activity and provide protection against further oxidant-mediated lung injury. To test this hypothesis, the AOA of BAL fluid was measured in patients with ARDS (n = 11) and normal subjects (n = 12). When compared with normal subjects, BAL fluid from ARDS patients had a significantly higher concentration of total protein (2,536.8 +/- 408.2 micrograms/ml vs. 77.3 +/- 7.0 micrograms/ml, P less than 0.005). When compared at several volumes, BAL fluid from ARDS patients was a more potent inhibitor of lipid peroxidation than BAL fluid from normals. In addition, when AOA was determined on equal milligram amounts of BAL fluid protein from ARDS patients and normal subjects, ARDS BAL fluid protein had a significantly higher AOA. Consistent with its higher AOA, ARDS BAL fluid contained increased concentrations of both transferrin (77.8 +/- 15.3 micrograms/ml vs. 2.78 +/- 0.3 micrograms/ml, P less than 0.05) and ceruloplasmin (36.5 +/- 5.6 micrograms/ml vs. 0.26 +/- 0.02 micrograms/ml, P less than 0.005) compared with normal subjects. The importance of both ceruloplasmin and transferrin in the enhanced AOA of ARDS BAL fluid was further demonstrated by studies that showed a significant decrement in AOA when the antioxidant functions of these two proteins were selectively blocked.(ABSTRACT TRUNCATED AT 250 WORDS)

Black-white differences in blood pressure among participants in NHANES II: the contribution of blood lead.

Separate studies using data from the second National Health and Nutrition Examination Survey, 1976-1980 (NHANES II) have shown that blacks in the United States have higher blood lead levels than whites and that blood lead is positively related to blood pressure. Based on these reports, we examined data from NHANES II to determine the extent to which race differences in blood pressure were explained by elevated blood lead levels in blacks. Regression analyses, with race as an indicator variable, were used to estimate the contribution of blood lead to black-white differences in blood pressure. The overall effect of blood lead on race differences in blood pressure was small, reflecting, in part, the magnitude of race differences in blood lead and in the association of blood lead and blood pressure. Nevertheless, a pronounced and consistent effect of lead on race differences in blood pressure was found among the poor, particularly women.