The species-dependent metabolism of efavirenz produces a nephrotoxic glutathione conjugate in rats.

Efavirenz, a potent nonnucleoside reverse transcriptase inhibitor widely prescribed for the treatment of HIV infection, produces renal tubular epithelial cell necrosis in rats but not in cynomolgus monkeys or humans. This species selectivity in nephrotoxicity could result from differences in the production or processing of reactive metabolites, or both. A detailed comparison of the metabolites produced by rats, monkeys, and humans revealed that rats produce a unique glutathione adduct. The mechanism of formation and role of this glutathione adduct in the renal toxicity were investigated using both chemical and biochemical probes. Efavirenz was labeled at the methine position on the cyclopropyl ring with the stable isotope deuterium, effectively reducing the formation of the cyclopropanol metabolite, an obligate precursor to the glutathione adduct. This substitution markedly reduced both the incidence and severity of nephrotoxicity as measured histologically. Further processing of this glutathione adduct was also important in producing the lesion and was demonstrated by inhibiting gamma-glutamyltranspeptidase with acivicin pretreatment (10 mg/kg, IV) prior to dosing with efavirenz. Again, both the incidence and severity of the nephrotoxicity were reduced, such that four of nine rats given acivicin were without detectable lesions. These studies provide compelling evidence that a species-specific formation of glutathione conjugate(s) from efavirenz is involved in producing nephrotoxicity in rats. Mechanisms are proposed for the formation of reactive metabolites that could be responsible for the renal toxicity observed in rats.

Reversible drug-induced oxyntic atrophy in rats.

BACKGROUND & AIMS: Oxyntic atrophy is the hallmark of chronic gastritis. Many studies have sought to develop animal models for oxyntic atrophy, but none of them are reversible. We now report that rats administered high doses of DMP 777 demonstrate reversible oxyntic atrophy. METHODS: DMP 777 was administered to CD-1 rats by oral gavage (200 mg. kg(-1). day(-1)). Serum gastrin level, in vivo acid secretion, and gastric histological changes were evaluated in DMP 777-dosed animals. Direct effects of DMP 777 on parietal cells were evaluated by assessment of aminopyrine accumulation into isolated rabbit parietal cells, as well as by assessment of DMP 777 effects on acridine orange fluorescence and H(+),K(+)-adenosine triphosphatase (ATPase) activity in isolated tubulovesicles. RESULTS: Oral dosing with DMP 777 caused a rapid increase in serum gastrin levels and severe hypochlorhydria. DMP 777 inhibited aminopyrine accumulation into rabbit parietal cells stimulated with either histamine or forskolin. DMP 777 reversed a stimulated proton gradient in isolated parietal cell tubulovesicles. Oral dosing with DMP 777 led to rapid loss of parietal cells from the gastric mucosa. In response to the acute loss of parietal cells, there was an increase in the activity of the progenitor zone along with rapid expansion of the foveolar cell compartment. DMP 777 treatment also led to the emergence of bromodeoxyuridine-labeled cells and cells positive for periodic acid-Schiff in the basal region of fundic glands. With extended dosing over 3-6 months, foveolar hyperplasia and oxyntic atrophy were sustained while chief cell, enterochromaffin-like cell, and somatostatin cell populations were decreased. No histological evidence of neoplastic transformation was observed with dosing up to 6 months. Withdrawal of the drug after 3 or 6 months of dosing led to complete restitution of the normal mucosal lineages within 3 months. CONCLUSIONS: DMP 777 acts as a protonophore with specificity for parietal cell acid-secretory membranes. DMP 777 in high doses leads to the specific loss of parietal cells. Foveolar hyperplasia, loss of normal gland lineages, and the emergence of basal mucous cells appear as sequelae of the absence of parietal cells. The results suggest that parietal cells are critical for the maintenance of the normal mucosal lineage repertoire.

Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine.

Dexfenfluramine was approved in the United States for long-term use as an appetite suppressant until it was reported to be associated with valvular heart disease. The valvular changes (myofibroblast proliferation) are histopathologically indistinguishable from those observed in carcinoid disease or after long-term exposure to 5-hydroxytryptamine (5-HT)(2)-preferring ergot drugs (ergotamine, methysergide). 5-HT(2) receptor stimulation is known to cause fibroblast mitogenesis, which could contribute to this lesion. To elucidate the mechanism of "fen-phen"-associated valvular lesions, we examined the interaction of fenfluramine and its metabolite norfenfluramine with 5-HT(2) receptor subtypes and examined the expression of these receptors in human and porcine heart valves. Fenfluramine binds weakly to 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors. In contrast, norfenfluramine exhibited high affinity for 5-HT(2B) and 5-HT(2C) receptors and more moderate affinity for 5-HT(2A) receptors. In cells expressing recombinant 5-HT(2B) receptors, norfenfluramine potently stimulated the hydrolysis of inositol phosphates, increased intracellular Ca(2+), and activated the mitogen-activated protein kinase cascade, the latter of which has been linked to mitogenic actions of the 5-HT(2B) receptor. The level of 5-HT(2B) and 5-HT(2A) receptor transcripts in heart valves was at least 300-fold higher than the levels of 5-HT(2C) receptor transcript, which were barely detectable. We propose that preferential stimulation of valvular 5-HT(2B) receptors by norfenfluramine, ergot drugs, or 5-HT released from carcinoid tumors (with or without accompanying 5-HT(2A) receptor activation) may contribute to valvular fibroplasia in humans.

Atypical antipsychotic-induced neutropenia in dogs.

DMP 406 is an atypical antipsychotic, antischizophrenic drug, biochemically related to clozapine, which exerts its desired pharmacologic effects through selective antagonism of 5-hydroxytryptamine and dopamine-receptor subtypes. Clozapine therapy is clinically associated with severe granulocytopenia in a small subset of patients. In the course of a 3-month toxicity study in dogs, severe neutropenia, thrombocytopenia, marked myeloid and erythroid left-shifted bone marrow hyperplasia with increased erythrophagocytosis, positive Coombs' tests, and hypergammaglobulinemia occurred in individual females dosed with 30 mg/kg/day of DMP 406. Related but less severe changes were also observed in males. Sera or purified immunoglobulins from affected and control dogs were tested in methylcellulose-based, canine hematopoietic colony-forming unit (CFU) assays with or without DMP 406. Neither size nor number of erythroid or myeloid CFUs differed between cultures containing control or affected dog serum components. Sera from individual affected dogs but not controls resulted in moderate numbers of fibroblast-like CFUs, suggesting DMP 406-associated marrow stromal cell-modifying, serum activities to be present. DMP 406 alone resulted in a concentration-dependent reduction of erythroid and myeloid CFUs with an approximate IC50 of 3.0 microg/mL. Taken together, DMP 406-induced granulocytopenia and bone marrow dyscrasia appear likely to result from both immune-mediated and direct drug-induced myelotoxicity.

Bile duct obstruction is not a prerequisite for type I biliary epithelial cell hyperplasia.

Biliary obstruction, produced by common bile duct ligation or alpha-naphthylisothiocyanate (ANIT) treatment in rats, has been associated with the development of type I biliary epithelial cell (BEC) hyperplasia. However, the exact mechanism(s) by which bile duct obstruction lead(s) to this proliferative lesion are not clear. The present studies were designed to determine if cholestasis, in the absence of biliary obstruction, would result in type I BEC hyperplasia. Male Sprague-Dawley rats were given a single oral dose of 150 mg/kg ANIT or i.v. doses of estradiol glucuronide (E2-17G; 21 mumol/kg/h for 48 h) to produce obstructive and non-obstructive cholestasis, respectively. E2-17G treatment resulted in cholestasis that was comparable in extent and duration to that observed following ANIT treatment. E2-17G and ANIT treatments produced comparable increases in serum bile acids (55- to 60-fold) and activities of ALT (36- to 38-fold), ALP (4- to 5-fold), and 5'-nucleotidase (7- to 11-fold), respectively, compared to controls. Both ANIT and E2-17G also increased serum bilirubin concentrations. ANIT treatment resulted in significant increases in biliary glucose concentrations that were associated with BEC damage/necrosis and obstruction of the bile duct lumen. Conversely, no evidence of BEC damage was observed in E2-17G-treated rats. Nonetheless, BEC hyperplasia was observed in the majority of rats following treatment with either ANIT or E2-17G, assessed by light microscopy and by BrdU immunohistochemistry. These data indicate that E2-17G treatment produces nonobstructive cholestasis and type I BEC hyperplasia, suggesting that biliary obstruction is not a prerequisite for type I BEC hyperplasia in rats. Differences in the time of onset of hyperplasia were observed: hyperplasia was noted immediately following 48 h of E2-17G-induced cholestasis but occurred several days after ANIT-induced cholestasis had subsided. Since the magnitude/duration of cholestasis was similar in the two models but the temporal association between cholestasis and type I BEC hyperplasia were different, these data suggest that the proliferative stimulus may be different in the two models and that E2-17G-induced type I BEC hyperplasia may not be attributed solely to cholestasis.

Inhibition of kinases impairs neutrophil activation and killing of Staphylococcus aureus.

Intracellular phosphorylations polymorphonuclear neutrophils are mediated by kinases, including mitogen activated-protein (MAP) kinases and phosphatidylinositol 3-kinase. In the present study we demonstrate their effector functions upon both ligation of cell-surface seven-transmembrane-spanning receptors by bacterial peptide formylmethionyl-leucylphenylalanine as well as in the process of destruction of Staphylococcus aureus. To regulate neutrophil MAP kinases p38 and p44/42, specifically, we made use of their specific inhibitors 10 microM SK&F 86002 (for p38) and PD 098059 (for activating kinase of p44/42). SK&F 86002 was a potent inhibitor (by 70%) of induced antimicrobial oxygen-radical generation compared with PD 098059 (by 20%). SK&F 86002 and PD 098059 inhibited mobilization of a dominant neutrophil adhesion molecule, beta2 integrin, from cytoplasmic granules to the plasma membrane by 40 and 10% respectively, and the combination of the two drugs resulted in a 90% effect. The combined effect of both drugs was moderate inhibition of bacterial destruction, despite the fact that neither compound had detectable effect on bactericidal activity if applied individually. Bacterial destruction was also inhibited by wortmannin (0.1 microM), the specific inhibitor of phosphatidylinositol 3-kinase, which had previously been described to target various other activations of the neutrophil, including oxygen-radical generation. Although the relative contribution of p38 and p44/42 MAP kinases varied, the marked effects of the combined inhibition of the kinases revealed their concerted actions to be critical for normal neutrophil function.

Temporal Changes in State Transitions and Photosystem Organization in the Unicellular, Diazotrophic Cyanobacterium Cyanothece sp. ATCC 51142.

The unicellular Cyanobacterium Cyanothece sp. ATCC 51142, grown under alternating 12-h light/12-h dark conditions, temporally separated N2 fixation from photosynthesis. The regulation of photosynthesis was studied using fluorescence spectra and kinetics to determine changes in state transitions and photosystem organization. The redox poise of the plastoquinone (PQ) pool appeared to be central to this regulation. Respiration supported N2 fixation by oxidizing carbohydrate granules, but reduced the PQ pool. This induced state 2 photosystem II monomers and lowered the capacity for O2 evolution. State 2 favored photosystem I trimers and cyclic electron transport, which could stimulate N2 fixation; the stimulation suggested an ATP limitation to N2 and CO2 fixation. The exhaustion of carbohydrate granules at around 6 h in the dark resulted in reduced respiratory electron flow, which led to a more oxidized PQ pool and produced a sharp transition from state 2 to state 1. This transient state 1 returned to state 2 in the remaining hours of darkness. In the light phase, photosystem II dimerization correlated with increased phycobilisome coupling to photosystem II (state 1) and increased rates of O2 evolution. However, dark adaptation did not guarantee state 2 and left photosystem I centers in a mostly monomeric state at certain times.

ACAT inhibitors derived from hetero-Diels-Alder cycloadducts of thioaldehydes.

Acyl-CoA:cholesterol acyltransferase (ACAT) is the enzyme largely responsible for intracellular cholesterol esterification. A systemic inhibitor of ACAT is believed to be able to slow or even reverse the atherosclerotic process. Towards that goal, a series of cyclic sulfides, derived from the hetero-Diels-Alder reaction of thioaldehydes with 1,3-dienes, and bearing carboxamide substituents, were prepared and evaluated for in vitro (in several tissues and species) and ex vivo ACAT inhibition. Minor changes in subsequent structure were found to have a significant effect in optimization of the biological activity of this series of compounds.

Improved 5-step modeling of the Photosystem II S-state mechanism in cyanobacteria.

We present a model of the S-state mechanism, as well as an improved eigenvalue analysis, that integrate into a coherent ensemble several features found since the S-state model was initially developed. These features include the presence of S-1, deactivations in the dark interval between flashes, and the change in the number of active PS II centers by photoinhibition or photoactivation. A new feature is the capacity to predict the steady-state distribution of S-states under conditions of steady photoinhibition or photoactivation. The improved eigenvalue analysis allowed the calculation of the initial S-state distribution. In addition, the model resolved 'true' photochemical misses from apparent misses due to deactivations in the dark interval between flashes. The model suggested that most of the misses that are commonly reported are due to deactivations, and not to an intrinsic inefficiency of the photochemical mechanism of PS II. Because models that allow double-hits encompassing the S2 to S3 transition often predict negative initial quantities of S2 in cyanobacteria, our proposed model specifically prohibited them. The model accounts for inhomogeneous misses and a steady-state distribution of the type (S2)≈(S1)>(S3)≈(S0). This 5-step model uses only 4 probabilities, and is therefore easy to handle. The use of this model is critical for the analysis of several cyanobacterial strains, as well as for any species that show non-negligible deactivations in the dark interval between flashes.

Biliary epithelial cell proliferation following alpha-naphthylisothiocyanate (ANIT) treatment: relationship to bile duct obstruction.

These studies were designed to evaluate the importance of bile duct obstruction in the pathogenesis of alpha-naphthylisothiocyanate (ANIT)-induced biliary epithelial cell (BEC) hyperplasia in rats. Hepatobiliary function and morphology were evaluated in adult male Sprague-Dawley rats 16, 24, 48, 72, 120, and 168 hr after a single oral dose of ANIT (0, 25, 75, or 150 mg/kg). After 75 or 150 mg/kg ANIT, multifocal bile duct obstruction was observed at 48 and 72 hr and preceded BEC hyperplasia which occurred at 120 and 168 hr. BEC proliferation, reflected by 5-bromo-2'-deoxyuridine (BrdU) incorporation, occurred at doses and at time points coinciding with BEC necrosis and/or bile duct obstruction. In contrast, 25 mg/kg ANIT produced minimal BEC damage and no evidence of bile duct obstruction or BEC hyperplasia. In a separate experiment, BEC proliferation was evaluated following bile duct ligation or ANIT treatment (150 mg/kg). The onset and peak of BEC proliferation occurred 24 and 48 hr, respectively, following bile duct obstruction resulting from either ligation or ANIT treatment. Furthermore, BEC proliferation occurred at all levels of the biliary tree in both bile duct-ligated and ANIT-treated rats. These data indicate that (a) dose-response curves for ANIT-induced bile duct obstruction and BEC hyperplasia are similar; (b) ANIT-induced BEC proliferation and bile duct obstruction precedes BEC hyperplasia; (c) BEC proliferation occurred at doses/timepoints associated with BEC damage and bile duct obstruction; and (d) once ANIT-induced bile duct obstruction occurs, the spatial and temporal aspects of BEC proliferation are comparable to those following biliary obstruction induced by bile duct ligation. Collectively, these data suggest that ANIT-induced BEC hyperplasia is secondary to intrahepatic bile duct obstruction.

Interaction of the photosynthetic and respiratory electron transport chains producing slow O2 signals under flashing light in Synechocystis sp. PCC 6803.

We investigated the slow signal of apparent O2 release under brief light flashes by using mutants of Synechocystis sp. PCC 6803 which lacked CP43 and D1. The slow signal was present at higher amplitudes in the mutants. It was inhibited by starving the mutants of glucose (>90%), by 10 mM NaN3 (85%) and by boiling samples for 2 min (100%). In the mutants and in the wild-type, the slow signal was 95% inhibited by the combination of DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). In the wild type, the addition of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) or CCCP (carbonylcyanide m-chlorophenylhydrazone) completely inhibited photosynthetic O2 evolution, yet failed to inhibit the slow signal. We explain the kinetics of the wild-type signal as a positive deflection due to the inhibition of respiration by PS I activity, and a negative deflection due to the stimulation of respiration by electrons originating from PS II. We found no evidence of a 'meta-stable S3' in Synechocystis sp. PCC 6803 that could contribute to the slow signal of apparent O2 release. We present a calculation which involves only averaging, division and subtraction, that can remove the contribution of the slow signal from the true photosynthetic O2 signal and provide up to a 10-fold improved accuracy of the S-state models.

Cholestatic potentials of alpha-naphthylisothiocyanate (ANIT) and beta-naphthylisothiocyanate (BNIT) in the isolated perfused rat liver.

Previous studies in rats have shown that a single oral dose of alpha-naphthylisothiocyanate (ANIT), but not the regioisomer beta-naphthylisothiocyanate (BNIT), results in intrahepatic cholestasis. The present studies were designed to evaluate the intrinsic cholestatic potential of ANIT and BNIT in the isolated perfused rat liver. Livers from male Sprague-Dawley rats (300-450 g) were isolated and perfused with Krebs-Henseleit buffer supplemented with 50 microM taurocholate and ANIT or BNIT (0, 5, 15 or 50 microM). Rates of bile flow, bile acid uptake and bile acid excretion were monitored for up to 70 min. Permeability of tight junctions also was evaluated. At concentrations of 5 microM, neither ANIT nor BNIT altered hepatobiliary function or tight junction permeability. In contrast, perfusion with 50 microM ANIT or BNIT for 35 min resulted in decreases in bile flow rates of 19 +/- 8 and 13 +/- 4%, respectively. After 70 min of perfusion with ANIT or BNIT, rates of bile flow were decreased by 78 +/- 5 and 71 +/- 4%, respectively. Bile acid excretion also was decreased following perfusion with 50 microM ANIT or BNIT. Perfusion with 50 microM ANIT or BNIT decreased bile acid uptake by 51 +/- 13 and 46 +/- 6%, respectively, at 60 min. Bile/plasma (B/P) ratios of [3H]sucrose were not affected by ANIT or BNIT at any time during perfusion, indicating that changes in bile flow and bile acid excretion in the isolated perfused liver were not associated with increased hepatocyte tight junction permeability. These data demonstrate that the direct portal infusion of a 50 microM concentration of either ANIT or BNIT produced marked decreases in bile flow, indicating that these isomers have a comparable intrinsic cholestatic potential in the isolated perfused liver.

Temporal relationship of changes in hepatobiliary function and morphology in rats following alpha-naphthylisothiocyanate (ANIT) administration.

These studies were designed to evaluate ANIT-induced changes in both hepatobiliary function and morphology during the onset, progression, and recovery of ANIT-induced cholestasis. A single oral dose of 150 mg/kg of ANIT or vehicle was administered by gavage to male Sprague-Dawley rats and hepatobiliary structure and function were evaluated 16, 24, 48, 72, and 168 hr later. Increased hepatocellular tight junction permeability, increased serum bile acids, and decreased bile acid excretion were observed 16 hr after ANIT administration. At 24 hr, bile flow was decreased in ANIT-treated rats, an effect accompanied by increased tight junction permeability, decreased bile acid excretion, and decreased erythritol clearance (estimate of canalicular flow). In addition, scattered small loci of hepatocellular necrosis accompanied by an inflammatory cell response were observed in ANIT-treated rats at this time, with no microscopic evidence of bile duct obstruction (BDO). These data suggest that the onset of ANIT-induced cholestasis was associated with hepatocanalicular changes and not BDO. In contrast, at 48 and 72 hr after ANIT treatment, cholestasis was more profound and was accompanied by mild hepatocellular necrosis and widespread BDO. Hepatocyte tight junction permeability in ANIT-treated rats was not different from controls at 72 hr. These data suggest that the pathogenesis of ANIT-induced cholestasis is biphasic; the onset of cholestasis appears to be associated with changes in hepatocanalicular function and increased tight junction permeability whereas the later and more profound phase of cholestasis appears to be related to a combination of BDO and hepatocellular dysfunction. The time course of biochemical and morphologic changes following ANIT treatment further suggests that the pathophysiologic changes during the onset or initiation phase of cholestasis differ from those involved in the later and more profound phase of ANIT-induced cholestasis.

On the rates of cyclic electron transport around Photosystem II in the presence of donor side limitation.

Photosystem II cyclic electron transport was investigated at low pH in spinach thylakoids and PS II preparations from the cyanobacteriumPhormidium laminosum. Variable fluorescence (Fv) quenching at a very low light intensity was examined as an indicator of cyclic electron flow. A progressive quenching of Fv was observed as the pH was lowered; however, this was shown to be mainly due to an inhibition of oxygen evolution. Cyclic electron flow in the uninhibited centres was estimated to occur at a rate comparable to or smaller than 1 µ mole O2 mg Chl(-1) h(-1) in the pH range 5.0 to 7.8.The quantum yeeld of oxygen production is known to decrease at low pH and has been taken to indicate cyclic electron flow (Crofts and Horton (1991) Biochim Biophys Acta 1058: 187-193). However, a direct all-or-none inhibition of oxygen production at low pH has also been reported (Meyer et al. (1989) Biochim Biophys Acta 974: 36-43). We have analysed the effects of light intensity on the rates of oxygen evolution in order to calculate ΦU, the quantum yield of open and uninhibited centres. ΦU was found to be constant over a broad pH range, and by using ferricyanide and phenyl-p-benzoquinone as electron acceptors the maximum possible rate of cyclic electron transport was equivalent to no more than 1 µmole O2 mg Chl(-1) h(-1). The rate was no greater when the acceptor was adjusted to provide the most favourable conditions for cyclic flow.

Oxygen evolution by Photosystem II: The contribution of backward transitions to the anomalous behaviour of double-hits revealed by a new analysis method.

Backward transitions in the analysis of oxygen production under flashing light were introduced by Packham et al., 1988, Photosynth. Res. 15: 221-232. In order to take backward transitions into account, a new method of analysis is presented: the 'eigenvalue method'. This method is based on the recurrence relation of oxygen production with four coefficients (also known as the four 'sigma' coefficients). It shows less susceptibility to round-off errors than other methods and permits the computation of double-hits directly from the coefficients, which was not possible before. With it we discovered that the inconsistent behaviour of double-hits observed previously under low flash intensities or low flash frequencies was mainly due to the inclusion of the backward transitions into the double-hit probability. In these conditions backward transitions seemed to be due either to the combination of an S-state deactivation and a miss, or to two S-state deactivations and a single-hit.In the presence of 3-(3, 4-Dichlorophenyl)-1, 1-dimethylurea (DCMU), the previous methods of 'sigma' analysis failed. In contrast, the new method resolved all four S-state probabilities; thus it has the further advantage of being more 'robust' (robustness being defined as the ability to yield a meaningful answer under difficult conditions).

Analysis of fluorescence induction in thylakoids with the method of moments reveals two different active Photosystem II centres.

There is presently a debate concerning the number of phases in fluorescence induction and on the identification of the several possible heterogeneities in PS II centres. However, the usual methods of analysis present numerical problems, including a lack of 'robustness' (robustness being defined as the ability to give the correct answer in the presence of distortions or artefacts). We present here the adaptation of the method of moments, which was developed for robustness, to the analysis of fluorescence induction. We were thus able to identify three phases in the fluorescence induction in the presence of DCMU. The slowest phase was attributed to the centres inactive in plastoquinone reduction by using duroquinone as electron acceptor. In order to compare fluorescence with and without DCMU, we introduced the 'rate of photochemistry', defined as the product of the area times the rate constant of an exponential. This quantity is invariant for a given centre no matter what the size of the electron acceptor pool is. The two fastest phases in the presence of DCMU were attributed to active centres because their rate of photochemistry was the same as that of the plastoquinone-reducing phases in the absence of DCMU. Because their reduction of plastoquinone showed different kinetics, these two types of active centres were either separated by more than 250 nm or were associated with discrete plastoquinone pools having restricted diffusion domains.

The time for oxygen release in photosynthesis: reconciliation of flash polarography with other measurement techniques.

The time for oxygen release in photosynthesis has been reported to be 30-130 ms when measured by flash polarography under low polarization voltages (Plijter et al. 1988), in opposition to 1-3 ms with light modulated oxygen polarography (Jolio et al. 1966), with the detection of produced oxygen in a flowing sample (Etienne 1968) or with photoacoustic detection of oxygen evolution (Canaani et al. 1988). However, we show here that flash polarographic measurements require properly cleaned electrodes, a precise polarization voltage, as well as a short polarization time of the electrodes. When these criteria were met, an oxygen release in less than 2 ms could be measured by flash polarography under low polarization voltages, in accordance with the other techniques. But under high polarization voltages, the interpretation of the polarographic response to oxygen production must take into account the diffusion of oxygen, the capacitance of the platinum electrode and the oxygen release time. We present a model of the electrode response taking into account these factors; by interpreting the response of the electrodes with this model, we found an oxygen release time of 1.7 ms. These evidences support strongly a short oxygen release time of 1-3 ms.

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II.

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163-171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.

Identification and isolation of a phospholipase A2 activating protein in human rheumatoid arthritis synovial fluid: induction of eicosanoid synthesis and an inflammatory response in joints injected in vivo.

Eicosanoids are important mediators of the destructive arthropathy observed in rheumatoid arthritis. The rate-limiting step in the eicosanoid synthesis pathway is the availability of free arachidonic acid. The phospholipase enzymes release arachidonic acid from membrane phospholipids and thus play an important role in the regulation of eicosanoid production. We have previously demonstrated enhanced phospholipase A2 and C enzyme activities in cells from patients with rheumatoid arthritis and have also described a phospholipase A2 activating protein (PLAP) in mammalian cell lines. In an attempt to determine the biochemical basis of enhanced phospholipase A2 activity found in patients with inflammatory joint disease, we examined synovial fluid from patients with rheumatoid arthritis for PLAP. To determine whether PLAP was specific for rheumatoid disease, we assayed specimens from patients with other arthropathies. Histologic examination of rheumatoid joint tissue, with the use of immunohistochemical techniques, demonstrated high concentration of PLAP in monocytes, macrophages, chondrocytes, vascular smooth muscle, and endothelial cells. Human PLAP could be biochemically isolated from synovial fluid from patients with rheumatoid arthritis and was found to be similar to PLAP previously isolated from murine and bovine sources. To determine whether PLAP could directly mediate any aspect of inflammatory disease, purified PLAP was injected into rabbit knee joints. This resulted in an acute inflammatory arthritis with synovial cell proliferation and synovial fluid leukocytosis. Purified PLAP also induced eicosanoid formation both in vivo and in vitro. With enzyme-linked immunosorbent assays, we found more PLAP in synovial fluid specimens from patients with rheumatoid arthritis compared with samples from patients with other inflammatory arthropathies as well as osteoarthritis, a noninflammatory arthropathy. These data suggest that PLAP may be responsible, at least in part, for some aspects of the destructive inflammatory arthropathy that is observed in patients with rheumatoid arthritis.

Activation of rat pulmonary lavage cells by intratracheal administration of polyinosinic-polycytidilic acid.

Systemic administration of biologic response modifiers (BRMs) is often limited by serious adverse effects. Local delivery of a BRM may provide a means to avoid systemic side effects while enhancing host defense. To test this hypothesis, the effects of intratracheal administration of the interferon inducer polyinosinic-polycytidilic acid (Poly-I:C) were examined. We demonstrated that intratracheal administration of Poly-I:C in rats results in alterations in pulmonary lavage cell number, population distribution, and function, suggestive of cellular activation. Following intratracheal instillation of 15, 75, or 150 mg of Poly-I:C, we observed a dose-dependent increase in Fc receptor-mediated phagocytosis, a dose-independent tumoricidal activity directed against xenogeneic P815 murine mastocytoma target cells, and a dose-independent decrease in superoxide anion (O2-) generation. With the exception of O2- generation, these functions returned to normal control levels by 7 days after a single dose of Poly-I:C. Thus, localized administration of Poly-I:C enhanced the host defense capability of pulmonary lavage cells, providing a rationale for compartmentalized immunoprophylaxis in the lung.