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Meptyldinocap is a dinitrophenol fungicide used to control powdery mildew. Although other dinitrophenol pesticides have been found to exhibit reproductive toxicity, studies of meptyldinocaps are scarce. This study investigated the adverse effects of meptyldinocap on porcine trophectoderm (pTr) and porcine endometrial luminal epithelial (pLE) cells, which play crucial roles in implantation. We confirmed that meptyldinocap decreased cell viability, induced apoptosis, and inhibited proliferation by decreasing proliferation-related gene expression and inducing changes in the cell cycle. Furthermore, meptyldinocap treatment caused mitochondrial dysfunction, endoplasmic reticulum stress, and disruption of calcium homeostasis. Moreover, it induces alterations in mitogen-activated protein kinase signaling cascades and reduces the migration ability, leading to implantation failure. Our findings suggest that meptyldinocap reduces the cellular functions of pTr and pLE cells, which are important for the implantation process, and interferes with interactions between the two cell lines, potentially leading to implantation failure. We also propose a mechanism by which the understudied fungicide meptyldinocap exerts its cytotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Atopic dermatitis (AD) is a common inflammatory skin disorder. Mast cells play an important role in AD because they regulate allergic reactions and inflammatory responses. However, whether and how the modulation of mast cell activity affects AD has not been determined. In this study, we aimed to determine the effects and mechanisms of 3-O-cyclohexanecarbonyl-11-keto-β-boswellic acid (CKBA). This natural compound derivative alleviates skin inflammation by inhibiting mast cell activation and maintaining skin barrier homeostasis in AD. CKBA markedly reduced serum IgE levels and alleviated skin inflammation in calcipotriol (MC903)-induced AD mouse model. CKBA also restrained mast cell degranulation both in vitro and in vivo. RNA-seq analysis revealed that CKBA downregulated the extracellular signal-regulated kinase (ERK) signaling in BM-derived mast cells activated by anti-2,4-dinitrophenol/2,4-dinitrophenol-human serum albumin. We proved that CKBA suppressed mast cell activation via ERK signaling using the ERK activator (t-butyl hydroquinone) and inhibitor (selumetinib; AZD6244) in AD. Thus, CKBA suppressed mast cell activation in AD via the ERK signaling pathway and could be a therapeutic candidate drug for AD., (© 2023 Wiley-VCH GmbH.)

Therapeutic antibodies should cover particular physicochemical and functional requirements for successful entry into clinical practice. Numerous experimental and computational approaches have been developed for early identification of different unfavourable features of antibodies. Immune repertoires of healthy humans contain a fraction of antibodies that recognize nitroarenes. These antibodies have been demonstrated to manifest antigen-binding polyreactivity. Here we observed that >20 % of 112 clinical stage therapeutic antibodies show pronounced binding to 2,4-dinitrophenol conjugated to albumin. This interaction predicts a number of unfavourable functional and physicochemical features of antibodies such as polyreactivity, tendency for self-association, stability and expression yields. Based on these findings we proposed a simple approach that may add to the armamentarium of assays for early identification of developability liabilities of antibodies intended for therapeutic use., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F 1 F O -ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neurons depend on mitochondria for homeostasis and survival, and thus, mitochondrial dysfunction has been implicated in neurodegenerative diseases, including Parkinson's disease (PD). Increasing evidence indicates the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), protects neurons against neurodegeneration and enhances neural plasticity. Here, the authors evaluated the protective effects of intraperitoneally (i.p.) administered low dose DNP in an acute mouse model of PD. Mice were administered DNP (1 or 5mg/kg) for 12 consecutive days, and then on day 13, MPTP (20mg/kg, i.p.) was administered four times (with 2h intervals between injections) to induce PD. It was found that MPTP-induced motor dysfunction was ameliorated in the DNP-treated mice versus vehicle-treated controls. Additionally, DNP effectively attenuated dopaminergic neuronal loss observed in MPTP treated mice. Moreover, in primary cultured neurons, DNP at 10μM, but not at 100μM, prevented MPP + -induced cell death and mitochondrial membrane potential (MMP) reduction. In addition, DNP was observed to cause the nuclear translocation of Nrf2 in primary neurons. Taken together, these findings of the present study suggest that DNP protects dopaminergic neurons against neurodegeneration and maintains MMP integrity in PD by activating adaptive stress responses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The calcium paradox was first mentioned in 1966 by Zimmerman et al. Thereafter gained great interest from the scientific community due to the fact of the absence of calcium ions in heart muscle cells produce damage similar to ischemia-reperfusion. Although not all known mechanisms involved in cellular injury in the calcium paradox intercellular connection maintained only by nexus seems to have a key role in cellular fragmentation. The addition of small concentrations of calcium, calcium channel blockers, and hyponatraemia hypothermia are important to prevent any cellular damage during reperfusion solutions with physiological concentration of calcium.

Objectives: Oxidative stress is a major etiology of obstructed bladder dysfunction. The major goal of the current study was to correlate the level of oxidative stress with both the severity and duration of obstruction., Methods: A total of 32 New Zealand White rabbits were divided into four equal groups. Groups 1-3 received partial bladder outlet obstructions by standard methods and survived for 4, 8 or 12 weeks. Group 4 received sham surgery at the end of each time period, isolated strips were taken for contractility studies and the balance of the bladder was frozen as muscle and mucosa for quantification of nitrotyrosine and carbonyl-oxidized proteins derivatized into dinitrophenyl. For each duration, the eight rabbits were divided into three severity groups: mild, intermediate or severe decompensation., Results: Contractile responses decreased in proportion to both severity and duration. The level of both oxidative products correlated to a much higher degree with the level of severity than the duration. There were significant decreases in the contractile responses in the mild decompensation group, whereas the level of derivatized into dinitrophenyl and nitrotyrosine of the muscle remained at control levels. This was not the case for the 4 weeks obstructed group., Conclusions: These findings suggest that the etiology for the mechanism of contractile dysfunction is not an oxidative stress., (© 2012 The Japanese Urological Association.)

Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 μM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ethnopharmacological Evidence: Agrimonia pilosa Ledeb (Rosaceae, AP) has long been used as a traditional medicine in Korea and other Asian countries to treat various diseases., Aim of the Study: In the present study, the anti-inflammatory and anti-allergic effects of AP extract in in vitro cell lines and in vivo mouse model of inflammation and the molecular mechanisms involved were reported., Materials and Methods: Using Raw 264.7 murine macrophages the effects of methanol extract of AP in lipopolysaccharide (LPS)-induced production of inflammatory mediators were measured. Further IgE-DNP-induced interleukin (IL)-4 production and degranulation in RBL-2H3 rat basophilic cell lines was also estimated. To investigate the anti-asthmatic effect of AP in vivo, airway inflammation in ovalbumin (OVA)-induced mouse model was used., Results: AP attenuated the production of inflammatory mediators such as NO, PGE(2) and pro-inflammatory cytokines in LPS-induced Raw 264.7 cells. Further, AP inhibited IL-4 production and degranulation in IgE-DNP-induced RBL-2H3 cells. Furthermore, AP attenuated the infiltration of immune cells into lung, cytokines production in broncho-alveolar lavage fluid (BALF) and airway-hyperresponsiveness (AHR) on OVA-induced mouse model of inflammation., Conclusion: Our results showed that AP attenuated the activation of macrophages, basophils, and inhibited the OVA-induced airway inflammation. The molecular mechanisms leading to AP's potent anti-inflammatory and anti-allergic effects might be through regulation of TRIF-dependent and Syk-PLCγ/AKT signaling pathways, suggesting that AP may provide a valuable therapeutic strategy in treating various inflammatory diseases including asthma., (Copyright © 2012. Published by Elsevier Ireland Ltd.)

Wasabi is a plant of Japanese origin. It belongs to the family Brassicaceae and produces various isothiocyanates (ITCs). To clarify the type I allergies inhibited by wasabi ITCs, we investigated the inhibitory effect on chemical mediator release from dinitrophenylated bovine serum albumin (DNP-BSA)-stimulated RBL-2H3 rat basophilic leukemia cells. Allyl ITC (AITC), sec-butyl ITC (s-BuITC), and 3-butenyl ITC (3-BuITC), which have 3 or 4 carbon chains, inhibited histamine release but did not inhibit the release of leukotriene B4 (LTB4) or cysteinyl LTs (CysLTs). 4-Pentenyl ITC (4-PeITC) and 5-hexenyl ITC (5-HeITC), which have 5 or 6 carbon chains and an unsaturated bond at the end, inhibited LTB4 release but did not inhibit the release of histamine or CysLTs. 6-Methylthiohexyl ITC (6-MTITC), 6-methylsulfinylhexyl ITC (6-MSITC), and 6-methylsulfonylhexyl ITC (6-MSFITC), which have a sulfur atom inserted at the end of a 6-carbon chain, inhibited the release of histamine, LTB4, and CysLTs and the elevation in intracellular Ca(2+). These results suggest that wasabi ITCs inhibited type I allergies by inhibiting chemical mediator release and that the inhibitory effects on each chemical mediator were due to differences in the side chain structure of the wasabi ITCs.

Stability and resistance to dissolution are key features of microbial biofilms. How these macroscopic properties are determined by the physiological state of individual biofilm cells in their local physical-chemical and cellular environment is largely unknown. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is an energy-dependent process and whether transcription and/or translation is required for biofilm dissolution. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or by addition of the inhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), dinitrophenol (DNP), or CN(-), resulted in massive dissolution. In 60-hour-old biofilms, the extent of uncoupler-induced cell loss was strongly attenuated, indicating that the integrity of older biofilms is maintained by means other than those operating in younger biofilms. In experiments with 12-hour-old biofilms, the transcriptional and translational inhibitors rifampin, tetracycline, and erythromycin were found to be ineffective in preventing energy starvation-induced detachment, suggesting that neither transcription nor translation is required for this process. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to an extent similar to that in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and this may represent a common but not ubiquitous mechanism for stability of microbial biofilms.

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

Introduction and Hypothesis: There are a number of lower urinary tract dysfunctions (LUTD) that occur primarily in women. Our hypothesis is that cyclical estrogen will produce LUTD in part by the generation of free radicals and oxidative damage to cellular and subcellular membranes., Methods: Twenty female rabbits were divided into five groups: control, ovariectomized (Ovx), Ovx receiving continuous estrogen, Ovx receiving cyclical estrogen ending off estrogen, and Ovx receiving cyclical estrogen ending on estrogen. Statistical analyses used ANOVA followed by the Tukey analysis for individual differences., Results: High estrogen increased bladder mass, contraction, compliance, and blood flow and decreased oxidative damage. Low estrogen decreased bladder mass, contraction, compliance, and blood flow and increased oxidative damage., Conclusions: The decreased blood flow associated with increased oxidative damage demonstrates that cyclical damage to cellular membranes occurs. This supports the hypothesis that cycling estrogen may play a role in the etiology of LUTD of women.

Twenty-five aromatic nitro, dinitro and trinitro compounds were isolated in low yields of less than 1 mg l(-1) from a Salegentibacter sp. strain T436 derived from Arctic pack ice. Their structures were elucidated by MS and NMR techniques. Seven of these compounds, namely, 2-hydroxy-3-(4'-hydroxy-3'-nitrophenyl)-propionic acid methyl ester (6), 2-chloro-3- (4'-hydroxy-3'-nitrophenyl)propionic acid methyl ester (7), 3-(4'-hydroxy-3',5'-dinitrophenyl)-propionic acid methyl ester (14), 4'-hydroxy-3',5'-dinitrophenylethylchloride (16), (4'-hydroxy-3',5'-dinitrophenyl)-2-chloropropionic acid methyl ester (17), N-acetyl-3',5'-dinitrotyramine (18) and 2,6-dinitro-4-(2'-nitroethenyl)phenol (19) are new, and five are reported in this study from a natural source for the first time.

Skeletal muscle is the major store and consumer of fatty acids and glucose. Glucose enters muscle through glucose transporter 4 (GLUT4). Upon insufficient oxygen availability or energy compromise, aerobic metabolism of glucose and fatty aids cannot proceed, and muscle cells rely on anaerobic metabolism of glucose to restore cellular energy status. An increase in glucose uptake into muscle is a key response to stimuli requiring rapid energy supply. This chapter analyses the mechanisms of the adaptive regulation of glucose transport that rescue muscle cells from mitochondrial uncoupling. Under these conditions, the initial drop in ATP recovers rapidly, through a compensatory increase in glucose uptake. This adaptive response involves AMPK activation by the initial ATP drop, which elevates cell surface GLUT4 and glucose uptake. The gain in surface GLUT4 involves different signals and routes of intracellular traffic compared with those engaged by insulin. The hormone increases GLUT4 exocytosis through phosphatidylinositol 3-kinase and Akt, whereas energy stress retards GLUT4 endocytosis through AMPK and calcium inputs. Given that energy stress is a component of muscle contraction, and that contraction activates AMPK and raises cytosolic calcium, we hypothesize that the increase in glucose uptake during contraction may also involve a reduction in GLUT4 endocytosis.

We demonstrate here that the transient receptor potential melastatin subfamily channel, TRPM4, controls migration of bone marrow-derived mast cells (BMMCs), triggered by dinitrophenylated human serum albumin (DNP-HSA) or stem cell factor (SCF). Wild-type BMMCs migrate after stimulation with DNP-HSA or SCF whereas both stimuli do not induce migration in BMMCs derived from TRPM4 knockout mice (trpm4(-/-)). Mast cell migration is a Ca(2+)-dependent process, and TRPM4 likely controls this process by setting the intracellular Ca(2+) level upon cell stimulation. Cell migration depends on filamentous actin (F-actin) rearrangement, since pretreatment with cytochalasin B, an inhibitor of F-actin formation, prevented both DNP-HSA- and SCF-induced migration in wild-type BMMC. Immunocytochemical experiments using fluorescence-conjugated phalloidin demonstrate a reduced level of F-actin formation in DNP-HSA-stimulated BMMCs from trpm4(-/-) mice. Thus, our results suggest that TRPM4 is critically involved in migration of BMMCs by regulation of Ca(2+)-dependent actin cytoskeleton rearrangements.

Helminthic infections, which are particularly common in the developing world, are associated with the accumulation of mucosal mast cells (MMCs) in the epithelial layer of the gut. Although intestinal parasite infection models argue that IL-18 plays a role in MMC differentiation and function, the direct effect of IL-18 on MMCs is still not well understood. To clarify the role of IL-18 in mast cell biology, we analyzed gene expression changes in MMCs in vitro. DNA microarray technology uncovered a group of chemokines regulated by IL-18, among which Ccl1 (I-309, TCA-3) showed the highest up-regulation. Ccl1 induction was only transient in mast cells and was characteristic for both immature and mature MMCs, but not for connective tissue-type mast cells. IL-18 exerts its Ccl1-inducing effect in MMCs primarily via the activation of NFkappaB. Moreover, IL-18 was effective both in the absence and the presence of IgE-antigen complex. The Ccl1 receptor (CCR8) is known to be expressed by T(h)2 cells and is involved in their recruitment. Our present findings suggest that IL-18 may contribute to mast cell-influenced Th2 responses by inducing Ccl1 production.

This article describes a new method for direct fluorometric immunoassay with a liposome array using pH-sensitive dye (BCECF [2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein])-encapsulating liposomes immobilized on an avidin slip and gramicidin channels. The liposomes were composed of phosphatidylcholine (PC), cholesterol (Chol), biotinylated phosphatidylethanolamine (B-cap-PE), and recognition sites (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(2,4-dinitrophenyl) [DNP-PE], Fab' fragment of anti-substance P, and Fab' of anti-neurokinin A). The addition of gramicidin induced release of H(+) ions from the inner solution (pH 5.5) to the outer one (pH 7.8), enhancing fluorescence of BCECF (1.0mM) encapsulated in liposome. The binding of an analyte (anti-dinitrophenyl [anti-DNP], avidin, substance P, or neurokinin A) to the membrane-bound recognition sites caused further enhancement of fluorescence of BCECF due to a local distortion of the bilayer structure that affects the channel kinetics of gramicidin. The intensity of fluorescence from the immobilized liposomes 60 min after the addition of gramicidin (10 ng/ml) increased with an increase in the concentration of anti-DNP ranging from 1.2 x 10(-8) to 1.2 x 10(-6)g/ml, avidin ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml, substance P ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml, and neurokinin A ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml. The direct fluorometric immunoassay with a liposome array is simple and easy to carry out. The intensity of fluorescence emitted from the immobilized liposomes is directly measured after incubation with a sample solution and a gramicidin solution in sequence without washing steps. The assay allows simultaneous quantification of multiple components without labeling of antibody or antigen with a fluorescent tag. The liposome-based assay is discussed in terms of principle, sensitivity, and selectivity.

Previous studies of secretion from basophils have demonstrated the phenomenon called nonspecific desensitization, the ability of one IgE-mediated stimulus to alter the cell's response to other non-cross-reacting IgE-mediated stimuli, and a process that would modify phosphatidylinositol 3,4,5-phosphate levels was speculated to be responsible for nonspecific desensitization. The current studies examined the changes and characteristics of SHIP1 phosphorylation as a measure of SHIP1 participation in the reaction. Based on the earlier studies, two predictions were made that were not observed. First, the kinetics of SHIP1 phosphorylation were similar to reaction kinetics of other early signals and returned to resting levels while nonspecific desensitization remained. Second, in contrast to an expected exaggerated SHIP phosphorylation, cells in a state of nonspecific desensitization showed reduced SHIP phosphorylation (compared with cells not previously exposed to a non-cross-reacting Ag). Discordant with expectations concerning partial recovery from nonspecific desensitization, treatment of cells with DNP-lysine to dissociate bound DNP-HSA, either enhanced or had no effect on SHIP phosphorylation following a second Ag. These experiments also showed a form of desensitization that persisted despite dissociation of the desensitizing Ag. Recent studies and the results of these studies suggest that loss of early signaling components like syk kinase may account for some of the effects of nonspecific desensitization and result in a form of immunological memory of prior stimulation. Taken together, the various characteristics of SHIP phosphorylation were not consistent with expectations for a signaling element involved in nonspecific desensitization, but instead one which itself undergoes nonspecific desensitization.

The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.

Neutralization of 4,6-dinitro-o-cresol, of which the best-before date had expired and which could not be used any longer, was carried out by means of the detonative combustion method. The pesticide comprises two nitro groups and is called DNOC. Composition of the applied explosive was designed on the basis of porous ammonium nitrate as an oxidizer and the pesticide as fuel. The thermodynamic parameters of such an explosive were estimated, and then three series of experiments with detonative decomposition of the material under a shield of sand were performed: in a steel chamber, in shot-holes in soil, and in shot-holes in a quarry. The efficiency of the pesticide decomposition was examined by determination of the residual remainder of the pesticide in the environment by means of gas chromatography. It was stated that pesticide content in the analyzed samples was below the determination threshold of the applied method. Biological tests with plants were carried out as well. Suggestion was made that the energy being produced during detonative combustion can be utilized for useful work, e.g. rock mining in a quarry.

Chronic exposure of pubertal male rats to ethanol results in a decline in serum testosterone and decreased or inappropriately normal serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels suggesting a functional defect in the pituitary. The molecular mechanisms behind this disorder are undefined. A role for ethanol-induced oxidative damage in the pathophysiology is supported by studies in liver, muscle, and heart of experimental animals, but there is limited evidence in the pituitary. We examined markers of oxidative damage to lipids and proteins in pituitaries from rats consuming ethanol for 5, 10, 20, 30, and 60 days in addition to markers of damage to nucleic acids in pituitaries after 60 days of ethanol exposure. There were increases in 8-oxo-deoxyguanosine immunoreactivity, a marker of oxidative damage to nucleic acids, and an overall increase in malondialdehyde and 4-hydroxynonenal, markers of lipid peroxidation. Protein carbonylation and protein nitrotyrosination, markers of protein oxidation, were significantly increased after 30 days and 60 days of ethanol consumption, respectively. After 60 days of ethanol exposure, TUNEL assay revealed that cell death in the ethanol-treated pituitaries was not significantly different from that in the pair-fed controls at the time of examination. We also measured serum testosterone, FSH, and LH after ethanol consumption for 5, 10, 20, 30, and 60 days. Through 5 to 60 days of ethanol exposure, testosterone levels were consistently lower whereas LH and FSH were inappropriately unchanged, suggesting pituitary malfunction. These results provide evidence for ethanol-induced oxidative damage at the pituitary level, which may contribute to pituitary dysfunction.

Background: Oxidative and nitrosative stress have been involved in gentamicin-induced nephrotoxicity. The purpose of this work was to study the effect of S-allylmercaptocysteine, a garlic derived compound, on gentamicin-induced oxidative and nitrosative stress and nephrotoxicity. In addition, the in vitro reactive oxygen species scavenging properties of S-allylmercaptocysteine were studied., Results: S-allylmercaptocysteine was able to scavenge hydroxyl radicals and singlet oxygen in vitro. In rats treated with gentamicin (70 mg/Kg body weight, subcutaneously, every 12 h, for 4 days), renal oxidative stress was made evident by the increase in protein carbonyl content and 4-hydroxy-2-nonenal, and the nitrosative stress was made evident by the increase in 3-nitrotyrosine. In addition, gentamicin-induced nephrotoxicity was evident by the: (1) decrease in creatinine clearance and in activity of circulating glutathione peroxidase, and (2) increase in urinary excretion of N-acetyl-beta-D-glucosaminidase, and (3) necrosis of proximal tubular cells. Gentamicin-induced oxidative and nitrosative stress and nephrotoxicity were attenuated by S-allylmercaptocysteine treatment (100 mg/Kg body weight, intragastrically, 24 h before the first dose of gentamicin and 50 mg/Kg body weight, intragastrically, every 12 h, for 4 days along gentamicin-treatment)., Conclusion: In conclusion, S-allylmercaptocysteine is able to scavenge hydroxyl radicals and singlet oxygen in vitro and to ameliorate the gentamicin-induced nephrotoxicity and oxidative and nitrosative stress in vivo.

The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.

The ability of allergens to induce hyperalgesia in immunoglobulin E (IgE)-sensitized rats was investigated. The left hind paws of Wistar rats were sensitized with intraplantar injections of IgE anti-dinitrophenylated bovine serum albumin monoclonal antibody, and challenged with dinitrophenylated bovine serum albumin 24 h later. Allergen challenge yielded rapid thermal hyperalgesia and oedema formation in the ipsilateral paws, both reaching a plateau from 15 min to 3 h, and both diminishing thereafter. Allergen-evoked hyperalgesia was inhibited by intraperitoneal treatment with meclizine or methysergide, histamine and 5-hydroxytryptamine receptor antagonists. There was also sensitivity to local treatment with either bradykinin B(1) or B(2) receptor antagonists, des-Arg(9)-[Leu(8)]-bradykinin or D-arginyl-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140). Anaphylactic hyperalgesia was mimicked by the combined administration of histamine, 5-hydroxytryptamine and bradykinin at doses which were ineffective when injected alone. This synergistic effect was abolished by treatment with either meclizine, methysergide, Hoe 140 or des-Arg(9)-[Leu(8)]-bradykinin. Our findings show that local thermal hyperalgesia is a feature of allergen-evoked inflammation, and that a synergistic interaction among bradykinin, 5-hydroxytryptamine and histamine plays a critical role in this phenomenon.

Aims: To investigate the possibility of reducing excess sludge production in activated sludge processes by the addition of chemical uncouplers to greatly dissociate anabolism from catabolism., Methods and Results: Ortho-chlorophenol (oCP), 2,4-dichlorophenol (DCP), 3,3',4',5-tetrachlorosalicylanilide (TCS), para-dinitrophenol (pNP) and 2,4-dinitrophenol (DNP) were chosen for short-term tests for their ability to reduce sludge yield by shaking bottle test. The most effective chemicals, DNP and pNP, together with TCS were tested for various uncoupler concentrations and biomass concentrations. TCS was tested in a lab-scale completely mixed activated sludge batch culture. The model (demonstrated by Liu) was verified with experimental data in completely mixed activated sludge batch test, but was inconsistent with the results from the shaking bottle batch test. The observed growth yield (Yobs) decreased with increasing of the ratio of initial uncoupler concentration to initial biomass concentration (Cu/X0)., Conclusions: We suggest that the uncouplers oCP, DCP, TCS, pNP and DNP can cause a significant decrease in sludge production, the metabolism of which can explain the decline in sludge yield., Significance and Impact of the Study: The real strength of chemical uncoupler imposing on biomass should be Cu/X0, not initial uncoupler concentration (Cu) alone. Chemical uncouplers can be used to develop the activated sludge processes for minimizing excess sludge production.

We have earlier reported chemotaxis of a Gram-negative, motile Ralstonia sp. SJ98 towards p-nitrophenol (PNP), 4-nitrocatechol (NC), o-nitrobenzoate (ONB), p-nitrobenzoate (PNB), and 3-methyl-4-nitrophenol (MNP) that also served as sole source of carbon and energy to the strain [S.K. Samanta, B. Bhushan, A. Chauhan, R.K. Jain, Biochem. Biophy. Res. Commun. 269 (2000) 117; B. Bhushan, S.K. Samanta, A. Chauhan, A.K. Chakraborti, R.K. Jain, Biochem. Biophy. Res. Commun. 275 (2000) 129]. In this paper, we report chemotaxis of a Ralstonia sp. SJ98 toward seven different nitroaromatic compounds (NACs) by drop assay, swarm plate assay, and capillary assay. These NACs do not serve as sole carbon and energy source to strain SJ98 but are partially transformed in the presence of an alternate carbon source such as succinate. This is the first report showing chemotaxis of a bacterial strain toward co-metabolizable NACs.

Mitochondrial oxidative damage from free radicals may be a factor underlying aging. We investigated whether long-term administration of physiological levels of melatonin, a direct free radical scavenger and indirect antioxidant, influences mitochondrial respiratory activity in liver of senescence-accelerated mice (SAM). Liver was obtained in the middle of dark period of the daily light:dark cycle from SAMP8, a strain of mice prone to accelerated senescence, and from SAMR1, a senescence-resistant strain, at 6 and 12 months of age. Respiratory control index (RCI), adenosine-5-diphosphate (ADP)/O ratio, State 3 respiration and dinitophenol (DNP)-dependent uncoupled respiration exhibited significant age-associated decreases in SAMP8. SAMP8 also showed significant age-associated reductions in respiratory chain complex I and IV activities. No age-related effects were found in these parameters in SAMR1. Daily oral melatonin administration (2 microg/mL of drinking fluid) beginning at 7 months of age significantly increased RCI, State 3 respiration, DNP-dependent uncoupled respiration, and complex I and IV activities in both mouse strains when they were 12 months old. These results reveal age-related reductions in mitochondrial function in SAM mice which are modified by melatonin; the most likely explanation for the corrective actions of melatonin relate to its antioxidative actions in mitochondria and other portions of the cell. The implication of the findings is that melatonin may be beneficial during aging as it reduces the deteriorative oxidative changes in mitochondria and other portions of the cell associated with advanced age.

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.

The peptidomimetic drug gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine diethyl ester (TER199) is an analog of glutathione designed to be an isozyme-specific inhibitor of GSTP1-1 protein1-1. This compound (and the de-esterified moiety) is shown to be an effective inhibitor of multidrug resistance-associated protein1 (MRP1)-mediated drug resistance. Kinetic analyses revealed that gamma-glutamyl-S-(benzyl)cysteinyl-R-(-)-phenyl glycine reversibly inhibits the transport of 2,4-dinitrophenyl-S-glutathione with a K(i) of 752 microM. TER199 reversed the accumulation deficit of daunorubicin in MRP1-transfected NIH3T3 fibroblasts and maintained intracellular levels for >2 h after daunorubicin removal. Cytotoxicity assays revealed that TER199 significantly reversed the resistance of MRP1-transfected NIH3T3 cells for vincristine, doxorubicin, etoposide, and mitoxantrone. HL-60 cells made resistant to TER199 by chronic, long-term selection had increased mRNA and protein levels of multidrug resistance-associated protein, MRP1, and gamma-glutamyl cysteine synthetase heavy and light subunits (the rate-limiting enzyme in GSH synthesis). In spite of increased gamma-glutamyl cysteine synthetase, their glutathione content was reduced approximately 35% from that of parental HL-60 cells. These cells also exhibited a drug resistance profile commensurate with the previously described MRP1 overexpressing phenotype, with resistance to Vinca alkaloids, epipodophyllotoxins, and anthracyclines; additional cross-resistance to paclitaxel (Taxol), mitoxantrone, and 5-fluorouracil was observed.

We report an optimized in situ hybridization (ISH) protocol with a rapid signal amplification procedure based on catalyzed reporter deposition (CARD) to increase the sensitivity of non-isotopic mRNA ISH on formaldehyde-fixed and paraffin-embedded tissue. The CARD method is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. Commercially available and newly synthesized haptenized tyramides, including digoxigenin-, biotin-, di- and trinitrophenyl- as well as fluorescein-tyramide, were compared. The haptenized tyramides were visualized using peroxidase conjugated anti-hapten antibodies followed by the diaminobenzidine reaction. As a test system, we applied digoxigenin-labeled oligonucleotides to detect insulin and vasoactive intestinal polypeptide mRNA in pancreatic endocrine tumors and liver metastases. Our results indicate that specificity, sensitivity, and applicability of oligonucleotide mRNA ISH can be significantly improved by using chemically digoxigenin-labeled oligonucleotide probes and signal amplification by CARD. Furthermore, all tested tyramides provided approximately equal amplification efficiency. In conclusion, CARD signal amplification should further promote mRNA ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ.

Background: Poisoning with agricultural toxins is well known in the adult environment, but is rarely seen in children. Vancouver, British Columbia, borders on a large agricultural area, and the British Columbia Children's Hospital is the provincial referral center for major pediatric emergencies. In our experience, children from farming communities are vulnerable to a range of agriculturally related injuries, and exposure to toxic chemicals requiring hospitalization is not rare., Methods: This is a case report with literature review., Results: A child presenting in a coma proved later to have been exposed to a toxic agent. The agent, dinitrophenol, is a widely used agricultural herbicide and insecticide. The diagnosis resulted from a detailed history and physical examination, which revealed symptoms and signs that suggested poisoning. A search of the farm resulted, and the parents located open dinitrophenol containers. The toxic effects of dinitrophenol described in adults by Poison Control matched those present in the child, and specific treatment interventions existed that could be implemented to positively influence outcome., Conclusions: Awareness of the possibility of exposure to dinitrophenol is required, as death can result from exposure, and milder cases may go unrecognized. Appropriate treatment can improve outcome. Injuries with toxic agricultural agents are wholly preventable if the materials are handled appropriately in the farm environment, and parents whose children live on or visit farms are aware of the risks involved and take appropriate precautions.

Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to Fc epsilon RI to stimulate cellular responses, although key features of the binding process are unknown. Fluorescence quenching can be used to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number of DNP groups per BSA is less than one. The binding data are well described by a transient hapten exposure model in which most of the DNP groups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measurement. Additional experiments indicate that, for suboptimal to optimal concentrations of DNP-BSA, most of the FITC fluorescence quenching on the cell surface is due to cross-linking events. With these concentrations at 15 degrees C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine phosphorylation of Fc epsilon RI. At 35 degrees C, the phosphorylation kinetics are biphasic during the time period in which cross-linking continues to increase. Our results establish a quantitative relationship between the time-course for cross-linking by multivalent Ag and Fc epsilon RI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.

A mixed culture of microorganisms able to utilize 4,6-dinitro-ortho-cresol (DNOC) as the sole source of carbon, nitrogen and energy was isolated from soil contaminated with pesticides and from activated sludge. DNOC was decomposed aerobically in batch cultures as well as in fixed-bed column reactors. Between 65% and 84% of the substrate nitrogen was released as nitrate into the medium, and 61% of the carbon from uniformly 14C-labelled DNOC was recovered as 14CO2. The mixed microbial culture also decomposed 4-nitrophenol and 2,4-dinitrophenol but not 2,3-dinitrophenol, 2,6-dinitrophenol, 2,4-dinitrotoluene, 2,4-dinitrobenzoic acid or 2-sec-butyl-4,6-dinitrophenol (Dinoseb). Maximal degradation rates for DNOC by the bacterial biofilm immobilized on glass beads in fixed-bed column reactors were 30 mmol day-1 (1 reactor volume)-1, leaving an effluent concentration of less than 5 micrograms l-1 DNOC in the outflowing medium. The apparent Ks value of the immobilized mixed culture for DNOC was 17 microM. Degradation was inhibited at DNOC concentrations above 30 microM and it ceased at 340 microM, possibly because of the uncoupling action of the nitroaromatic compound on the cellular energy-transducing mechanism.

The title compounds, (Xylp beta (1-->4))nXylp beta-3,4-DNP (n = 0-4) have been made by selective anomeric deprotection of peracetylated xylose oligosaccharides with hydrazine, followed by formation of the trichloroacetimidate, uncatalysed reaction with 3,4-dinitrophenol, and Zemplén deacetylation. The values of k(cat)/K(m) for 3,4-dinitrophenol release from these substrates by xylanase III of Streptomyces cyaneus, expressed in Escherichia coli, increase with increasing n up to n = 2 and then slightly decrease. Since it is known from previous work that in its normal host, the enzyme is produced constitutively at low levels and excreted, these results suggest that the biological function of the enzyme may be to produce small molecule inducers, predominantly xylotriose, from the non-reducing end of the xylan. Activity on cellooligosaccharide glycosides (Glcp beta (1-->4))nGlcp beta-3,4-DNP (n = 0-3) was detected, at a rate about two-and-a-half orders of magnitude less than that observed on the corresponding xylooligosaccharides, indicating that the enzyme is a true xylanase.

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response.

Immune complexes were formed between dinitrophenylated human serum albumin (DNP-HSA) and polyclonal rabbit immunoglobulin G (IgG) anti-DNP antibodies at antibody excess. The antigen was labelled with isotope (125I-tyramine-cellobiose) or fluorochrome, (6-[fluorescein-5-(and-6)-carboxamido] hexanoic-acid, succinimidyl ester). The radiolabelled antigen, native or antibody complexed, was given intravenously to rats. Radioactivity was measured in various organs at 1 hour following injection. The liver was the main site for removal of the antigen as well as of the immune complexes. Within the liver, immune complexes were mainly associated with nonparenchymal liver cells, the total recovery from Kupffer cells being about 10 times greater than from the liver endothelial cells. The uncomplexed radiolabelled antigen was readily degraded by both cells types. After IgG complexing, the degradation decreased, both in Kupffer cells and in liver endothelial cells. In vitro experiments with isolated liver cells, showed that IgG complexing increased antigen uptake to about the same extent in Kupffer cells and in liver endothelial cells. The degradation of both antigen and immune complexes was less efficient in vitro than in vivo. Immune complex uptake in vitro was shown also by confocal fluorescence microscopy in Kupffer cells and in liver endothelial cells. Also in vitro, only minor uptake was found in the hepatocytes. We conclude that both liver endothelial cells and Kupffer cells are involved in the hepatic handling of soluble IgG immune complexes, but we found no evidence for substantial uptake by hepatocytes.

A strain of Clostridium bifermentans, KMR-1, degraded 2-sec-butyl-4,6-dinitrophenol (dinoseb) to a level below the limit of detection by high-performance liquid chromatography (0.5 mg/liter) within 96 h, with no accumulation of aromatic intermediates. KMR-1 could not utilize dinoseb as a sole carbon or energy source, and degradation occurred via cometabolism in the presence of a fermentable carbon source. KMR-1 mineralized some dinoseb in anaerobic cultures, evolving 7.2% of the radioactive label in U-ring 14C-labeled dinoseb as 14CO2. The remaining anaerobic degradation products were incubated with aerobic soil bacteria, and 35.4% of this residual radioactive label was evolved as 14CO2. During this mineralization experiment, 38.9% of the initial label was evolved as 14CO2 after both anaerobic and aerobic phases. This is the first demonstration of dinoseb degradation by a pure microbial culture.

To detect high density lipoprotein binding proteins, a new ligand blotting method that uses apolipoprotein AI-dinitrophenol as a ligand was established. Apolipoprotein AI-dinitrophenol was prepared by incubating the mixture of apolipoprotein AI and 2,4-dinitrobenzene sulfonic acid sodium salt at pH 8.5. Four to six dinitrophenol groups were found to be conjugated with an apolipoprotein AI molecule without any obvious impairment of its binding activity to the binding proteins. By this detection procedure, we detected two apolipoprotein AI binding proteins in plasma membrane fractions from bovine liver and adrenal cortex with molecular masses of 120 kDa and 95 kDa. Since this new ligand blotting method is simple and time-saving, it is expected to become a useful tool in the studies on high density lipoprotein binding proteins.

In the present study, we have evaluated the glomerular handling of circulating glycated albumin in the normal mouse kidney by quantitative immunocytochemistry. Bovine serum albumin (BSA) was glycated in vitro and dinitrophenylated. Glycated and nonglycated probes were introduced into the circulation of anesthetized mice and traced by postembedding immunogold cytochemistry after 10 and 30 min of circulation. Endogenous albumin, as well as dinitrophenylated native BSA (DNP-BSA) and glycated albumins (DNP-gBSA), were localized within the capillary lumen, glomerular and peritubular basement membranes, and the mesangial matrix. Morphometric evaluation of the labeling over the glomerular basement membrane (GBM) revealed a peak of labeling in the endothelial side for either endogenous albumin or DNP-BSA. In contrast, the labeling distribution for DNP-gBSA showed a shift toward the epithelial side, suggesting a further penetration of the glycated probe into the GBM. When coinjected with gBSA, DNP-BSA was found to display a labeling distribution similar to that displayed by DNP-gBSA. These results indicate that the glycated tracer penetrates the normal glomerular wall deeper than the nonglycated one. Moreover, glycated albumin increases the infiltration of the nonglycated tracer through the normal glomerular wall. Circulating glycated serum proteins thus appear to play an important role in the onset of the glomerular dysfunction and proteinuria, which take place in long-term hyperglycemic states.

The degradation pathway for dinoseb (2-sec-butyl-4,6-dinitrophenol) under reducing conditions was investigated. Cultures were inoculated with a dinoseb-degrading anaerobic enrichment culture used in field studies. Biotransformation intermediates were extracted with ethyl acetate and analyzed by high pressure liquid chromatography, gas chromatography, and mass spectrometry. Dinoseb degradation involves reduction of the nitro groups to amino groups followed by replacement with hydroxyl groups. Depending on the pH and redox potential in the culture, these intermediates may exist as quinones or hydroquinones.

The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain), isolated from a continuous anaerobic digester, used various nitroaromatic compounds such as 2,4-dinitrophenol, 2,4-dinitrotoluene, and 2,6-dinitrotoluene as sole nitrogen sources for growth and also used these compounds as electron acceptors in the absence of sulfate in the culture medium. More than 60% of the nitroaromatics were transformed within 6 days of incubation. The organism also used aniline as sole nitrogen source, but not as an electron acceptor. Desulfovibrio sp. (B strain) did not use nitroaromatics as sole source of carbon and energy. The nitro groups in the aromatic ring were reduced and reductively deaminated to ammonia, which was used as nitrogen source, leaving the aromatic ring intact. Even though this organism did not degrade the nitroaromatics completely, it may be useful in degrading nitroaromatics in contaminated soil and water containing other aromatic degraders in a syntrophic culture system under anaerobic conditions.

The secretion of a glutathione-S-conjugate, dinitrophenyl-glutathione (GS-DNP) was studied in the Caco-2 cells, a cultured human colonic adenocarcinoma cell line with many of the characteristics of enterocytes. The labelled glutathione conjugate was generated within the cell by incubation with 14C-labelled 1-chloro-2,4-dinitrobenzene (CDNB). This compound is hydrophobic and enters the cell by simple diffusion. Cells incubated with CDNB at 10 degrees C form only one metabolite, GS-DNP. After secretion into the medium GS-DNP is partly converted into one or two slightly more hydrophobic products. This must represent hydrolysis of the glutathione moiety by the action of gamma-glutamyltransferase (EC 2.3.2.2.; gamma-GT) because the reaction was completely inhibited by acivicin, an inhibitor of gamma-GT. Secretion of GS-DNP was a temperature-sensitive, saturable process with an apparent Km of 1.03 +/- 0.26 nmol/mg of protein and a Vmax of 111 +/- 17 pmol/min per mg of protein. The secretion was not sensitive to trans-stimulation by extracellular concentrations of GS-DNP up to 2.5 mM. Furthermore the initial GS-DNP secretion rate was sensitive to dissipation of the membrane potential and correlated closely with the cellular ATP content. Caco-2 cells cultured on nitrocellulose filters secreted GS-DNP significantly faster over the basolateral membrane than over the apical membrane (146 +/- 25 versus 90 +/- 18 pmol/min per mg respectively). Secretion over both membrane domains of the cell was sensitive to ATP depletion. In conclusion, Caco-2 cells contain an active-transport system that is primarily involved in the secretion of glutathione conjugates and that is present in both plasma membrane domains of the cell.

Objective: The aim was to assess the effects of various antiarrhythmic drugs on 2,4-dinitrophenol (DNP) induced outward current (IDNP), presumably the ATP sensitive K+ current (IK,ATP) of isolated cardiac cells and to discuss mechanisms involved in the hypoglycaemia which occurs in patients on these drugs., Methods: The quasi-steady state current-voltage relationship from the isolated guinea pig ventricular cells was measured using whole cell voltage clamp techniques with a ramp pulse programme. The effects of seven different antiarrhythmic drugs on IDNP were examined. Action potentials were elicited at a rate of 0.2 Hz by an intracellular current injection., Results: DNP (50 mumol.litre-1) increased the quasi-steady state outward current at potentials positive to about -60 mV. This current (IDNP) was completely inhibited by the subsequent application of glibenclamide (1 mumol.litre-1), thereby suggesting that the IDNP is probably IK,ATP. Cibenzoline (10 mumol.litre-1, class Ia), disopyramide (30 mumol.litre-1, class Ia), and procainamide (100 mumol.litre-1, class Ia) significantly inhibited the IDNP by 95.5(SD 11.3)%, 77.8(21.2)%, and 76.4(23.9)% respectively. Flecainide (class 1c) inhibited the IDNP by 66.9(23.9)% at 10 mumol.litre-1 but not at 2 mumol.litre-1. Mexiletine (30 mumol.litre-1, class Ib), pilsicainide (50 mumol.litre-1, class Ic), and E4031 (10 mumol.litre-1, class III) at concentrations as high as approximately fivefold the clinically effective blood levels, did not suppress IDNP. Except for 10 mumol.litre-1 flecainide, all the concentrations listed above which blocked IDNP were within twofold of the clinical blood concentrations documented to be effective for suppression of arrhythmias. Cibenzoline, disopyramide, and procainamide, but not flecainide, belong to class Ia antiarrhythmic drugs. All these class Ia antiarrhythmic drugs "shortened" the action potential duration of guinea pig ventricular cells, an opposite change to that noted for multicellular preparations, eg, guinea pig papillary muscles., Conclusions: Class Ia antiarrhythmic drugs (cibenzoline, disopyramide, and procainamide) inhibit IDNP (presumably IK,ATP) in guinea pig ventricular cells within a range of therapeutic concentrations. This inhibitory effect of IK,ATP can probably explain the hypoglycaemia which occurs in some patients receiving these drugs, and the prolongation of the action potential duration alleged to occur in "superfused" papillary muscles.

A series of nitrophenolic electron-transport inhibitors (2-substituted 4,6-dinitrophenols) of rat liver mitochondrial cytochrome-bc1 complex and of photosystem II (QB site) of spinach thylakoids was synthesized. The structure/inhibitory-activity relationship was examined to elucidate differences in the three-dimensional structure of the quinone redox site in the two systems. These inhibitors occupy the ubiquinone redox site of cytochrome-bc1 complex competitively with natural ubiquinol, probably at a Qo reaction center. The inhibitory activity tended to increase with the length of the 2-substituent, which may correspond to the isoprenoid side chain of ubiquinone and plastoquinone, increased in both experimental systems. However, the strict structural requirements of the 2-substituent for binding to the ubiquinone or plastoquinone redox site were not identical. The alkyl substituents with a branching structure at the alpha-position to the benzene ring were favorable for inhibition of the cytochrome-bc1 complex, but not of photosystem II. Molecular-orbital calculations indicated that the main chain of 2-substituents with an alpha-branching structure was almost perpendicular to the benzene-ring plane because of steric congestion between the alpha-methyl and phenolic OH groups. The main chain of 2-substituents without an alpha-branching structure was flexible. Molecular-orbital studies indicated that ubiquinol was most stable when the portion of the isoprenoid side chain adjacent to the quinol ring was perpendicular to the quinol-ring plane, because of steric congestion by the vicinal OH and methyl groups. The side chain of plastoquinol was flexible because of the lack of a vicinal methyl group. Thus, the difference in the inhibitory activities between the two systems seemed to reflect the difference in the configuration of the isoprenoid side chain of ubiquinone and plastoquinone. These results suggested that the quinone redox site of the cytochrome-bc1 complex may recognize the configuration of the side chain near the quinone ring in the strict sense, whereas that of photosystem II (QB site) may recognize it in a loose sense.

Two Rhodococcus erythropolis strains, HL 24-1 and HL 24-2, were isolated from soil and river water by their abilities to utilize 2,4-dinitrophenol (0.5 mM) as the sole source of nitrogen. Although succinate was supplied as a carbon and energy source during selection, both isolates could utilize 2,4-dinitrophenol also as the sole source of carbon. Both strains metabolized 2,4-dinitrophenol under concomitant liberation of stoichiometric amounts of nitrite and 4,6-dinitrohexanoate as a minor dead-end metabolite.