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Resveratrol is a phytoalexin abundantly found in red grape skin and is effective in antitumor and antiinflammation associated with immune responses. This study investigated whether resveratrol suppressed immunoglobulin (Ig)E-mediated allergic responses and passive cutaneous anaphylaxis (PCA) in rat RBL-2H3 mast cells and in BALB/c mice. The release of β-hexosaminidase and histamine was enhanced in mast cells sensitized with anti-dinitrophenyl (DNP)-IgE and subsequently stimulated by DNP-human serum albumin (HSA), indicative of mast cell degranulation. When mast cells were pretreated with nontoxic resveratrol at 1-25 μmol/L, such induction was dose dependently diminished. Spleen tyrosine kinase (Syk) and phospholipase Cγ (PLCγ) of sensitized mast cells were activated by stimulation with DNP-HSA antigen, which was dampened by ≥5 μmol/L resveratrol. The phosphorylation of protein kinase C (PKC)μ and PKCθ was attenuated by administering resveratrol to DNP-HSA-exposed mast cells, whereas quiescent PKCζ/λ in sensitized cells was dose-dependently activated by resveratrol. Male BALB/c mice were sensitized for 24 h with DNP-IgE and orally administered with resveratrol 1 h before the DNP-HSA challenge. The histamine concentration was enhanced in sensitized mice challenged to DNP-HSA, which was reversed by administration of 10 mg/kg resveratrol. Additionally, it encumbered the tissue activation of Syk, PLCγ, and PKCμ in antigen-exposed mice. Resveratrol decreased IgE-mediated PCA and alleviated allergic edema of mouse ear and dorsal skin. Mast cell degranulation and allergic inflammation, accompanying the induction of monocyte chemotactic protein-1 and macrophage inflammatory protein-2, were inhibited by supplementing resveratrol to antigen-challenged mice. Resveratrol inhibited mast cell-derived, immediate-type allergic reactions, and these responses of resveratrol suggest possible therapeutic strategies in preventing allergic inflammatory diseases.

Two sulfur-containing amino acids, DL-cysteine (Cys) and DL-penicillamine (PenA), were condensed with ninhydrin to form their spirothiazolidine derivatives. These were separated by HPLC using alpha-acid glycoprotein (AGP) and beta-cyclodextrin (beta-CD) columns. The resolution conditions were optimized and the results were compared. Since the method provided resolution greater than 2 it was also applied to preparative separation. After separation, each of them was detagged using Zn dust and 10% aqueous trifluoroacetic acid. For analytical purposes dinitrophenyl (DNP) derivatives of DL-Cys and DL-PenA were also prepared and were resolved on both the columns. The detection was carried out using photodiode array detection system at 231 nm. The limits of detection were found to be 0.01% and 0.004% for spirothiazolidine carboxylic acid and DNP derivatives, respectively.

Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor dl-threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a beta isoform of Class II phosphatidylinositol 3-kinase (PI3KC2beta). In such clones including clone 5A4C, PI3KC2beta was overexpressed throughout the cell, although endogenous PI3KC2beta was normally expressed only in the ER. Overexpression of PI3KC2beta in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcvarepsilonRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.

Inflammation underlines all major bladder pathologies and represents a defense reaction to injury involving a mandatory participation of mast cells and sensory nerves. Mast cells are particularly frequent in close proximity to epithelial surfaces where they are strategically located in the bladder and release their mediators in response to inflammation. Tryptase is specifically produced by mast cells and modulates inflammation by activating protease-activated receptors (PARs). We recently found that PAR-4 mRNA is up-regulated in experimental bladder inflammation regardless of the initiating stimulus. Because it has been reported that PAR-1, PAR-2, and PAR-3 may also be involved in the processes of inflammation, we used immunohistochemistry to characterize the expression of all known PARs in normal, acute, and chronic inflamed mouse bladder. We found that all four PARs are present in the control mouse bladder, and follow a unique distribution. All four PARs are co-expressed in the urothelium, whereas PAR-1 and PAR-2 are predominant in the detrusor muscle, and PAR-4 is expressed in peripheral nerves and plexus cell bodies. The strong expression of PARs in the detrusor muscle indicates the need for studies on the role of these receptors in motility whereas the presence of PAR-4 in nerves may indicate its participation in neurogenic inflammation. In addition, PARs are differentially modulated during inflammation. PAR-1 and PAR-2 are down-regulated in acute inflammation whereas PAR-3 and PAR-4 are up-regulated. Bladder fibroblasts were found to present a clear demarcation in PAR expression secondary to acute and chronic inflammation. Our findings provide evidence of participation of PARs in the urinary system, provide a working model for mast cell tryptase signaling in the mouse bladder, and evoke testable hypotheses regarding the roles of PARs in bladder inflammation. It is timely to understand the role of tryptase signaling and PARs in the context of bladder biology.

Metabolism of alkyl halides and some organophosphorous compounds by glutathione S-transferases (EC 2.5.1.18) leads to formation of an S-alkylglutathione as a common product. We have developed an HPLC assay for formation of S-methylglutathione and S-ethylglutathione that is applicable to measuring enzyme activity toward a variety of xenobiotic substrates. The conjugates are derivatized with 1-fluoro-2,4-dinitrobenzene to form the corresponding N-2,4-dinitrophenyl derivatives, which are then separated by reverse-phase HPLC with gradient elution. The utility of the method is illustrated by the use of partially purified preparations of rat liver glutathione S-transferases and several prototypic substrates including iodomethane, iodoethane, dichlorvos, and methyl parathion. The limit of detection is about 50 pmol of N-(2,4-dinitrophenyl)-S-alkylglutathione. Advantages of the method over other assays of S-alkyl transferase activity are discussed.

DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.

The capability of a simple countercurrent chromatographic scheme for obtaining high-resolution preparative-scale separations was demonstrated with the separations of a series of dinitrophenyl (DNP) amino acids and peptides. Basic studies on stationary phase retention and partition efficiency with a low-viscosity chloroform-acetic acid-0.1 N hydrochloric acid (2:2:1) phase system together with the results previously obtained with a vascous n-butanol system suggest a general applicability of low-interfacial tension phase systems by the present method.

A microtiter latex antiglobulin test (LAGT) with heavy-and-stable latex particles was developed. Antibodies of weak avidity, such as rabbit antisera to DNP or digoxin were much enhanced by the LAGT, whereas the method only slightly promoted strong avidity antibody, such as that found in rabbit antisera to human chorionic gonadotrophin. A microtiter latex agglutination-inhibition test (LAGIT) proved to be an effective tool for detection of weak antigenic substances such as digoxin and DNP.

A method is described by which different antigens and antibodies in solutions containing deoxycholate (DOC) may be coated to glutaraldehyde (GA)-fixed erythrocytes. This method, based on the use of CrCl3, yields erythrocytes which preserve the antigenicity and antibody activity of the coated molecules and can thus be applied to various assays in which indicator red cells are required. The sensitivity of the assays increases when these red cells are used. The cells form rosettes with the appropriate receptor positive cells. They sediment in Ficoll-Isopaque as do native erythrocytes and do not aggregate spontaneously even when coated at very high or low concentrations of CrCl3 or antigen. This coupling method, which is performed in the presence of DOC, and which requires only a small amount of antigen, is proposed for the coupling of cell membrane dissolved antigens.

Physical changes are observed in the feathers of chickens fed diets with high levels of methionine or cysteine. Chicks were fed diets chemically analyzed to contain 21% crude protein, 0.35% methionine and 0.37% cystine (basal) supplemented with DL-methionine [0.063 (control), 0.25 or 1.45%] or L-cysteine (0.203%). At 3 wk of age, the birds were weighed and the feathers scored for softness. Feather strength (i.e., force-displacement curves) was determined on feathers from the pectoral tract. A significant (P less than 0.05) reduction in body weight and an increase in feather softness were seen when chicks fed the control diet and those fed the diet with 1.45% added methionine were compared. Chicks fed the diet supplemented with 0.203% L-cysteine produced the strongest feathers; those fed diets supplemented with 1.45% DL-methionine produced the weakest feathers. Volatile sulfur compounds released from the feathers were trapped as dinitrophenyl (DNP) thioethers and were analyzed by thin-layer chromatography, high pressure liquid chromatography and mass spectrometry. More bound sulfide (P less than 0.05) was recovered from feathers of chickens fed the diet supplemented with 1.45% methionine than from feathers of chickens fed the other diets. The feather softness score was correlated (r = 0.5; P less than 0.05) with bound sulfide. Thus, consumption of a diet with a level of methionine that is approximately three times the requirement resulted in decreased growth, elevated sulfide bound to the feathers and soft, weak feathers.

In vitro generation of secondary anti-viral H-2 restricted cytotoxic T cells (Tc cells) alloreactive Tc cells, alloantigen-induced T cell proliferation, bacterial lipopolysaccharide (LPS)-induced B cell proliferation and anti-dinitrophenyl IgM and IgG antibody responses were used to evaluate pools of mouse sera (MS) and other animal sera as medium supplements by comparison with 5-10% fetal calf serum (FCS). Some batches of normal MS were comparable with FCS while others inhibited Tc cell responses when used at 1%, particularly with limiting stimulator cell numbers. In contrast, 2 pools of nude mouse sera and several pools of MS from thioglycollate-injected mice (TMS) used at 1% were consistently comparable with FCS at 5-10% in terms of in vitro generation of effector Tc cells and antibody secreting B cells. TMS at 1% was satisfactory for Tc cell responses either fresh, after heat inactivation at 56 degrees C for 30 min, or after storage for 6 months at -70 degrees C. 0.5% or 2% heat-inactivated rat and rabbit sera (but not guinea pig sera) were also satisfactory for Tc cell generation. Total thymidine incorporation into proliferating B or T cells was 5-15-fold lower in cultures with 1% normal MS, nude MS or TMS than with 5% FCS, but stimulation indices with 1% nude MS and TMS were similar to those with 5% FCS, whereas 1% normal MS apparently completely inhibited stimulation. One percent syngeneic TMS is a convenient, reliable alternative to 5-10% FCS in culture medium, supporting all the murine responses listed above.

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.

Dinitrophenyl phosphotidylethanolamine-containing bilayers have been immobilized on carbon-shadowed support films by UV irradiation of the first monolayer transferred to the support film. The immobilized bilayer is capable of allowing bound protein (anti-DNP antibody) to organize into 2-D arrays in the presence of organic solvents such as acetonitrile and dilute concentrations of detergents such as beta-octyl glucoside. The ability of the bilayers to remain attached to supports under various conditions that include organic solvents and detergents as well as divalent ions is of potential interest in the study of protein crystallization and particularly in the study of membrane proteins.

Circulating dinitrophenylated human serum albumin (DNP-HSA) was shown by indirect immunofluorescence microscopy to adhere to non-parenchymal liver cells and capillaries of striated muscle in mice. The DNP-HSA at these sites could be removed by surface-tension-lowering agents such as Triton X-100 and ethylene glycol. The adherence of DNP-HSA to white blood cells in vitro was inhibited in the presence of 0.3 M ethylene glycol. The findings support the hypothesis that hydrophobic interaction with different tissue structures may be important for the fate of circulating antigens and immune complexes. Indirect immunofluorescence microscopy offers a simple means of checking the role of hydrophobic bonding for the tissue adherence of various substances in vivo.

The migration inhibitory factor (MIF) production of peripheral lymphocytes following exposure to dinitrophenylated microsomes derived from both human (DNP-hy-Mic) and guinea pig (DNP-gp-Mic) epidermis was quantitated to detect human contact sensitivity to dinitrochlorobenzene (DNCB). The lymphocytes from nonsensitized subjects did not generate MIF following exposure to either antigen. With DNP-gp-Mic as the antigen, MIF production was noted in only 1 out of 6 DNCB-sensitized subjects and was not significant statistically. With DNP-hu-Mic as the antigen, highly significant MIF production was observed in all 12 sensitized subjects ( p less than 0.0005). In order to confirm MIf production by sensitized lymphocytes following stimulation by DNP-hu-Mic, the subjects were actively sensitized with DNCB and MIF production was assessed before and after sensitization. Remarkable MIf production was noted in the lymphocyte cultures after sensitization, although no significant MIF production was observed before sensitization. MIF production of the sensitized lymphocytes cultured in the presense of DNP-hu-Mic generally correlated well with the results of patch testing, but not with the intensity of the skin test.

A simple and sensitive method for the estimation of polymer-supported amino groups is reported. The polymer support is treated either with N-succinimidyl-4-O-(4,4'-dimethoxytrityl)-butyrate or 2,4-dinitrophenyl-4-O-(4,4'-dimethoxytrityl)-butyrate and a catalytic amount of 4-dimethylaminopyridine. After removal of the excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the solid support into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption (epsilon 498 = 70,000/M) at 498 nm, is determined spectrophotometrically. A comparative study of these reagents with N-succinimidyl-3-(2-pyridyldithio)-propionate, 4,4'-dimethoxytrityl chloride, and sodium 2,4,6-trinitrobenzenesulfonate methods is also included.

Volumes of agglutinates produced by the microtiter latex antiglobulin test (LAGT) were measured by an electronic data processing (EDP) method using a particle volume distribution analyzer. This method proved highly sensitive for quantitating weak antibodies, such as anti-digoxin and anti-DNP. EDP measurement of the microtiter latex antiglobulin-inhibition test (LAGIT) is also a suitable method for quantitating HCG, digoxin and DNP.

Two uncouplers of mitochondrial oxidative phosphorylation [2,4-dinitrophenol (DNP) and carbonylcyanide-m-chlorophenylhydrazone (CCCH)] were infused intra-arterially into a branch of the lower abdominal aorta of anesthetized rats over a 60-180 min period. An acute, severe, hypermetabolic state with systemic lactic acidosis and stiffness of the lower extremeities developed. In the plantaris muscles, by histochemistry, numerous "ragged red" fibers were present after the infusion. The "ragged red" areas presumably represented an absolute increase in mitochondrial mass in affected muscle fibers. By electron microscopy, linear inclusions were present in the intracristal space of many mitochondria. Simultaneous infusion of DNP and chloramphenicol, an inhibitor of mitochondrial protein synthesis, prevented the formation of ragged red fibers but not the intracristal inclusions. Infusion of relatively large amounts of oleic acid produced histochemical and electron-microscopic changes similar to those caused by the uncouplers. A possible pathogenesis of these reversible mitochondrial changes was discussed and their potential relevance to morphologic abnormalities of skeletal muscle mitochondria in human diseases was reviewed.

1-Fluoro-2,4-dinitrobenzene can be used to selectively label histidine, tyrosine, and cysteine residues in maleylated proteins. The usefulness of the resulting chromophores for peptide mapping by high-performance liquid chromatography was demonstrated with the lectin from sainfoin (Onobrychis viciifolia). The 2,4-dinitrophenyl (Dnp) label also can be used in a hydrophobic modulation approach as the mobility of a labeled model peptide changes considerably when its Dnp group is removed by thiolysis. Application of the method for checking sequences obtained by DNA or amino acid methods was shown by experiments with Viciae lectins. The probable cleavage site that generates the pea lectin's beta-chain from the alpha-beta precursor was identified and the sequence differences between the lentil and pea lectin beta-chains were examined.

The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-epsilon-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.

A cloned murine mast cell MC9 expresses phospholipase and lipoxygenase activity when stimulated with IgE and hapten. Addition of DNP-BSA to sensitized MC9 cells causes release of 58% of the cell histamine and 127 pmoles LTC4/10(6) cells. Prelabelling studies with [1-14C]-arachidonic acid showed that LTC4 production was proceeded by the release of arachidonic acid from membrane phospholipids. Approximately 8.7% of the cell arachidonic acid was released and half of this was converted to LTC4. The remaining radioactivity was converted to diHETES including LTB4 (15%), 5-HETE (10%), free arachidonic acid (10%), reesterified 5-HETE and arachidonic acid (8%) and prostaglandins (7%). This stimulation was dependent on hapten (DNP-BSA) and extracellular Ca++. Under identical conditions the calcium ionophore A23187 stimulated the release of 10.3% of the total cell arachidonic acid, and 51% of this was metabolized to LTC4. In addition the ionophore stimulated the release of 61% of the total cellular histamine.

A sorbent obtained by treatment of cyanogen bromide-activated Sepharose 4B with mono-N-DNP-hexamethylenediamine has been shown to be effective in the affinity chromatography of pepsin, pepsinogen and acid proteinase from Aspergillus awamori. It is considered that 2,4-dinitrophenyl residues of the sorbent interact specifically with the hydrophobic zone of the enzyme, which may belong to the substrate binding site. The chromatography of chymotrypsin on the same sorbent supports this assumption.