CD100 boosts the inflammatory response in the challenge phase of allergic contact dermatitis in mice.

BACKGROUND: Allergic contact dermatitis (ACD) is an inflammatory disease with a complex pathophysiology in which epidermal-resident memory CD8+ T (TRM ) cells play a key role. The mechanisms involved in the activation of CD8+ TRM cells during allergic flare-up responses are not understood. METHODS: The expression of CD100 and its ligand Plexin B2 on CD8+ TRM cells and keratinocytes before and after allergen exposure was determined by flow cytometry and RT-qPCR. The role of CD100 in the inflammatory response during the challenge phase of ACD was determined in a model of ACD in CD100 knockout and wild-type mice. RESULTS: We show that CD8+ TRM cells express CD100 during homeostatic conditions and up-regulate it following re-exposure of allergen-experienced skin to the experimental contact allergen 1-fluoro-2,4-dinitrobenzene (DNFB). Furthermore, Plexin B2 is up-regulated on keratinocytes following exposure to some contact allergens. We show that loss of CD100 results in a reduced inflammatory response to DNFB with impaired production of IFNγ, IL-17A, CXCL1, CXCL2, CXCL5, and IL-1ß and decreased recruitment of neutrophils to the epidermis. CONCLUSION: Our study demonstrates that CD100 is expressed on CD8+ TRM cells and is required for full activation of CD8+ TRM cells and the flare-up response of ACD.

Inhibition of Mast Cell Degranulation in Atopic Dermatitis by Celastrol through Suppressing MRGPRX2.

Background: Atopic dermatitis is a common dermatological disease, and mast cell degranulation is believed to be related with the progression of atopic dermatitis. Mas-related G protein-coupled receptor-X2 (MRGPRX2), and calcium release-activated calcium channel protein 1-2 (ORAI-1, ORAI-2) are involved in mast cell degranulation. Celastrol is an active monomer of Tripterygium wilfordii, and it presents an antiatopic role. Methods: 2,4-Dinitrofluorobenzene (DNFB) and compound 48/80 (C 48/80) were used to establish a slow and acute scratching animal model, respectively. Hematoxylin-eosin and toluidine blue staining was used to investigate tissue injury. Inflammatory factor concentration was measured with ELISA. The expression of MRGPRX2, ORAI-1, and ORAI-2 was detected with immunohistochemistry (IHC) staining. Gene expression profiling and microRNA array were performed to investigate gene differential expression. Results: Celastrol greatly inhibited atopic dermatitis-related tissues injury, mast cell production, histamine release, scratching level, inflammatory factor expression, and activation of MRGPRX2/ORAI axis in the DNFB-induced atopic dermatitis model. The influence of Celastrol on atopic dermatitis was remarkably reversed by overexpression of MRGPRX2. Conclusion: We found that the improvements of atopic dermatitis caused by Celastrol were reversed by treatment with MRGPRX2OE, indicating that Celastrol might affect atopic dermatitis through MRGPRX2. This study might provide a novel thought for the prevention and treatment of atopic dermatitis by regulating MRGPRX2.

Myricetin treatment has ameliorative effects in DNFB-induced atopic dermatitis mice under high-fat conditions.

Atopic dermatitis (AD) is a chronic, inflammatory cutaneous disorder. Obesity is associated with increased prevalence and severity of AD for reasons that remain poorly understood. Myricetin, a dietary flavonoid found in fruits and vegetables, is known to have anti-inflammatory effects, but its role in AD is unclear. Thus, we investigated the effects of obesity on exacerbation AD lesions and evaluated the effects of myricetin on obese AD. Mice were fed normal diet (ND) or high-fat diet, and then 2,4-dinitrofluorobenzene was used to induce AD-like lesions. We found that obesity exacerbated AD lesions, and myricetin topical administration ameliorated symptoms and skin lesions of obsess AD mice, such as dermatitis scores, scratching behavior, epidermal thickness, and mast cell infiltration. In addition, myricetin reduced the levels of immunoglobulin E and histamine, inhibited the infiltration of CD4+T cells, and modulated the expression of Th1, Th2, Th17, and Th22 cytokines and pro-inflammatory factors (CCL17, CCL22, IL-1ß, and TGF-ß). Moreover, myricetin restored impaired barrier function by reducing transepidermal water loss, increasing lamellar body secretion, as well as upregulating the mRNA and protein expression of filaggrin. Western blot results showed that significantly increased levels of phosphorylated IκB and NF-κB p65 was observed in the obese AD mice compared with the AD mice fed ND, whereas the myricetin could downregulated the phosphorylations of IκB and NF-κB, and inhibited mRNA expression of iNOS and COX2. Taken together, our results suggest that myricetin treatment exhibits potentially protective effects against the obeseassociated AD by inhibiting inflammatory response and restoring skin barrier function.

High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units.

The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C(vi)(JATP)) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C(vi)(JATP) were for system A: Complex I - 0.19, Complex III - 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) - 0.11, ATP synthase - 0.01, Pi carrier - 0.20, and the sum of C(vi)(JATP) was 0.75. In the presence of 10mM creatine (system B) the C(vi)(JATP) were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C(vi)(JATP) were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C(vi)(JATP) was 1.33 and 3.84, respectively. Thus, C(vi)(JATP) were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin ßII which restricts permeability of the mitochondrial outer membrane.

Structure-immune response relationships of hapten-modified collagen II peptides in a T-cell model of allergic contact dermatitis.

Allergic contact dermatitis (ACD) is mediated by T cells that specifically recognize hapten-modified peptides. T cells are known to recognize antigens as short processed peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). It has previously been demonstrated that T cells can specifically recognize carbohydrates on the lysine at position 264 of the immunodominant (256-273) sequence from type II collagen (CII) and that such recognition is critical for the development of arthritis in mice and may play a role in rheumatoid arthritis in humans. In the present study, we have used this approach in modeling ACD, but instead of the carbohydrate, the strong sensitizer 2,4-dinitrofluorobenzene (DNFB) is bound to the epsilon-amine of the lysine at position 264. Specific T-cell hybridomas of this antigenic peptide, with dinitrophenyl (Dnp) on the epsilon-amine of lysine at position 264 (CIILysDnp 3), were established from mice immunized with CIILysDnp 3. In an immune response assay, these T-cell hybridomas were tested with a series of new synthetic hapten-modified peptides, all chemically identical except for the stereochemimistry (D, L) and the length of the position-264 amino acid side chain bonding the hapten. The T-cell hybridomas recognized the CIILysDnp 3 peptide used for immunization; interestingly, they also recognized the CII peptide with a one-carbon-longer side chain (homolysine), CIIhLysDnp 6, and CIIAlaPipDnp 11, having a ring structure analogous to that of lysine with the same number of carbons in the bonding chain as in the CIILysDnp 3 peptide used for immunization. Dnp-modified CII peptides with a shorter bonding chain produced no immune response. These data demonstrate that the T-cell recognition of the Dnp-modified peptides is highly specific and moreover dependent on the length of the amino acid side chain that bonds the Dnp.

Covalent linkage of a synthetic peptide to a fluorescent phospholipid and its incorporation into supported phospholipid monolayers.

A number of fluorescent peptide-lipid conjugates have been synthesized. Peptides with ten or eleven amino acids are linked through a single lysine residue to the headgroup of phosphatidylethanolamine, fluorescently labelled on one acyl chain, using homobifunctional disuccinimidyl crosslinking reagents. Peptide-lipids can be further derivatized with the hapten dinitrophenyl. Purified peptide-lipids have been incorporated into dimyristoylphosphatidylcholine monolayers at the interface of air and phosphate-buffered saline, at concentrations of up to 11 mol%. For equal average molecular areas, monolayers containing peptide-lipids have higher surface pressures than pure lipid monolayers; for equal surface pressures, peptide-lipid monolayers have higher average molecular areas than pure lipid monolayers. When the peptide-lipid monolayers are transferred to hydrophobic glass slides, the fluorescence appears uniformly distributed. Fluorescence recovery after photobleaching measurements indicate that peptide-lipids diffuse in the monolayer with coefficient 1.5 X 10(-9) cm2/s, which is much smaller than that of typical lipids in fluid membranes. In addition, the diffusion coefficient of peptide-lipids decreases with increasing peptide-lipid concentration. We conclude that the peptide portion of the peptide-lipid associates with the lipid monolayer and/or that peptide-lipids oligomerize.

Peptide fragments of the heavy-chain region of phosphorylated and dinitrophenylated gizzard myosin.

Sulfopropyl (SP) Sephadex chromatography of trypsin-pepsin digests of dinitrophenylated gizzard myosin, pretreated with and without the myosin light-chain kinase calcium-calmodulin phosphorylating system, yielded similar elution patterns and nine peptide fractions were found. From a comparison with trypsin-pepsin digests of the heavy chains of dinitrophenylated myosin, pretreated with and without the phosphorylating system, it was established that peptide I, a major peptide fraction, was part of the 17-kDa dinitrophenylated light chain. Phosphorylation of myosin did not change the dinitrophenyl group content of peptide I but it did result in a significant increase in the dinitrophenylation of other peptides. The peptides contained only S-dinitrophenyl cysteine. Peptide III, previously considered to be part of the light-chain region (Bailin, G. and Lopez, F. (1982) J. Biol. Chem. 257, 264-270), was shown to originate in the heavy chains of myosin based on a comparison of the elution patterns of the digests of modified myosin and its heavy chains. Several neutral and basic peptides (peptides III to IX) originated in the heavy-chain region and they were different from those from the heavy chains of rabbit skeletal myosin. Phosphorylation of the 20-kDa light chain shifted the dinitrophenylation of the sulfhydryl groups from the 17-kDa light chain to the heavy chains of myosin, predominantly. These thiol groups do not resemble the fast-reacting -SH groups of rabbit skeletal myosin. The light chains are involved, in part, in making sites available on myosin that are necessary for actin-myosin interaction.

Proteins associated with rRNA in the Escherichia coli ribosome.

Ribosomal proteins located near the rRNA have been identified by cross linking to [14C]spermine with 1,5-difluoro-2,4-dinitrobenzene. The polyamine binds to double-stranded rRNA; those proteins showing radioactivity covalently bound after treatment with the bifunctional reagent should therefore be located in the vicinity of these regions of rRNA. Six proteins from the small subunit, S4, S5, S9, S18, S19 and S20 and ten proteins from the large subunit L2, L6, L13, L14, L16, L17, L18, L19, L22 and L27 preferentially take up the label. The results obtained with three proteins from the large subunit, L6, L16 and L27, show a high degree of variability that could reflect differences of conformation in the subunit population. Several proteins were drastically modified by the cross-linking agent but were not detected in the two-dimensional gel electrophoresis (e.g., S1, S11, S21, L7, L8 and L12) and therefore could not be studied.

Crosslinking of troponin complex with 1,3-difluoro-4,6-dinitrobenzene. Identification of the crosslink formed between troponin C and troponin I in the absence of Ca2+.

The single SH-group of rabbit skeletal muscle troponin C (Cys-98) was reacted with the bifunctional reagent, 1,3-difluoro-4,6-dinitrobenzene. This labelled troponin C was used to reconstitute the troponin complex by the addition of equimolar amounts of troponin T and troponin I. The second function of the bifunctional reagent was triggered in the complex by an increase of pH. A crosslink was formed between troponin C and troponin I both in the presence and absence of Ca2+, but the probability of crosslinking was decreased by Ca2+. Covalently linked troponin C-troponin I was isolated from the complex crosslinked without Ca2+, and cleaved by CNBr. The analysis of crosslinked peptides has revealed that in the presence of Mg2+ and absence of Ca2+ the crosslink in the troponin complex is formed between Cys-98 of troponin C and Cys-133 of troponin I.

Interaction of chemical probes with sarcoplasmic reticulum membranes.

The labelling of the sarcoplasmic reticulum membranes by the chemical probes, trinitrobenzenesulfonate (TNBS) and fluorodinitrobenzene (FDNB) has been investigated. The incorporation of TNBS, but not of FDNB, depends on the binding of Ca2+ or Mg2+ to the membranes. The labelling of lipids and of the various reticulum proteins by TNBS is increased by those agents, but the effect is not uniform for all membrane proteins. The Ca2+ -ATPase contributes only 2.2% for the total labelling of the sarcoplasmic reticulum proteins, whereas the proteins of molecular weight 90 000 and 30 000 contribute about 34 and 56%, respectively. However, the Ca2+-ATPase isolated from the membrane reacts with an amount of TNBS 5-fold higher than that which reacts with the enzyme in situ. Both probes, TNBS and FDNB, inhibit the Ca2+-ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum, whereas the Mg2+-ATPase remains unaffected. The results indicate that FDNB is maximally incorporated into the sarcoplasmic reticulum membrane, whereas only some of the membrane amino groups are accessible to TNBS in the absence of Ca2+, Mg2+ or ATP which, when present, make additional amino groups available to TNBS. The highest degree of TNBS incorporation takes place into proteins, other than the ATPase, but sufficient reaction occurs with the enzyme to inhibit its activity.

A comparison of the conjugation of DNTB and other dinitrobenzenes with free protein radicals and their ability to sensitize or tolerize.

Of several dinitrobenzenes tested, 2,4-dinitrothiocyanatebenzene (DNTB) was found to be the only one that did not induce contact sensitivity when applied to the guinea pig ear epicutaneously, but when applied epicutaneously it induced tolerance to 2,4-dinitrofluorobenzene (DNFB). The manner in which DNFB, DNTB, and other dinitrobenzene compounds conjugated in vitro to soluble proteins, at physiologic pH, was examined. By measuring the free amino and sulfydryl radicals in the protein before and after conjugation, it was possible to determine to which groups the hapten was bound. It was found that although all the haptens bound to the free sulfydryl groups, DNTB was the only one that did not bind to amino groups. It is suggested that to be an epicutaenous tolerizer, as opposed to sensitizer, a hapten should bind to sulfydryl groups exclusively. It is hoped that a search for agents binding in a similar manner will reveal epicutaneous tolerizers for important industrial sensitizers.

Drug-protein conjugates--X. The role of protein conjugation in the disposition of dinitrofluorobenzene.

The metabolism and irreversible protein binding of 2,4-[3,5-3H]dinitrofluorobenzene (3H-DNFB), a model chemically reactive compound, were studied in the rat. 3H-DNFB given intravenously (5 micrograms, 5 mg or 25 mg per kg) to anaesthetized cannulated rats was rapidly metabolized via the mercapturic acid pathway. The metabolites were extensively eliminated in bile and urine: predominantly as the glutathione conjugate and mercapturate in bile, and as the mercapturate in urine. Only ca. 3-10% of the doses remained in the liver, kidneys, spleen, heart and lungs at 3 hr. Dinitrophenyl mercapturate was the principal urinary metabolite in conscious rats dosed i.v. (5 mg or 25 mg per kg). Only 15-25% of the radiolabelled material in liver and kidney at 3 hr was irreversibly bound to protein, but 45-99% of that in the other organs and 49-88% in plasma was irreversibly bound. Preliminary evidence for the metabolism of 3H-DNFB (5 mg/kg and 25 mg/kg doses) to N2-acetyl-N6-DNP-lysine, a novel conjugate and metabolite of dinitrophenylated proteins in vivo, is presented.

Drug-protein conjugates--XI. Disposition and immunogenicity of dinitrofluorobenzene, a model compound for the investigation of drugs as haptens.

The conjugation of drugs to autologous proteins is thought to be a key step in the hapten mechanism of drug hypersensitivity. We have studied the mild arylating agent dinitrofluorobenzene (DNP-F) as a model compound with which to investigate the relationship between the disposition and immunogenicity of drug haptens in two species, the rat and rabbit. Intramuscular administration of DNP-F (0.027-27 mumol/kg/day) for 4 days to male Wistar rats produced a dose-dependent (ED50 2.7 mumol/kg) IgG anti-DNP antibody response, measured by enzyme-linked immunosorbent assay. Subsequent monthly administrations (for 4 days) increased both the frequency and titre of antibody response. Intravenous administration of [3H]DNP-F (0.27 or 2.7 mumol/kg) for 9 days to male New Zealand White rabbits produced an IgG and IgM anti-DNP response in all animals from day 9 onwards. Formation of circulating (serum) DNP-protein conjugates was determined by radiometric analysis, and found to reach steady state (0.12-0.17% dose/ml) between days 6 and 8 and decline with a half-life of 7.4 days. The immunogenicity of fully characterized haptenated, autologous proteins was investigated in further experiments in which dinitrophenylated serum protein conjugates (DNP-RSP) and albumin conjugates (DNP-RSA) were prepared ex vivo and then administered (50 mg/kg; i.v.) to the rabbit from which the protein had been obtained. The plasma clearance and immunogenicity of DNP-RSA conjugates was dependent on epitope density. Anti-DNP antibodies were detected after administration of an RSA-DNP15 conjugate but not after either RSA-DNP0.5 or RSA-DNP5. Plasma concentrations of RSA-DNP15 conjugate declined slowly initially, but then fell rapidly between days 8 and 10. The plasma clearance of DNP-RSP conjugates showed a dependence on epitope density from day 1 onwards and anti-DNP antibodies were detectable after administration of all conjugates investigated (range of epitope densities 0.5-30 DNP/albumin equivalent). Thus conjugates derived from proteins other than albumin are likely to be the effective immunogens, for the model hapten DNP. These studies show that DNP-F is a useful model compound in studies of the disposition and immunogenicity of drugs acting as haptens, and may therefore be used as a positive control in experiments designed to assess the potential immunogenicity of drugs and other xenobiotics.

Location and identification of substituents with free amino group in lipopolysaccharides of gram-negative bacteria with the use of chromophore labelling.

Interaction of ten different lipopolysaccharides (LPS) with 2,4-dinitrofluorobenzene yielded quantitatively yellow dinitrophenyl derivatives (DNP-LPS) to show the presence of substituents with free amino group. The DNP-LPS samples were degraded with 1% acetic acid, and after removal of lipid A precipitates the supernatants were separated on a Sephadex G-25 column to give coloured polysaccharide, oligosaccharide and monomeric fractions monitored at lambda DNP = 365 nm. The coloured materials, including DNP-derivative of lipid A, were dephosphorylated with hydrofluoric acid followed by identification of the released DNP-amines by thin layer chromatography (TLC) on silica gel. Subsequently, the dephosphorylated materials were hydrolysed with hydrochloric acid followed by TLC analysis. The approach allowed to detect, locate and identify the substituents with free amino group within the LPS molecules. Moreover, two types of core structures within LPS preparation from one strain were discovered for five microorganisms.

Interaction of membrane aminophospholipids of E. coli with fluorodinitrobenzene and trinitrobenzenesulfonate.

E. coli cells were reacted with TNBS in bicarbonate-NaCl buffer, pH 8.5 (buffer A) and in phosphate-NaCl buffer, pH 7.0 (buffer B). In buffer A, DNP-GPE is the major product when FDNB is used. DNP-PE and DNP-LPE are formed in lesser amounts. Phospholipase A activity is high in buffer A. When TNBS is used, the labeling of the lipid components is less than with FDNB and more TNP-PE is formed relative to TNP-GPE. This data suggests that the phospholipases which are located primarily on the outer L-membrane of the cell wall act to a lesser extent on TNP-PE than on DNP-PE. E. coli cells were prelabeled with TNBS and FDNB in buffer A, washed and incubated in buffer A. The endogenous labeled DNP-PE gradually decreased with time with a concomitant increase in DNP-LPE and DNP-GPE due to phospholipase A activity. In contrast, the endogenous labeled TNP-PE also decreased with time as did the endogenous labeled TNP-LPE but a new orange lipid was produced. This lipid is believed to be a derivative of TNP-PE in which one of the nitro groups has been reduced to an amino group by nitroreductase. E. coli cells were prelabeled with TNBS and FDNB in buffer A, washed and incubated in buffer B. Under these conditions with both TNBS and FDNB there is an increase in TNP-PE and DNP-PE with a concomitant decrease in TNP-LPE, TNP-GPE, DNP-LPE and DNP-GPE. These results show that at neutral pH acylation occurs to regenerate TNP-PE and DNP-PE. E. coli cells were incubated with exogenous DNP-GPE or TNP-GPE in buffer A. The DNP-GPE and TNP-GPE were rapidly hydrolyzed by a phosphodiesterase to DNP-ethanolamine and TNP-ethanolamine. An orange derivative was formed which was provisionally identified as a derivative of DNP-ethanolamine or TNP-ethanolamine in which a nitro group has been reduced to an amino group by nitroreductase. The phospholipases and acylating enzymes present in the cell wall of E. coli are active on the dinitrophenyl and trinitrophenyl derivatives of PE and LPE and may act in concert to model and repair the plasma membrane.

Reaction of amino-phospholipids of the inner mitochondrial membrane with fluorodinitrobenzene and trinitrobenzenesulfonate.

Mitoplasts from rat liver mitochondria and ETPH particles from beef heart mitochondria were reacted with TNBS and FDNB in sucrose-mannitol-bicarbonate buffer pH 8.2 (BUFFER A) and in sodium chloride-bicarbonate buffer pH 8.5 (buffer B). Mitoplasts and ETPH particles are more stable in buffer A and very little hydrolysis of phospholipids occurs during the reaction period. In this buffer TNBS reacts to a lesser extent with phosphatidylethanolamine (PE) than does FDNB. The data suggests that with mitoplasts 65% of the total PE is localized on the outer surface of the membrane. With mitoplasts the labeling of membrane proteins is much more extensive with FDNB and suggests that 66% of the proteins are localized on the inner membrane surface. Thus a dual type of asymmetry occurs in the mitoplast membrane with more PE localized on the outer surface and more protein localized on the inner surface. In buffer B, extensive degradation of the dinitrophenylated and trinitrophenylated PE and LPE occurs to yield DNP-GPE and TNP-GPE respectively. DNP-GPE and TNP-GPE are degraded by a phosphodiesterase to DNP-ethanolamine and TNP-ethanolamine. When ETPH particles are labeled with TNBS and FDNB, washed, and incubated in buffer A and buffer B, a resynthesis of TNP-PE and DNP-PE occurs in buffer A by acylation of TNP-LPE whereas DNP-PE continues to be formed, primarily from DNP-GPE. These studies provide evidence for an asymmetric arrangement of PE in the inner mitochondrial membrane and demonstrate the presence of membrane-bound phospholipases which act on dinitrophenylated and trinitrophenylated amino-phospholipids. A membrane bound phosphodiesterase is also present which degrades dinitrophenylated or trinitrophenylated GPE. The degradative reactions prevail in bicarbonate-NaCl buffer B whereas acylation reactions prevail in sucrose-mannitol buffer A.

Cellular and subcellular distribution of 2,4-dinitrophenyl groups in mouse epidermis and regional lymph nodes after epicutaneous application of 2,4-dinitrofluorobenzene.

The cellular and subcellular distribution of 2,4-dinitrophenyl (DNP) groups in the epidermis and regional lymph nodes of the mouse was investigated after epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to sensitized and non-sensitized mice. The peroxidase-antiperoxidase method and the immunogold technique were used to visualize the DNP groups at both light and electron microscopic levels. The highest intensity of immunolabelling was found on tonofilaments of keratinocytes present in the upper layers of the epidermis. On the other hand, in vitro experiments showed that DNFB has the capacity to bind keratin which, together with immunocytochemistry, suggests that this molecule may be one of the skin protein carriers for DNFB. In addition, intense immunostaining for DNP was observed in the Golgi area of some epidermal Langerhans cells. Cells immunoreactive to DNP were also observed in the marginal sinus of cervical lymph nodes 6, 12 and 24 h after challenge. Immunoelectron microscopy revealed immunoreactive DNP groups in phagosomes of Langerhans cells at this site. The present findings support the hypothesis that the hapten DNFB penetrates passively into the cytoplasm of Langerhans cells, concentrates in the Golgi area and, during the migration of Langerhans cells to the lymph nodes, it is probably processed in the lysosomes before its presentation to T lymphocytes.

The role of fluoroacetate-specific dehalogenase and glutathione transferase in the metabolism of fluoroacetamide and 2,4-dinitrofluorobenzene.

2,4-Dinitrofluorobenzene (DNFB) reacts with glutathione to form a stable product similar to that formed with the model glutathione-S-transferase (GST) substrate, 1-chloro-2,4-dinitrobenzene (CDNB). DNFB is approx. 40 times als reactive as CDNB in this chemical reaction. The enzymatic defluorination of DNFB also proceeds at a more rapid rate than that of CDNB in the GST assay. Fluoroacetamide (FAM), like fluoroacetate (FAC), undergoes no discernable chemical defluorination. Its enzymatic defluorination is approx. 10% of that observed for FAC and only 0.2% of the rate for DNFB. An antibody raised to the fluoroacetate specific dehalogenase (FSD) precipitated both FAC and FAM defluorinating activity but had no effect on either CDNB or DNFB activity. The data are consistent with the hypothesis that DNFB is metabolized by the GST while FAM is metabolized by the FSD.

Suppressive effects on delayed type hypersensitivity by fasting and dietary restriction in ICR mice.

Dietary restriction improves declining physiologic functions, prevents or lessens the severity of neoplasms and autoimmune diseases, and attenuates various inflammatory reactions. In the present study, we compared the effect on allergic dermatitis from repeated short-term fasting (every 3 days), and from moderate dietary restriction receiving 60% of the amount of food consumed by an ad libitum feeding group. In addition, we attempted to verify the involvement of corticosteroids and oxidative stress during nutritional deprivation. The overall food intake in mice undergoing moderate dietary restriction was less than that in mice undergoing repeated fasting. Nonetheless, moderate dietary restriction and repeated fasting showed similar suppressive effects on dermatitis. Furthermore, both the restricted-diet and fasted mice showed less oxidative stress than the mice fed ad libitum. In RU486 (a glucocorticoid receptor antagonist)-injected mice, no suppressive effect of fasting on dermatitis was seen. In conclusion, repeated fasting and moderate dietary restriction suppressed dermatitis in similar ways. Hypercorticism and reduced oxidative stress is associated with the suppression of dermatitis.

Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg(2+).

For over four decades free Mg(2+) ions, that is, those in excess of MgATP, have been reported to affect a wide variety of properties of phosphorylase kinase (PhK), including its affinity for other molecules, proteolysis, chemical crosslinking, phosphorylation, binding to certain monoclonal antibodies, and activity, which is stimulated. Additionally, for over three decades Mg(2+) has been known to act synergistically with Ca(2+) , another divalent activator of PhK, to affect even more properties of the enzyme. During all of this time, however, no study has been performed to determine the overall effects of free Mg(2+) ions on the physical properties of PhK, even though the effects of Ca(2+) ions on PhK's properties are well documented. In this study, changes in the physicochemical properties of PhK induced by Mg(2+) under nonactivating (pH 6.8) and activating (pH 8.2) conditions were investigated by circular dichroism spectroscopy, zeta potential analyses, dynamic light scattering, second derivative UV absorption, negative stain electron microscopy, and differential chemical crosslinking. The effects of the activator Mg(2+) on some of the properties of PhK measured by these techniques were found to be quite different at the two pH values, and displayed both differences and similarities with the effects previously reported to be induced by the activator Ca(2+) (Liu et al., Protein Sci 2008;17:2111-2119). The similarities may reflect the fact that both cations are activators, and foremost among their similarities is the dramatically less negative zeta potential induced by their binding to PhK.