Hiroyuki Mizuguchi

Regulation of Nuclear Import/Export of Carbohydrate Response Element-binding Protein (ChREBP) INTERACTION OF AN α-HELIX OF ChREBP WITH THE 14-3-3 PROTEINS AND REGULATION BY PHOSPHORYLATION

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.

PRESEASONAL PROPHYLACTIC TREATMENT WITH ANTIHISTAMINES SUPPRESSES NASAL SYMPTOMS AND EXPRESSION OF HISTAMINE H1 RECEPTOR mRNA IN THE NASAL MUCOSA OF PATIENTS WITH POLLINOSIS

Administration of antihistamines 2-4 weeks before the pollen season showed a greater inhibitory effect on nasal allergy symptoms in patients with seasonal allergic rhinitis. However, the mechanism of slow-onset effects of preseasonal treatment with antihistamines remains unclear. Here, we investigated the effect of preseasonal prophylactic treatment with antihistamines on nasal symptoms and the expression of histamine H₁ receptor (H1R) mRNA of the nasal mucosa in patients with cedar pollen pollinosis. During the peak pollen period, the expression of H1R mRNA in the nasal mucosa and the scores of sneezing and watery rhinorrhea in patients receiving preseasonal prophylactic treatment with antihistamines were significantly suppressed in comparison with those in the patients without treatment. Moreover, there was a significant correlation between the nasal symptoms and the expression of H1R mRNA in both patients with or without preseasonal prophylactic treatment. These findings suggest that preseasonal prophylactic treatment with antihistamines is more effective than on-seasonal administration to patients with pollinosis in reducing nasal symptoms during the peak pollen period by suppressing H1R gene expression in the nasal mucosa.

Sho-seiryu-to Suppresses Histamine Signaling at the Transcriptional Level in TDI-Sensitized Nasal Allergy Model Rats

BACKGROUNDnThe therapeutic use of Kampo medicine, Sho-seiryu-to (SST) in allergic disorders is well known. As histamine plays a central role in allergic diseases, it is possible that SST affects the allergy-related histamine signaling. In this study, we investigated the effect of SST on allergy-related histamine signaling in the nasal mucosa of toluene 2, 4-diisocyanate (TDI)-sensitized nasal allergy model rats.nnnMETHODSnSix-week-old male, Brown Norway rats were sensitized for 2 weeks with 10 microl of 10% TDI, and after a 1 week interval, provocation was initiated with the same amount of TDI. SST (0.6g/rat) was given orally 1 hour before TDI treatment began for a period of 3 weeks. Nasal symptoms were scored for 10 minutes immediately after TDI-provocation. The genes expression in nasal mucosa was determined using real-time quantitative RT-PCR.nnnRESULTSnSST significantly suppressed TDI-induced nasal allergy-like symptoms. TDI provocation showed a significant up-regulation of histamine H(1) receptor (H1R) and histidine decarboxylase (HDC) gene expressions. Prolonged pre-treatment of SST significantly suppressed the mRNA levels of H1R and HDC that was up-regulated by TDI. SST also suppressed TDI-induced interleukin (IL)-4 and IL-5 mRNA elevation. However, SST showed no significant effect for TDI-induced mRNA elevation of IL-13.nnnCONCLUSIONSnThese results demonstrate that SST alleviates nasal symptoms by the inhibition of histamine signaling through suppression of TDI-induced H1R and HDC gene up-regulation. SST also suppresses cytokine signaling through suppression of IL-4 and IL-5 gene expression. Suppression of histamine signaling may be a novel mechanism of SST in preventing allergic diseases.

Strain and catalysis in aspartate aminotransferase

The notion of ground-state destabilization has been well documented in enzymology. It is the unfavourable interaction (strain) in the enzyme-substrate complex, and increases the k(cat) value without changing the k(cat)/K(m) value. During the course of the investigation on the reaction mechanism of aspartate aminotransferase (AAT), we found another type of strain that is crucial for catalysis: the strain of the distorted internal aldimine in the unliganded enzyme. This strain raises the energy level of the starting state (E+S), thereby reducing the energy gap between E+S and ES(++) and increasing the k(cat)/K(m) value. Further analysis on the reaction intermediates showed that the Michaelis complex of AAT with aspartate contains strain energy due to an unfavourable interaction between the main chain carbonyl oxygen and the Tyr225-aldimine hydrogen-bonding network. This belongs to the classical type of strain. In each case, the strain is reflected in the pK(a) value of the internal aldimine. In the historical explanation of the reaction mechanism of AAT, the shifts in the aldimine pK(a) have been considered to be the driving forces for the proton transfer during catalysis. However, the above findings indicate that the true driving forces are the strain energy inherent to the respective intermediates. We describe here how these strain energies are generated and are used for catalysis, and show that variations in the aldimine pK(a) during catalysis are no more than phenomenological results of adjusting the energy levels of the reaction intermediates for efficient catalysis.

Suplatast Tosilate Inhibits Histamine Signaling by Direct and Indirect Down-Regulation of Histamine H1 Receptor Gene Expression through Suppression of Histidine Decarboxylase and IL-4 Gene Transcriptions

Allergic rhinitis (AR) is an inflammatory disorder typified by symptoms such as sneezing, congestion, and rhinorrhea. Histamine plays important roles in eliciting AR symptoms. Up-regulation of the histamine H1 receptor (H1R) and histidine decarboxylase (HDC) mRNAs was observed in AR patients. Th2 cytokines are also involved in the pathogenesis of AR. We examined the effect of suplatast tosilate on nasal symptoms, and H1R, HDC, and IL-4 gene expression using toluene-2,4-diisocyanate (TDI)-sensitized rats and HeLa cells expressing endogenous H1R. Provocation with TDI increased nasal symptoms, HDC activity, the histamine content of nasal lavage fluid, and the expression of H1R, HDC, and IL-4 mRNAs in TDI-sensitized rats. Pretreatment with suplatast for 2 wk significantly suppressed TDI-induced nasal symptoms and elevation of H1R, HDC, and IL-4 mRNAs. Suplatast also suppressed HDC activity in the nasal mucosa and the histamine content of the nasal lavage fluid. Bilateral injection of IL-4 into the nasal cavity of normal rats up-regulated H1R mRNA, while intranasal application of histamine up-regulated IL-4 mRNA. Suplatast suppressed IL-4-induced up-regulation of H1R mRNA in HeLa cells. However, it did not inhibit histamine-induced H1R mRNA elevation. These results suggest that suplatast alleviates nasal symptoms by inhibiting histamine signaling in TDI-sensitized rats through the suppression of histamine- and IL-4-induced H1R gene expression by the inhibitions of HDC and IL-4 gene transcriptions, respectively.

Anti-allergic activity of standardized extract of Albizia lebbeck with reference to catechin as a phytomarker.

Background and aim: The major groups of phytonutrients found in plants include polyphenols, flavonoids, terpenes, amines, etc., all of which are observed to have potential anti-allergic activity. In this study, we evaluated the anti-allergic activity of the standardized extract of Albizia lebbeck with respect to the catechin, a polyphenolic phytomarker. Material and methods: The percentage of catechin in the ethanolic extract was found to be 14.72u2009mg/g. We administered Albizia lebbeck (50–300u2009mg/kg) and 50u2009mg/kg of catechin to mice to evaluate the mast cell stabilization and estimation of histamine elevation in the plasma. Results and conclusion: Results support the conclusion that Albizia lebbeck at different concentrations has got potent mast cell stabilizing property and the IC50 value of Albizia lebbeck was found to be 85u2009μg/ml. This inhibitory potential of catechin from Albizia lebbeck is perhaps due to modulation of two important effector’s functions, histamine release and cytokine expression of antigen -IgE activated mast cells.

Mast cell stabilization and antihistaminic potentials of Curculigo orchioides rhizomes.

AIM OF THE STUDYnTo investigate the mast cell stabilization and antihistaminic activities of the rhizomes of Curculigo orchioides (COR). Extract of Curculigo orchioides Gaertn. (Fam. Amaryllidaceae) has been reported to possess immunostimulant, and anti-inflammatory potentials. In Indian traditional system of medicine it is also used as anti-asthmatic and anti-inflammatory.nnnMATERIALS AND METHODSnEstimation of histamine release is key parameter for evaluating any target for its anti-allergic potential. The stabilization potential of the alcoholic extract of COR (100-400mg/kg) against mast cell degranulation was studied on isolated mice peritoneal mast cells. The antihistaminic activity was performed by determining the mortality rate of mice upon exposure to compound 48/80 and effect on inhibition of histamine release upon degranulation.nnnRESULTSnThe raised number of intact mast cells intimates that the COR stabilized the mast cell degranulation (60.96+/-1.96%) and percentage antihistaminic potential of the extract (63.58+/-1.8 inhibition at dose of 400mg/kg) and it virtues further work towards the isolation of phytoconstituents from this plant.nnnCONCLUSIONnThis finding provides evidence that COR inhibits mast cell-derived immediate-type allergic reactions and mast cell degranulation.

Albizia lebbeck suppresses histamine signaling by the inhibition of histamine H1 receptor and histidine decarboxylase gene transcriptions

Histamine plays major roles in allergic diseases and its action is mediated mainly by histamine H(1) receptor (H1R). We have demonstrated that histamine signaling-related H1R and histidine decarboxylase (HDC) genes are allergic diseases sensitive genes and their expression level affects severity of the allergic symptoms. Therefore, compounds that suppress histamine signaling should be promising candidates as anti-allergic drugs. Here, we investigated the effect of the extract from the bark of Albizia lebbeck (AL), one of the ingredients of Ayruvedic medicines, on H1R and HDC gene expression using toluene-2,4-diisocyanate (TDI) sensitized allergy model rats and HeLa cells expressing endogenous H1R. Administration of the AL extract significantly decreased the numbers of sneezing and nasal rubbing. Pretreatment with the AL extract suppressed TDI-induced H1R and HDC mRNA elevations as well as [(3)H]mepyramine binding, HDC activity, and histamine content in the nasal mucosa. AL extract also suppressed TDI-induced up-regulation of IL-4, IL-5, and IL-13 mRNA. In HeLa cells, AL extract suppressed phorbol-12-myristate-13-acetate- or histamine-induced up-regulation of H1R mRNA. Our data suggest that AL alleviated nasal symptoms by inhibiting histamine signaling in TDI-sensitized rats through suppression of H1R and HDC gene transcriptions. Suppression of Th2-cytokine signaling by AL also suggests that it could affect the histamine-cytokine network.

Preseasonal prophylactic treatment with antihistamines suppresses IL−5 but not IL-33 mRNA expression in the nasal mucosa of patients with seasonal allergic rhinitis caused by Japanese cedar pollen

Abstract Conclusions: These findings suggest that the down-regulation of interleukin (IL)-5 gene expression in collaboration with the suppression of histamine H1 receptor (H1R) gene expression in the nasal mucosa provides the basis for better therapeutic effects of preseasonal prophylactic treatment with antihistamines in patients with seasonal allergic rhinitis caused by Japanese cedar pollen. Objectives: The effects of prophylactic administration of antihistamines on the expression of IL-5 and IL-33 mRNA in the nasal mucosa of the patients with pollinosis were investigated. Methods: Eight patients had already visited the hospital before the peak pollen period and started preseasonal prophylactic treatment with antihistamines. Seventeen patients who first visited the hospital during the peak pollen period were designated as the no treatment group. After local anesthesia, nasal mucosa was obtained by scraping the inferior concha with a small spatula during the peak pollen period. Results: During the peak pollen period, the expression of IL-5 mRNA, but not that of IL-33 mRNA, in the nasal mucosa of patients receiving preseasonal prophylactic treatment with antihistamines was significantly lower in comparison with that of patients without treatment. Moreover, there was a significant correlation between the expression of IL-5 mRNA and the nasal symptoms or the expression of H1R mRNA.

Quercetin inhibits transcriptional up-regulation of histamine H1 receptor via suppressing protein kinase C-δ/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 signaling pathway in HeLa cells

It has been reported that the histamine H1 receptor (H1R) gene is up-regulated in patients with allergic rhinitis and H1R expression level strongly correlates with the severity of allergy symptoms. Accordingly compounds that suppress the H1R gene expression are promising as useful anti-allergic medications. Recently, we demonstrated that histamine or phorbol-12-myristate-13-acetate (PMA) stimulation induced the up-regulation of H1R gene expression through the protein kinase Cδ (PKCδ)/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 signaling pathway in HeLa cells expressing H1R endogenously. Quercetin is one of the well-characterized flavonoids and it possesses many biological activities including anti-allergic activity. However, effect of quercetin on H1R signaling is remained unknown. In the present study, we examined the effect of quercetin on histamine- and PMA-induced up-regulation of H1R gene expression in HeLa cells. We also investigated its in vivo effects on the toluene-2,4-diisocyanate (TDI)-sensitized allergy model rats. Quercetin suppressed histamine- and PMA-induced up-regulation of H1R gene expression. Quercetin also inhibited histamine- or PMA-induced phosphorylation of Tyr(311) of PKCδ and translocation of PKCδ to the Golgi. Pre-treatment with quercetin for 3weeks suppressed TDI-induced nasal allergy-like symptoms and elevation of H1R mRNA in the nasal mucosa of TDI-sensitized rats. These data suggest that quercetin suppresses H1R gene expression by the suppression of PKCδ activation through the inhibition of its translocation to the Golgi.