The components of the AhR-molecular chaperone complex differ depending on whether the ligands are toxic or non-toxic.

The aryl hydrocarbon receptor (AhR) forms a complex with the HSP90-XAP2-p23 molecular chaperone when the cells are exposed to toxic compounds. Recently, 1,4-dihydroxy-2-naphthoic acid (DHNA) was reported to be an AhR ligand. Here, we investigated the components of the molecular chaperone complex when DHNA binds to AhR. Proteins eluted from the 3-Methylcolanthrene-affinity column were AhR-HSP90-XAP2-p23 complex. The AhR-molecular chaperone complex did not contain p23 in the eluents from the DHNA-affinity column. In 3-MC-treated cells, AhR formed a complex with HSP90-XAP2-p23 and nuclear translocation occurred within 30 min, while in DHNA-treated cells, AhR formed a complex with AhR-HSP90-XAP2, and translocation was slow from 60 min. Thus, the AhR activation mechanism may differ when DHNA is the ligand compared to toxic ligands.

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

An open-label prospective randomized multicenter study of intensive versus weekly granulocyte and monocyte apheresis in active crohn's disease.

BACKGROUND: Granulocyte and monocyte adsorptive apheresis (GMA) has shown efficacy in patients with active Crohn's disease (CD). However, with routine weekly therapy, it may take several weeks to achieve remission. This study was performed to assess clinical efficacy and safety of intensive GMA in patients with active CD. METHODS: In an open-label, prospective, randomized multicentre setting, 104 patients with CD activity index (CDAI) of 200 to 450 received intensive GMA, at two sessions per week (n = 55) or one session per week (n = 49). Clinical remission was defined as a CDAI score <150. Patients in each arm could receive up to 10 GMA sessions. However, GMA treatment could be discontinued when CDAI decreased to <150 (clinical remission level). RESULTS: Of the 104 patients, 99 were available for efficacy evaluation as per protocol, 45 in the weekly GMA group, and 54 in the intensive GMA group. Remission was achieved in 16 of 45 patients (35.6 %) in the weekly GMA and in 19 of 54 (35.2 %) in the intensive GMA (NS). Further, the mean time to remission was 35.4 ± 5.3 days in the weekly GMA and 21.7 ± 2.7 days in the intensive GMA (P = 0.0373). Elevated leucocytes and erythrocyte sedimentation rate were significantly improved by intensive GMA, from 8005/µL to 6950/µL (P = 0.0461) and from 54.5 mm/hr to 30.0 mm/hr (P = 0.0059), respectively. In both arms, GMA was well tolerated and was without safety concern. CONCLUSIONS: In this study, with respect to remission rate, intensive GMA was not superior to weekly GMA, but the time to remission was significantly shorter in the former without increasing the incidence of side effects. UMIN registration # 000003666.

Geranylgeranylacetone selectively binds to the HSP70 of Helicobacter pylori and alters its coccoid morphology.

Geranylgeranylacetone (GGA) is used to treat patients suffering from peptic ulcers and gastritis. We examined the effect of GGA on Helicobacter pylori, which is a causative factor of gastrointestinal diseases. Previously, we have reported that GGA binds specifically to the molecular chaperone HSP70. In this paper, we report that GGA bounds to H. pylori HSP70 (product of the DnaK gene) with 26-times higher affinity than to human HSP70, and induced large conformational changes as observed from surface plasmon resonance and circular dichroism. Binding of GGA suppressed the activity of the H. pylori chaperone. GGA also altered several characteristics of H. pylori cells. GGA-treated cells elicited enhanced interleukin-8 production by gastric cancer cell lines and potentiated susceptibility to complement as compared to untreated cells. GGA also caused morphological alterations in H. pylori as reflected in fewer coccoid-like cells, suggesting that GGA converts H. pylori to an actively dividing, spiral state (vegetative form) from a non-growing, coccoid state. This morphological conversion by GGA resulted in accelerated growth of H. pylori. These results suggest a model in which GGA sensitizes H. pylori to antibiotic treatment by converting the cells to an actively growing state.

The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90.

The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or ß-naphthoflavone (ß-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to ß-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90. We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with ß-NF.

ATPase activity and ATP-dependent conformational change in the co-chaperone HSP70/HSP90-organizing protein (HOP).

Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.

Zinc-L-carnosine binds to molecular chaperone HSP70 and inhibits the chaperone activity of the protein.

In this study, we have investigated the specific binding proteins of Zinc-L-carnosine (Polaprezinc) using Polaprezinc-affinity column chromatography in vitro. A protein having a 70-kDa molecular mass was eluted by the linear gradient of 0-1.0 mM Polaprezinc from the affinity column and the protein was identified as the molecular chaperone HSP70 by immunoblotting. The chaperone activity of HSP70 was completely suppressed by Polaprezinc. The ATPase activity of HSP70 was affected to some extent by the reagent. In the circular dichroism (CD) spectrum, the secondary structure of HSP70 was changed in the presence of Polaprezinc, i.e. it decreased in the α-helix. We have determined the Polaprezinc-binding domain of HSP70 by using recombinant HSP70N- and C-domains. Although Polaprezinc could bind to both the N-terminal and the C-terminal of HSP70, the HSP70N-domain has a high affinity to the drug. Regarding the peptide cleavage of the HSP70N- and C-domains with proteinase K, the intact HSP70N still remained in the presence of Polaprezinc. On the other hand, the quantity of the intact C-domain slightly decreased under the same conditions along with the newly digested small peptides appeared. It has been suggested that Polaprezinc binds to HSP70 especially in the N-domains, suppresses the chaperone activity and delays an ATPase activities of HSP70.

The relationship between physical activity level and completion rate of small bowel examination in patients undergoing capsule endoscopy.

OBJECTIVE: Capsule endoscopy (CE) allows direct examination of the small bowel in a safe, noninvasive and well-tolerated manner. Nonetheless, experience indicates failure to reach the cecum in 20-30% of patients within the 8 hour battery life. Attempts to improve the completion rate (CR) as defined by reaching the cecum have been unsuccessful. This study was to investigate the relationship between patients' physical activity and CR. METHODS: Between January 2009 and January 2010, 76 patients (44 men, 32 women; median age 64.5 yr) underwent CE for the diagnosis of small intestinal disorders. Indications for CE were obscure gastrointestinal bleeding/anemia (62 cases), others (14 cases). Patients were divided into an outpatient group (n=23), mild bed rest group (n=35) and strict bed rest group (n=18). RESULTS: For all patients, the average gastric transit time was 65.5 minutes, small bowel transit time was 301.4 minutes and the CR was 86.8%. However, the CR was 100% (23/23) in the outpatient group, an 85.7% (30/35) in the mild bed rest group, and 72.2% (13/18) in the strict bed rest group. The CR increased with physical activity of patients by Cochran-Armitage Trend Test (p=0.009). In multivariate logistic regression analyses, low physical activity was a significant risk factor for failure to reach the cecum during CE examination; adjusted OR: 3.39, 95% CI: 1.01-11.42 (p=0.048). CONCLUSION: Our observations suggested that increasing physical activity would increase the likelihood of a complete bowel examination by CE. Further, for CE, inconvenient bowel preparations like the use of polyethylene glycol may be avoided.

Traditional Herbal Medicine, Rikkunshito, Induces HSP60 and Enhances Cytoprotection of Small Intestinal Mucosal Cells as a Nontoxic Chaperone Inducer.

Increasing incidence of small intestinal ulcers associated with nonsteroidal anti-inflammatory drugs (NSAIDs) has become a topic with recent advances of endoscopic technology. However, the pathogenesis and therapy are not fully understood. The aim of this study is to examine the effect of Rikkunshito (TJ-43), a traditional herbal medicine, on expression of HSP60 and cytoprotective ability in small intestinal cell line (IEC-6). Effect of TJ-43 on HSP60 expression in IEC-6 cells was evaluated by immunoblot analysis. The effect of TJ-43 on cytoprotective abilities of IEC-6 cells against hydrogen peroxide or indomethacin was studied by MTT assay, LDH-release assay, caspase-8 activity, and TUNEL. HSP60 was significantly induced by TJ-43. Cell necrosis and apoptosis were significantly suppressed in IEC-6 cells pretreated by TJ-43 with overexpression of HSP60. Our results suggested that HSP60 induced by TJ-43 might play an important role in protecting small intestinal epithelial cells from apoptosis and necrosis in vitro.

Relationship between endoscopic findings and clinical severity in ischemic colitis.

BACKGROUND AND AIM: The incidence of ischemic colitis (IC) in Japan has been increasing due to the westernization of diet and the aging population. The aim of this study was to evaluate the relationship between endoscopic findings and clinical severity in IC. METHODS: This retrospective analysis included 106 cases diagnosed with IC that were divided into two groups based on endoscopic findings in the acute stage: redness and erosion (RE) versus longitudinal and circumferential ulcers (LCU). The clinical variables were compared between the two groups. In addition, we investigated the risk factors of IC associated with the severity of the endoscopic findings by multivariate logistic regression analysis. RESULTS: The percentage of cases presenting abdominal pain was significantly higher in the LCU group than that in the RE group (p=0.002), as were the baseline serum CRP levels (p=0.0001). The periods of hospitalization in LCU group were longer than in the RE group (p=0.0001). Multivariate logistic regression analysis indicated that ischemic heart disease (IHD) and connective tissue disease were the independent explanatory factor associated with the endoscopic severity of IC (p<0.05). CONCLUSION: We showed clearly that the two endoscopic classifications were accurate indicators of severity and could be used to anticipate severity of IC. Furthermore, we confirmed that IHD and connective tissue disease were the exacerbating factor associated with the severity of endoscopic findings in IC.

Colonic mucosal lesions associated with low-dose aspirin: a case control study.

OBJECTIVE: Low-dose aspirin (LDA) is widely used because it reduces the risk of vascular events in patients with atherosclerosis. Recently, there has been a substantial increase in prescriptions for LDA. We analyzed the risk of colonic mucosal lesions associated with the long-term use of LDA. MATERIAL AND METHODS: Among Japanese patients who underwent a colonoscopy between January 2004 and December 2006, 199 colitis cases and 5764 non-colitis controls were identified after excluding 749 patients based on study criteria. The history of LDA use was compared between the cases and controls and the multivariate (age-, sex- and underlying diseases-) adjusted odds ratio (OR) was estimated using a multiple logistic regression model. RESULTS: The adjusted OR for colonic mucosal lesions associated with LDA use versus non-use was 1.45 [95% confidence interval (CI), 0.87-2.42; p = 0.152]. In terms of gender differences, the OR for LDA-induced colitis in females was significantly increased at 2.55 (95% CI, 1.31-4.94; p = 0.006) but was not significantly increased in males at 0.70 (95% CI, 0.34-1.45; p = 0.334). CONCLUSIONS: In females, LDA increased the risk of colonic mucosal lesions, suggesting that LDA may contribute to the pathogenesis of colonic ulceration or colitis. Therefore, it is essential that prescribing physicians be aware of the risk of LDA-induced colonic lesions.

Metastatic renal cell carcinoma diagnosed by capsule endoscopy and double balloon endoscopy.

BACKGROUND: Renal cell carcinoma commonly metastasizes to lung, liver, and bone. Small intestinal metastases are exceedingly rare. CASE REPORT: A 75-year-old man presented at our hospital with tarry stools. He had undergone a right nephrectomy for renal cell carcinoma (RCC) 6 years previously; in addition, he had received antiplatelet treatment for ischemic heart disease. Esophagogastroduodenoscopy, total colonoscopy, and computed tomography did not identify any cause for the gastrointestinal bleeding. He underwent capsule endoscopy (CE), which revealed an ulcerated submucosal tumor in the jejunum. We performed a double-balloon endoscopy (DBE), and histological findings identified a clear cell carcinoma. We diagnosed metastasis from the RCC. We performed a jejunectomy to resect the tumor and thus eliminate the source of the bleeding. CONCLUSIONS: CE and DBE are useful diagnostic tools. We recommend investigating the possibility of small intestinal metastases in cases of intestinal bleeding or anemia in patients with a history of malignant tumor.

Evidence for enhanced cytoprotective function of HSP90-overexpressing small intestinal epithelial cells.

BACKGROUND: In the clinical field, increasing incidence of small intestinal ulcers associated with nonsteroidal anti-inflammatory drugs (NSAIDs) has become a topic with the advances of capsule endoscopy and balloon enteroscopy technology for the detection of small intestinal lesions. However, the pathogenesis of NSAID-induced mucosal damage, defensive mechanism of intestinal epithelial cells, and therapy for small intestinal mucosal lesion have not been fully understood. Heat shock proteins (HSPs) are involved in cytoprotection mediated by their function as a molecular chaperone. Since the function of HSP90 in the intestinal epithelial cells has not been well investigated, we examined the cytoprotective ability of HSP90-overexpressing small intestinal epithelial cells against hydrogen peroxide-induced or indomethacin-induced cell damage. METHODS: cDNA of human HSP90 gene was transfected to rat small intestinal epithelial cells (IEC-6 cells), and HSP90-overexpressing cells (IEC-6-90 cells) were selected and cloned. Anti-necrotic abilities and anti-apoptotic abilities of IEC-6-90 cells were compared with IEC-6-mock cells (transfected with vector alone). To examine the specific contribution of HSP90 on cytoprotection of IEC-6-90 cells, cytoprotective ability of IEC-6-90 cells was analyzed with or without pretreatment with functional inhibitor of HSP90, geldanamycine analog, followed by hydrogen peroxide-challenge or indomethacin-challenge. RESULTS: Hydrogen peroxide-induced or indomethacin-induced cell necrosis and apoptosis were significantly suppressed in IEC-6-90 cells. The cytoprotective ability of IEC-6-90 cells was suppressed by HSP90 inhibitor. CONCLUSIONS: Our results suggest that HSP90 might play an important role in protecting small intestinal epithelial cells from hydrogen peroxide-induced or indomethacin-induced cell injury in vitro, and raised the possibility of protection of small intestinal epithelial cells by manipulation of HSP90 expression.

Establishment of a reflux esophago-laryngitis model in rats.

BACKGROUND: To investigate the pathophysiology of reflux laryngitis, an experimental model is required. AIM: The aim of this study is to establish an animal model of reflux esophago-laryngitis, modifying our previously reported model of chronic acid reflux esophagitis. METHODS: The modified chronic acid reflux esophagitis (m-RE) group (n = 10), in which the duodenum was wrapped with 2.5 mm of Nelaton catheter, was not treated with any drugs. Also postoperatively, two treatment groups (n = 10 in each) received different dosages of rabeprazole (RPZ): 1.0 mg/kg/day (RPZ 1.0 group) or 10.0 mg/kg/day (RPZ 10.0 group). As a control group (n = 5), other rats underwent sham operation. The esophagus and larynx were resected on day 14 after the operation, and ulcer score of the esophagus was assessed. The epithelial thickness and leukocyte infiltration of the supraglottic and subglottic laryngeal mucosae were investigated. The number of interleukin (IL)-1ß-positive cells was also counted and defined as the IL-1ß labeling index. RESULTS: In the m-RE group, the epithelial thickness, leukocyte infiltration, and IL-1ß labeling index of the supraglottic and subglottic laryngeal mucosae were increased compared with controls (P < 0.01). In the RPZ groups, not only the ulcer score of esophagus but also the epithelial thickness, leukocyte infiltration, and IL-1ß labeling index of both the supraglottic and subglottic laryngeal mucosae were decreased dose-dependently relative to the m-RE group (P < 0.05). CONCLUSIONS: Our modified chronic acid reflux esophagitis model proved useful in establishing a rat reflux esophago-laryngitis model, with both pathological laryngeal findings and reflux esophagitis shown to be improved by administration of a proton pump inhibitor.

Observational comparative trial of the efficacy of proton pump inhibitors versus histamine-2 receptor antagonists for uninvestigated dyspepsia.

BACKGROUND AND AIMS: It is still controversial which drugs, proton pump inhibitors (PPI) or histamine-2 receptor antagonists (H2RA), are more effective for dyspepsia in the Japanese population. METHODS: Patients with uninvestigated dyspepsia (n = 104; male/female 41/63) were treated with either rabeprazole 10 mg o.d. (n = 62) or lafutidine 10 mg b.i.d. (n = 42) for 4 weeks. Questionnaires (modified Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease [mFSSG] and quality of life [QOL], SF-8) were administered before and after therapy. The mFSSG was classified into a total score (Q-T), reflux score (Q-R), dyspepsia score (Q-D) and pain score (Q-P). The SF-8 had a physical component summary (PCS) and mental component summary (MCS). The predominant type of symptom was reflux (R-S), pain (P-S) or dysmotility (D-S). RESULTS: R-S was 19.2%, P-S 48.1%, D-S 24.0% and overlap 8.7%. In the R-S, Q-T and Q-R significantly improved with rabeprazole, but neither scale improved with lafutidine. MCS significantly improved with rabeprazole. In P-S, Q-T, Q-R, Q-D and Q-P significantly improved with both drugs. PCS significantly improved with both, whereas the MCS significant improved with rabeprazole. In D-S, Q-R and Q-D significant improved with rabeprazole, but neither improved with lafutidine. QOL did not improve with either. With overlap, neither scale nor the QOL reached a significant difference. CONCLUSION: Both PPI and H2RA have a positive effect on P-S, but H(2)RA therapy is limited for R-S and D-S, whereas PPI therapy is generally effective. Therefore, careful prescription based on symptoms is important.

Clinicopathological characteristics of colorectal cancer less than 10 mm in diameter and invading submucosa and below.

BACKGROUND AND AIMS: Identifying the invasive depth of cancers less than 10 mm in diameter remains a challenge. This study examines the clinicopathological characteristics of colorectal cancers less than 10 mm in diameter and invading submucosal layer (SM)3 and below, which require surgery and must never be treated by endoscopic mucosal resection. METHODS: We studied 54 cases of colorectal cancer less than 10 mm in diameter and invading the submucosa and deeper tissues, by dividing them into two groups: those invading SM1 and SM2 versus those invading SM3 and below. We investigated the clinicopathological characteristics of cancers invading SM3 and below by comparing them with cancers invading SM1 and SM2. Similarly, 38 cases, whose endoscopic findings could be analyzed, were selected and examined. RESULTS: In cases invading SM3 and below, the rates of moderately to poorly differentiated adenocarcinoma, lymphatic and venous permeation and lymph node metastasis were significantly higher than those invading SM1 and SM2. Among cases invading SM3 and below, the presence of endoscopic findings-including white spots of the protruded type, and fullness, white spots, hardness and protruded lesions in the depressed area of the depressed type-was significantly higher than among those invading SM1 and SM2. CONCLUSION: Colorectal cancers less than 10 mm in diameter and invading SM3 and below have high malignant potential. Cancers of this invasive depth can be identified by looking for characteristics such as white spots, fullness, hardness and protruded lesions in the depressed area. Careful endoscopic observation for these signs aids in determining the appropriate treatment.

Correlation of heat shock protein expression to gender difference in development of stress-induced gastric mucosal injury in rats.

Recent studies have indicated that heat shock proteins (HSPs), which function as molecular chaperones, play important roles in cellular responses to stress-related events. However, the gender difference in the expression of HSP in the gastric mucosa remains unclear. In order to understand the mechanism of gender difference in the prevalence or severity of gastric mucosal lesions, the expression level of HSP and the correlation of estrogen to HSP induction in the gastric mucosa were evaluated in this study. The basal expression levels of HSP60 and HSP90 in the gastric mucosa were significantly higher in females than those in males. The gastric ulcer index was significantly higher in male rats compared to female rats observed after 12 h water immersion stress exposure. At this time point, the expression levels of HSP60 and HSP90 in the gastric mucosa were significantly higher in females than those in males. An estrogen-treatment significantly induced the expression of HSP60, HSP70 and HSP90 in the gastric mucosa. Inversely, an ovariectomy dramatically reduced the expression of HSP60, HSP70 and HSP90 in the gastric mucosa. Our results suggested that estrogen might play an important role in gastric mucosal protection with the induction of gastric mucosal HSPs.