Search

Cancer patients are regarded as highly vulnerable to severe acute respiratory syndrome coronavirus (SARS-CoV)-2. However, little is known regarding how cancer treatments should be restarted for cancer patients after coronavirus disease (COVID)-19. We herein report a pancreatic cancer case in which chemotherapy was able to be reinstituted after COVID-19. The patient was a 67-year-old man diagnosed with pancreatic cancer. On day 7 after first chemotherapy, he was infected with COVID-19. A SARS-CoV-2 test was negative after one month of treatment, and we reinstituted chemotherapy. The patient has received three cycles of chemotherapy without recurrence of COVID-19. It may be feasible to reinstitute chemotherapy for cancer patients after a negative SARS-CoV-2 test.

Background: The emergence of frequent hitters (FHs) remains a challenge in drug discovery. We have previously used in silico structure-based drug screening (SBDS) to identify antimycobacterial candidates. However, excluding FHs has not been integrated into the SBDS system. Methods: A dataset comprising 15,000 docking score (protein–compound affinity matrix) was constructed by multiple target screening (MTS): DOCK–GOLD two-step docking simulations with 154,118 compounds versus the 30 target proteins essential for mycobacterial survival. After extraction of 141 compounds from the protein–compound affinity matrix, compounds determined to be FHs or false positives were excluded. Antimycobacterial properties of the top nine compounds selected through SBDS were experimentally evaluated. Results: Nine compounds designated KS1–KS9 were selected for experimental evaluation. Among the selected compounds, KS3, identified as adenosylhomocysteinase inhibitor, showed a potent inhibitory effect on antimycobacterial growth (inhibitory concentration [IC]50 = 1.2 M). However, the compound also showed potent cytotoxicity. Conclusion: The MTS method is applicable in SBDS for the identification of enzyme-specific inhibitors.

A 15‐year‐old boy was referred to our hospital with elevated hepatobiliary enzyme levels and jaundice. Magnetic resonance cholangiopancreatography performed at the previous medical facility revealed a stricture of the intrahepatic and extrahepatic bile duct. Computed tomography showed dilatation and wall thickness of the intrahepatic bile ducts. Primary sclerosing cholangitis or cholangiocarcinoma was suspected. Endoscopic retrograde cholangiopancreatography (ERCP) showed stricture in the intrahepatic and extrahepatic bile duct. On admission, the eosinophil count in the peripheral blood was normal; however, rapid hypereosinophilia in the peripheral blood was observed after admission, leading us to suspect eosinophilic cholangitis (EC). A bile duct biopsy showed inflammatory cells and eosinophil infiltration during a second ERCP. The patient was diagnosed with EC based on histopathology., Eosinophilic cholangitis was diagnosed based on the elevated blood eosinophil count after admission and bile duct pathology.

The mechanism and physiological functions of heme oxygenase-2 (HO-2)-mediated carbon monoxide (CO) production, accompanied by heme metabolism, have been studied intensively in recent years. The enzymatic activity of constitutively expressed HO-2 must be strictly controlled in terms of the toxicity and chemical stability of CO. In this study, the molecular interaction between HO-2 and caveolin-1 and its effect on HO action were evaluated. An enzyme kinetics assay with residues 82-101 of caveolin-1, also called the caveolin scaffold domain, inhibited HO-2 activity in a competitive manner. Analytical ultracentrifugation and a hemin titration assay suggested that the inhibitory effect was generated by direct binding of caveolin-1 to aromatic residues, which were defined as components of the caveolin-binding motif in the HO-2 heme pocket. Herein, we developed a HO-2-based fluorescence bioprobe, namely EGFP-Δ19/D159H, which was capable of quantifying heme binding by HO-2 as the initial step in the CO production. The fluorescence of EGFP-Δ19/D159H decreased in accordance with 5-aminolevulinic acid-facilitated heme biosynthesis in COS-7 cells. In contrast, expression of the N-terminal cytosolic domain of caveolin-1 (residues 1-101) increased the probe fluorescence, suggesting that the cytosolic domain of caveolin-1 potently inhibits the binding of heme to the heme pocket of EGFP-Δ19/D159H. Taken together, our results suggest that caveolin-1 is a negative regulator of HO-2 enzymatic action. Moreover, our bioprobe EGFP-Δ19/D159H represents a powerful tool for use in future studies addressing HO-2-mediated CO production.

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH&ndash, cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (&Delta, TGEE) is tightly bound to heme&ndash, HO-1, whereas the reduction in heme&ndash, HO-1 coupled with &Delta, TGEE is considerably slow because the distance between FAD and FMN in &Delta, TGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme&ndash, HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme&ndash, HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of &Delta, TGEE in complex with heme&ndash, HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme&ndash, HO-1 in solution. We conclude that the &ldquo, open&ndash, close&rdquo, transition of CPR is indispensable for electron transfer from CPR to heme&ndash, HO-1.

Objective This study aims to elucidate the association between the clinical characteristics of post-colonoscopy colorectal cancer (PCCRC) and quality indicators (QIs) of colonoscopy. Methods Patients with PCCRC who underwent total colonoscopy (TCS) and were histologically diagnosed with adenocarcinoma within six months to five years of the last examination were included in this study. PCCRC and normally detected cancer (NDC) identified within the same period were compared in terms of their clinicopathological characteristics. Furthermore, the QIs at PCCRC detection were compared to those at the last examination. Results Patients with PCCRC had a significantly higher rate of colon surgery history than those with NDC (PCCRC: 25/76, 32.9%; NDC: 31/1,437, 2.2%; p

InhA or enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis (mtInhA), which controls mycobacterial cell wall construction, has been targeted in the development of antituberculosis drugs. Previously, our in silico structure-based drug screening study identified a novel class of compounds (designated KES4), which is capable of inhibiting the enzymatic activity of mtInhA, as well as mycobacterial growth. The compounds are composed of four ring structures (A-D), and the MD simulation predicted specific interactions with mtInhA of the D-ring and methylene group between the B-ring and C-ring; however, there is still room for improvement in the A-ring structure. In this study, a structure-activity relationship study of the A-ring was attempted with the assistance of in silico docking simulations. In brief, the virtual chemical library of A-ring-modified KES4 was constructed and subjected to in silico docking simulation against mtInhA using the GOLD program. Among the selected candidates, we achieved synthesis of seven compounds, and the bioactivities (effects on InhA activity and mycobacterial growth and cytotoxicity) of the synthesized molecules were evaluated. Among the compounds tested, two candidates (compounds 3d and 3f) exhibited superior properties as mtInhA-targeted anti-infectives for mycobacteria than the lead compound KES4.

The physical interaction of the human growth factor receptor-bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser358 , Ser362 , and Ser366 in hGrb14 by glycogen synthase kinase-3 repressed hGrb14-IR complex formation. In this study, we investigated phosphatase-mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14-derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser358 and Ser362 . Furthermore, coimmunoprecipitation experiments suggested that insulin-induced hGrb14-IR complex formation was repressed by the substitution of Ser358 or Ser362 with glutamic acid. These findings suggested that phosphate groups on Ser358 and Ser362 in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14-IR complex formation.

Background: Enzymes responsible for cell wall development in Mycobacterium tuberculosis are considered as potential targets of anti-tuberculosis (TB) agents. Mycobacterial cyclopropane mycolic acid synthase 1 (CmaA1) is essential for mycobacterial survival because of its critical role in synthesizing mycolic acids. Materials and Methods: We screened compounds that were capable of interacting with the mycobacterial CmaA1 active site using a virtual compound library with an in silico structure-based drug screening (SBDS). Following the selection of such compounds, their antimycobacterial activity was examined. Results: With the in silico SBDS, for which we also used DOCK-GOLD programs and screening methods that utilized the structural similarity between the selected active compounds, we identified two compounds with potent inhibitory effects on mycobacterial growth. The antimycobacterial effect of the compounds was comparable to that of isoniazid, which is used as a first-line anti-TB drug. Conclusion: The compounds identified through SBDS were expected to be a novel class of anti-TB pharmacophores.

Pigment epithelium-derived factor (PEDF) is a glycoprotein with complex neuroprotective, anti-angiogenic, and anti-inflammatory properties, all of which could potentially be exploited as a therapeutic option for vascular complications in diabetes. We have previously shown that PEDF-derived synthetic peptide, P5-3 (FIFVLRD) has a comparable ability with full PEDF protein to inhibit rat corneal neovascularization induced by chemical cauterization. However, the effects of PEDF peptide on experimental diabetic nephropathy remain unknown. To address the issue, we modified P5-3 to stabilize and administered the modified peptide ( d -Lys- d -Lys- d -Lys-Gln- d -Pro-P5-3-Cys-amide, 0.2 nmol/day) or vehicle to streptozotocin-induced diabetic rats (STZ-rats) intraperitoneally by an osmotic mini pump for 2 weeks. We further examined the effects of modified peptide on human proximal tubular cells. Renal PEDF expression was decreased in STZ-rats. Although the peptide administration did not affect blood glucose or blood pressure, it decreased urinary excretion levels of 8-hydroxy-2′-deoxyguanosine, an oxidative stress marker, and reduced plasminogen activator inhibitor-1 (PAI-1) gene expression, and suppressed glomerular expansion in the diabetic kidneys. High glucose or advanced glycation end products stimulated oxidative stress generation and PAI-1 gene expression in tubular cells, all of which were significantly suppressed by 10 nM modified P5-3 peptide. Our present study suggests that PEDF-derived synthetic modified peptide could protect against experimental diabetic nephropathy and inhibit tubular cell damage under diabetes-like conditions through its anti-oxidative properties. Supplementation of modified P5-3 peptide may be a novel therapeutic strategy for diabetic nephropathy.

AIM Protein and energy malnutrition is a severe problem for patients with liver cirrhosis (LC) and fasting often induces starvation which is a vitally important outcome. Dietary restriction is essential for endoscopic injection sclerotherapy (EIS) in patients with risky esophageal varices, thereby creating the possible exacerbation of nutritional state and inducing liver dysfunction. Whether EIS induces nutritional deficiency in LC patients and the effects of branched-chain amino acid (BCAA)-enriched nutrient are prospectively investigated. METHODS A total of 61 LC patients were randomly divided into an EIS monotherapy group (non-BCAA group, n = 31) and an EIS combined with BCAA therapy group (n = 30). Platelet count, blood chemistry and somatometry values were prospectively measured at five time points. RESULTS The platelet counts before treatment were at the same level in both groups (P = 0.72). Three months after treatment, the counts decreased in the non-BCAA group; however, they increased in the BCAA group (P = 0.019). Body mass index, triceps skin fold thickness and arm muscle circumference significantly decreased in both groups. The BCAA and tyrosine ratio value increased only in the BCAA group (P

The Mycobacterium tuberculosis (M. tuberculosis) enoyl-acyl carrier protein reductase (mtInhA) is an attractive enzyme and a thoroughly studied target for tuberculosis therapy. In this study, to identify novel structure-activity relationships (SARs) of mtInhA inhibitors, a series of diphenyl ether derivatives were designed based on the matched molecular pair (MMP) method, and the binding energies of these compounds were subsequently estimated by in silico structure-based drug screening (SBDS) to provide more useful data. Consequently, the 10 unique candidate compounds (KEM1-KEM10) were identified and assessed for the inhibition of mtInhA enzymatic activity, in vitro antibiotic effects against model mycobacteria and toxicity level on both intestinal bacteria and mammalian cells. Among the compounds tested, phenyl group (KEM4) and 2-fluorobenzyl group (KEM7) substitutions produced preferable inhibitory effects on mtInhA enzymatic activity relative to those provided by a furyl group (KES4: base compound) at the terminal of the compound, and KEM7 inhibited the growth of the mycobacteria strain with a lower IC50 value. Moreover, most of the candidate compounds exhibited neither inhibition of the growth of enterobacteria nor toxic effects on mammalian cells, though KEM10 exhibited toxicity against cultured MDCK cells. The structural and experimental information concerning these mtInhA inhibitors identified through MMP-based in silico screening will likely contribute to the lead optimisation of novel antibiotics for M. tuberculosis.

Tenascin-C is an adhesion modulatory matrix protein that is highly expressed in tumors; however, its biochemical activity involved in tumorigenesis is not fully understood. On the other hand, increasing evidence indicates the importance of integrin α5β1 in cancer development. We previously demonstrated that tenascin-C harbors a functional site that can be released as a proadhesive peptide such as TNIIIA2. Peptide TNIIIA2 is capable of inducing activation of β1-integrins including α5β1 via syndecan-4. In this study the proadhesive effect of TNIIIA2 was characterized by potentiated and sustained activation of integrin α5β1. Based on this effect, TNIIIA2 rendered nontransformed fibroblasts (NIH3T3) resistant to serum deprivation-elicited anoikis through activation of the Akt/Bcl-2 pathway. Moreover, TNIIIA2 hyperstimulated PDGF-dependent proliferation of NIH3T3 by activating integrin α5β1. Tenascin-C, a parental protein of TNIIIA2, also stimulated PDGF-dependent proliferation, which was blocked by a matrix metalloproteinase-2/9 inhibitor and an anti-TNIIIA2 function-blocking antibody, suggesting proteolytic exposure of the proadhesive effect of TNIIIA2. Mechanistic analyses revealed that TNIIIA2 induced a lateral association of PDGF receptor β with the molecular complex of activated integrin α5β1 and syndecan-4 in the membrane microdomains enriched with cholesterol/caveolin-1, resulting in prolonged activation of PDGF receptor β and the subsequent Ras/mitogen-activated protein kinase pathway in a PDGF-dependent manner. Of note, TNIIIA2 induced continuous proliferation in NIH3T3 in an integrin α5β1-dependent manner even after they formed a confluent monolayer. Thus, it was proposed that tenascin-C might be involved in deregulated cell growth through potentiated and sustained activation of integrin α5β1 after exposure of the proadhesive effect of TNIIIA2.

Phosphorylation of high mobility group box 1 (HMGB1) is involved in the subcellular translocation of this protein and its subsequent secretion. Two nuclear localization signals (NLSs), NLS1 and NLS2, in this protein regulate its nucleocytoplasmic relocation, and phosphorylation of both NLSs strongly promotes HMGB1 mobilization. However, the phosphorylation properties of serine residues in NLS1 and the kinases involved are not well known. In the present study, we predicted kinases that phosphorylate serine residues in NLS1 and performed an in vitro kinase assay utilizing NLS1-derived phosphopeptides. Among the predicted kinases, protein kinase C phosphorylated Ser46 of HMGB1-derived peptides, and a mutagenesis experiment confirmed that phosphorylation at this site could induce the translocation of the N-terminal region of NLS1-containing HMGB1 into the cytosol. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Aim The combination therapy of pegylated interferon-α and ribavirin (PEG IFN/RBV) is one of the effective treatments for chronic hepatitis C (CHC) patients. Natural killer (NK)-cell activity was reported to be impaired in patients with hepatitis C virus (HCV). The aim of this study was to examine whether PEG IFN/RBV therapy could restore NK activity in CHC patients. Methods In 19 CHC patients, PEG IFN/RBV therapy was performed. Just before (0M), at 3 months of the therapy (3M) and at 6 months after completion of the therapy (6M), NK activity and the frequency of NK cells, CD56dimNK cells and CD56brightNK cells in peripheral blood was estimated by creatinine release assay and flow cytometry. Statistical analysis was performed by anova and Mann–Whitney U-test. Results anova showed that NK activity significantly improved at 6M (vs 0M, P

Growth factor receptor-bound protein 14 (Grb14) interacts with insulin receptor (IR) through the between PH and SH2 (BPS) domain. Grb14-IR complex formation is initiated by insulin stimulation, and the binding event results in the inhibition of insulin signalling. Thus, Grb14 is regarded as an endogenous suppressor of insulin signal transduction; however, there are no studies describing the mechanism whereby Grb14-IR complex formation is suppressed in the absence of insulin stimulation. In the present study, multiple phosphorylation motifs for glycogen synthase kinase 3 (GSK-3) were identified within the Grb14 BPS domain (Ser(358), Ser(362) and Ser(366) of human Grb14). Pharmacological inhibition as well as knockdown of GSK-3 facilitated complex formation between Grb14 and IR, implicating GSK-3 activity in regulating Grb14-IR binding. In situ proximity ligation assay and in vitro kinase assays of phosphopeptides suggested that serine residues in the BPS domain would be substrates for GSK-3. The kinase assays also indicated phosphoserine 370 (in human Grb14) was required for the phosphorylation of Ser(358), Ser(362) and Ser(366) by GSK-3. Grb14-IR binding was also facilitated by replacement of the serines with Ala. We also observed that Ser(366) of endogenous Grb14 in Hep G2 cell was phosphorylated and the phosphorylation was influenced by treatments with insulin, as well as the GSK-3 inhibitor.

Background and Aim Liver cirrhosis (LC) is accompanied by hepatic arterializations, intrahepatic shunts, and hyperdynamic circulations. These changes shorten the arrival time (AT) of ultrasound contrast agents to the hepatic vein (HV). Whether treatment of gastric fundal varices (GVs) by balloon-occluded transvenous obliteration (B-RTO) improves the AT in LC patients was prospectively investigated. Methods A total of 32 LC patients with GVs and 10 normal controls (NCs) were enrolled. This study was approved by the clinical research ethics committee. Images of hepatic artery (HA), portal vein (PV), and HV were monitored after an injection of a contrast agent using quantification software. The AT before and after B-RTO in LC patients and that in NCs were compared. Results All GVs were treated effectively, and indocyanine green retention rate was improved (P

High mobility group box 1 (HMGB1), a non-histone chromosomal protein, is a proinflammatory cytokine. There are two known pathways for the release of HMGB1 into the extracellular milieu-passive and active. The passive pathway is attributable to cell death from damage or necrosis, and the active pathway is secretion from immunocompetent cells activated by proinflammatory stimuli. Recent studies have shown that post-translational modifications of HMGB1, including phosphorylation, are involved in the relocation of HMGB1 to the cytoplasm and subsequent secretion. With regard to the HMGB1 phosphorylation, Youn and Shin [Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion. J Immunol 2006;177:7889-97] reported that treatment of the murine macrophage RAW264.7 with okadaic acid resulted in nucleocytoplasmic translocation and secretion of HMGB1. Herein, we demonstrate the physical interaction between HMGB1 and protein phosphatase 2A (PP2A) in the RAW264.7. The results of in vitro phosphatase assay further indicate that PP2A dephosphorylates specific phosphoserine residues within one of the two nuclear localization signals (NLSs) of HMGB1. The cytoplasmic relocation of HMGB1 through PP2A inhibition was markedly suppressed by replacement of the Ser residues within the NLS with Ala. These consequences imply that PP2A correlates in the nucleocytoplasmic shuttling of HMGB1.

Summary PA3535 (EprS), an autotransporter (AT) protein of Pseudomonas aeruginosa, is predicted to contain a serine protease motif. The eprS encodes a 104.5 kDa protein with a 30-amino-acid-long signal peptide, a 51.2 kDa amino-terminal secreted passenger domain and a 50.1 kDa carboxyl-terminal outer membrane channel formed translocator. Although the majority of AT proteins have been reported to be virulence factors, little is known about the functions of EprS in the pathogenicity of P. aeruginosa. In this study, we performed functional analyses of recombinant EprS secreted by Escherichia coli. The proteolytic activity of EprS was markedly decreased by changing Ser to Ala at position 308 or by serine protease inhibitors. EprS preferred to cleave substrates that terminated with arginine or lysine residues. Thus, these results indicate that EprS, a serine protease, displays the substrate specificity, cleaving after basic residues. We demonstrated that EprS activates NF-κB-driven promoters through protease-activated receptor (PAR)-1, -2 or -4 and induces IL-8 production through PAR-2 in a human bronchiole epithelial cell line. Moreover, EprS cleaved the peptides corresponding to the tethered ligand region of PAR-1, -2 and -4 at a specific site with exposure oftheir tethered ligands. Collectively, these results suggest that EprS activates host inflammatory responses through PARs.

Background: Argon plasma coagulation (APC) is very useful as a consolidation treatment for reducing the recurrence of esophageal varices (EVs). However, repeated sedation in endoscopic treatment has the risk of prolonging hepatic encephalopathy and affects the respiratory state of liver cirrhosis (LC) patients, in whom pulmonary arteriovenous shunts are observed. We evaluated prospectively whether transnasal endoscopic APC without sedation is more effective than peroral endoscopic APC with sedation. Patients and Methods: LC patients (n = 101), treated by endoscopic injection sclerotherapy to eradicate EVs, were randomly divided into a transnasal APC group (n = 50) and a peroral APC group (n = 51). The primary efficacy endpoint was the cumulative recurrence rate of EVs. The secondary endpoints were blood pressure (BP), heart rate, oxygen saturation during APC and complications. Results: There was no significant difference in the cumulative recurrence rate of EVs at 36 months between the transnasal APC and peroral APC groups (35.0 vs. 40.8%, p = 0.39, log-rank test), indicating that transnasal APC is not inferior to peroral APC. The transnasal APC group showed more stable intraoperative BP and oxygen saturation values, and a lower incidence of epigastralgia (56.0 vs. 74.5%, p = 0.04). Conclusion: The efficacy of reducing the recurrence of EVs in the transnasal APC group was not significantly different from that in the peroral APC group. Transnasal APC caused less distress and required no sedation. Therefore, this method was more advantageous for LC patients at risk of suffering from prolongation of hepatic encephalopathy.

The enzymes responsible for biotin biosynthesis in mycobacteria have been considered as potential drug targets owing to the important role in infection and cell survival that the biotin synthetic pathway plays in Mycobacterium tuberculosis. Among the enzymes that comprise mycobacterium biotin biosynthesis systems, 7,8-diaminopelargonic acid synthase (DAPAS) plays an essential role during the stationary phase in bacterial growth. In this study, compounds that inhibit mycobacterial DAPAS were screened in the virtual chemical library using an in silico structure-based drug screening (SBDS) technique, and the antimycobacterial activity of the selected compounds was validated experimentally. The DOCK-GOLD programs utilized by in silico SBDS facilitated the identification of a compound, referred to as KMD6, with potent inhibitory effects on the growth of model mycobacteria (M. smegmatis). The subsequent compound search, which was based on the structural features of KMD6, resulted in identification of three additional active compounds, designated as KMDs3, KMDs9 and KMDs10. The inhibitory effect of these compounds was comparable to that of isoniazid, which is a first-line antituberculosis drug. The high antimycobacterial activity of KMD6, KMDs9 and KMDs10 was maintained on the experiment with M. tuberculosis. Of the active compounds identified, KMDs9 would be a promising pharmacophore, owing to its long-term antimycobacterial effect and lack of cytotoxicity.

Growth factor receptor-bound protein 14 (Grb14) is a negative regulator of insulin receptor (IR) and is involved in a negative feedback mechanism of insulin signaling. Grb14 associates with IR and inhibits its tyrosine kinase activity through the between pleckstrin homology and Src homology-2 (BPS) domain. We previously reported that the pharmacological inhibition and knockdown of glycogen synthase kinase-3 (GSK-3) facilitates the insulin-induced complex formation of human Grb14 (hGrb14) and IR, suggesting that GSK-3 suppresses hGrb14 recruitment to IR. This study further investigated a functional phosphorylation of the serine residues in hGrb14 BPS domain, identified as putative GSK-3 targets to verify an effect of GSK-3 on the hGrb14-IR complex formation. In vitro kinase assay using the motif-derived peptides showed that the serine residues located in N-terminal (Ser358, Ser362 and Ser366) and C-terminal (Ser419 and Ser423) regions of the BPS domain were phosphorylated by GSK-3. Co-immunoprecipitation and yeast two-hybrid (Y2H) experiments suggested that the negative charges genetically introduced on the Ser358, Ser362 and Ser366 suppressed the association of hGrb14 to IR. Surface plasmon resonance experiment gave Kd values of 8 nM for recombinant hGrb14 with respect to the interaction with IR β-subunit, and this affinity was lost after the replacements of the Ser358, Ser362 and Ser366 with glutamic acid residues. Y2H experiment with the BPS domain alone; however, did not show any difference owing to the same mutations. It is therefore evident that the N-terminus of the BPS domain plays an important role in the regulation of hGrb14-IR complex formation through phosphorylation, in addition to other domains.

Objective To clarify the usefulness of transnasal esophagogastroduodenoscope (N-EGD) with all flexible imaging color enhancement (FICE) patterns (0–9) for the diagnosis of esophageal varices (EV). Methods A total of 50 patients with EV were examined during the same period by N-EGD without sedation and by peroral endoscopy (O-EGD) with sedation. The visibility of treatment scars, microvessels, recurrent EV and red color sign (RCS) were measured. Visibility was evaluated by five blinded observers (poor–good: 0–10), and the effect of both endoscopic examinations on the cardiorespiratory function of the patients was measured. Results The image scores for O-EGD and N-EGD with ordinary mode and N-EGD with FICE were 7.3 ± 1.2, 6.1 ± 1.0 and 6.9 ± 1.0 for treatment scars; 7.2 ± 1.4, 6.2 ± 1.2 and 7.3 ± 1.0 for microvessels; 7.2 ± 1.2, 6.1 ± 1.0 and 7.1 ± 1.0 for recurrent EV and 7.2 ± 1.3, 6.1 ± 1.3, and 7.2 ± 1.2 for RCS, respectively (intraclass correlation coefficients > 0.6). When FICE patterns 2, 6 and 9 were used, visibility was much improved. Ten minutes after insertion, the double product values in the N-EGD with the FICE group were significantly better (P

To compare contrast-enhanced (CE) ultrasound with gadoxetate disodium-enhanced magnetic resonance (MR) imaging in the assessment of arterial hypervascularity of hepatocellular carcinoma (HCC) and dysplastic nodule (DN), with CT during hepatic arteriography (CTHA) as the reference standard.This study included 54 consecutively diagnosed patients, with 57 histologically confirmed HCCs and 3 DNs (high-grade). All patients underwent CE ultrasound, gadoxetate disodium-enhanced MR imaging and CTHA. Two trained diagnostic radiologists interpreted the CTHA images and rated the degree of intratumoral arterial vascularity by consensus using a five-point confidence scale as the reference standard. In the observer study, the degrees of vascularity on CE ultrasound and gadoxetate disodium-enhanced MR images were qualitatively analysed by four independent readers using a five-point confidence scale. Diagnostic accuracy was analysed by receiver-operating characteristic (ROC) analysis.The diagnostic accuracies of the average area under the ROC curve (AUC) were significantly greater with CE ultrasound (average AUC: 0.94; 95% confidence interval [CI]: 0.88-1.00) than with gadoxetate disodium-enhanced MR imaging (average AUC 0.84, 95% CI 0.74-0.93, P = 0.0014).Contrast-enhanced ultrasound yields a significantly higher AUC value than gadoxetate disodium-enhanced MR imaging in the assessment of arterial hypervascularity of HCC and DN.• Arterial hypervascularity is an important feature determining treatment options in hepatocellular carcinoma. • It can be assessed by contrast-enhanced (CE) ultrasound or magnetic resonance (MR) imaging. • CE ultrasound was more accurate than Gd-EOB-DTPA MRI in assessing intratumoral vascularity. • Hypovascular hepatic nodules should be further investigated using CE ultrasound.

Heme oxygenase (HO) catalyzes the O(2)-dependent degradation of heme to biliverdin IXα, carbon monoxide (CO), and free ferrous iron through a multistep mechanism. Electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase. Recently, Kim et al. reported for the first time that HO, especially inducible HO-1, appears in caveolae and showed that caveolin-1, a principal isoform of the caveolin family, physically interacts with HO-1 [ Jung , N. H. et al. ( 2003 ) IUBMB Life 55 , 525 - 532 ; Kim , H. P. et al. ( 2004 ) FASEB J. 18 , 1080 - 1089 ]. In the present study, we confirmed by immunoprecipitation experiments that rat HO-1 and rat caveolin-1 (residues 1-101) directly interact with each other and that the HO-1 activity is inhibited by caveolin-1 (1-101). The 82-101 residues of caveolin-1 (CAV(82-101)), called the caveolin scaffolding domain, play essential roles in caveolin-related protein-protein interactions. The HO-1 activity is also inhibited by CAV(82-101) in a competitive manner with hemin, and a hemin titration experiment showed that CAV(82-101) interferes with hemin binding to HO-1. The enzyme kinetics and surface plasmon resonance experiments gave comparable K(i) and K(D) values of 5.2 and 1.0 μM for CAV(82-101), respectively, with respect to the interaction with HO-1. These observations indicated that CAV(82-101) and hemin share a common binding site within the HO-1 protein. The identified caveolin binding motif (FLLNIELF) of rat HO-1 is incomplete compared to the proposed consensus sequence. The affinity between HO-1 and CAV(82-101), however, was almost completely or remarkably eliminated by replacement of Phe(207) and/or Phe(214) with Ala, indicating that HO-1 binds to caveolin-1 via this motif. Among the peptide fragments derived from CAV(82-101), i.e., CAV(82-91), CAV(87-96), CAV(92-101), and CAV(97-101), CAV(92-101) and CAV(97-101) are able to inhibit the HO-1 activity to a similar extent; thus, the five-amino acid sequence (residues 97-101) is considered to be a minimum sequence for binding to HO-1.

We report a case of a 28-year-old woman with hepatocellular adenoma and correlate findings of pathology and magnetic resonance (MR) imaging with gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) enhancement. In the hepatobiliary phase, the peripheral region of the tumor that corresponded with proliferating hepatocytes with steatosis showed slight hypointensity compared with the surrounding liver parenchyma, and the central region of the tumor that corresponded with cellular areas showed isointensity.

Characteristic motifs have been identified in natural channel forming peptides though critical roles of such motifs are not well understood. In this paper, the helix‐kink motif found in peptaibols was embedded into the α-aminoisobutyric acid (Aib) rich template to explore its roles in peptide structure and ion channel functions. According to circular dichroism studies and single channel measurements, the motif reduced helical contents of peptide whereas ion channel forming was facilitated and conductance value was increased.

The widespread natural sources-derived cationic peptides have been reported to reveal bacterial killing and/or growth-inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug-resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib-Arg-Aib-Ala sequences, showed strong antibacterial activity against both Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug-resistant Pseudomonas aeruginosa, known as proteases-secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib-derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices.

Several ion channel forming cyclic peptides have been reported over the past two decades and various ion conducting mechanisms have been proposed. In this article, we report on amino acid substitut...

It has been postulated that inactivated beta1-integrins are involved in the disordered growth of hematopoietic tumor cells. We recently found that TNIIIA2, a peptide derived from tenascin-C, strongly activates beta1-integrins through binding with syndecan-4. We show here that Ramos Burkitt's lymphoma cells can survive and grow in suspension but undergo apoptosis when kept adhering to fibronectin by stimulation with TNIIIA2. Other integrin activators, Mg(2+) and TS2/16 (an integrin-activating antibody), were also capable of inducing apoptosis. The inactivation of ERK1/2 and Akt and the subsequent activation of Bad were involved in the apoptosis. The results using other hematopoietic tumor cell lines expressing different levels of fibronectin receptors (VLA-4 and VLA-5) showed that potentiated and sustained adhesion to fibronectin via VLA-4 causally induces apoptosis also in various types of hematopoietic tumor cells in addition to Ramos cells. Because TNIIIA2 requires syndecan-4 as a membrane receptor for activation of beta1-integrins, it induced apoptosis preferentially in hematopoietic tumor cells, which expressed both VLA-4 and syndecan-4 as membrane receptors mediating the effects of fibronectin and TNIIIA2, respectively. Therefore, normal peripheral blood cells, such as neutrophils, monocytes, and lymphocytes, which poorly expressed syndecan-4, were almost insusceptible to TNIIIA2-induced apoptosis. The TNIIIA2-related matricryptic site of TN-C could contribute, once exposed, to preventing prolonged survival of hematopoietic malignant progenitors through potentiated and sustained activation of VLA-4.

症例は，66歳男性．肝外転移を有する進行肝癌治療目的でsorafenib 800 mg/day投与開始し，治療開始17日目に代謝性脳症で死亡した．治療前および治療後1週に，ソナゾイド造影超音波とMRI拡散強調画像をそれぞれ撮像した．造影超音波では，治療前と比べ治療後1週において，腫瘍部と健常腎実質部に著明な血流低下を認めた．一方，拡散強調画像では，腫瘍部のADCの著明な低下を認めた．sorafenib治療において，腫瘍部に血流低下などの，急激な画像所見の変化が生じる場合，治療効果良好の徴候とも考えられるが，重篤な有害事象の発現の可能性も念頭に置き，厳重な経過観察が必要であると考える．

The hydrophobic value and hydrophobic moment value of helical peptides correlate with membrane–peptide interactions such as ion channel forming or membrane perturbation. In this paper, two ...

An ultimate goal of synthetic ion-channel peptide design is to construct stable and functional ion-conducting pores. It is expected that specific interhelical interactions would facilitate the association of helices in phospholipid membranes and the successive helix-bundle formation. In the present study, we rationally designed helix-bundle ion channels using the synthetic hybrid peptide K20E20, a disulfide dimer of cationic- and anionic-amphiphilic helices Ac-CGG-(BKBA) 5-NH 2 and Ac-CGG-(BEBA) 5-NH 2. Circular dichroism (CD) measurements in aqueous media implied helix stabilization in the peptide caused by the interhelical electrostatic interactions. In addition, CD spectra recorded in the presence of DPPC liposomes and dye-leakage measurements suggested a high degree of association of peptide monomers in phospholipid membranes as well as high affinities between peptide and lipid bilayers. These features allowed ion-channel formation at extremely low peptide concentrations (as low as 1 nM). According to electrophysiological analyses, stable helix bundles were constructed of six peptide helices by association of three K20E20 molecules. Helix-helix association in lipid membranes, peptide-membrane interactions, and ion-channel formation of K20E20 peptides were all facilitated by intramolecular electrostatic interactions between the helices of the hybrid peptide and were pH-dependent. Conductance through K20E20 ion channels decreased under acidic conditions because of the interruption of the salt bridges.

Pseudomonas aeruginosa possesses an arsenal of both cell-associated (flagella, pili, alginate, etc.) and extracellular (exotoxin A, proteases, type III secretion effectors, etc.) virulence factors. Among them, secreted proteases that damage host tissues are considered to play an important role in the pathogenesis of P. aeruginosa infections. We previously reported that EprS, an autotransporter protease of P. aeruginosa, induces host inflammatory responses through protease-activated receptors. However, little is known about the role of EprS as a virulence factor of P. aeruginosa. In this study, to investigate whether EprS participates in the pathogenicity of P. aeruginosa, we characterized various pathogenic phenotypes of the wild-type PAO1 strain and its eprS-disrupted mutant. The growth assays demonstrated that the growth of the eprS mutant was somewhat lower than that of the wild-type strain in a minimal medium containing BSA as the sole carbon and nitrogen source. Thus, these results indicate that eprS would have a role in the growth of P. aeruginosa in the presence of limited nutrients, such as a medium containing proteinaceous materials as a sole nutrient source. Furthermore, disruption of eprS resulted in a decreased production of elastase, pigments, autoinducers and surfactants, and a reduction of swimming and swarming motilities. In addition, the eprS mutant exhibited a reduction in the ability to associate with A549 cells and an attenuation of virulence in leucopenic mice as compared with the wild-type strain. Collectively, these results suggest that EprS exerts pleiotropic effects on various pathogenic phenotypes of P. aeruginosa.