Brain p3-Alcß peptide restores neuronal viability impaired by Alzheimer's amyloid ß-peptide.

We propose a new therapeutic strategy for Alzheimer's disease (AD). Brain peptide p3-Alcß37 is generated from the neuronal protein alcadein ß through cleavage of γ-secretase, similar to the generation of amyloid ß (Aß) derived from Aß-protein precursor/APP. Neurotoxicity by Aß oligomers (Aßo) is the prime cause prior to the loss of brain function in AD. We found that p3-Alcß37 and its shorter peptide p3-Alcß9-19 enhanced the mitochondrial activity of neurons and protected neurons against Aßo-induced toxicity. This is due to the suppression of the Aßo-mediated excessive Ca2+ influx into neurons by p3-Alcß. Successful transfer of p3-Alcß9-19 into the brain following peripheral administration improved the mitochondrial viability in the brain of AD mice model, in which the mitochondrial activity is attenuated by increasing the neurotoxic human Aß42 burden, as revealed through brain PET imaging to monitor mitochondrial function. Because mitochondrial dysfunction is common in the brain of AD patients alongside increased Aß and reduced p3-Alcß37 levels, the administration of p3-Alcß9-19 may be a promising treatment for restoring, protecting, and promoting brain functions in patients with AD.

Role of a bacterial glycolipid in Sec-independent membrane protein insertion.

Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.

A decline in activities of daily living due to acute heart failure is an independent risk factor of hospitalization for heart failure and mortality.

BACKGROUND: Although activities of daily living (ADL) are recognized as being pertinent in averting relevant readmission of heart failure (HF) and mortality, little research has been conducted to assess a correlation between a decline in ADL and outcomes in HF patients. METHODS: The Kitakawachi Clinical Background and Outcome of Heart Failure Registry is a prospective, multicenter, community-based cohort of HF patients. We categorized the patients into four types of ADL: independent outdoor walking, independent indoor walking, indoor walking with assistance, and abasia. We defined a decline in ADL (decline ADL) as downgrade of ADL and others (non-decline ADL) as preservation of ADL before discharge compared with admission. RESULTS: Among 1253 registered patients, 923 were eligible, comprising 98 (10.6%) with decline ADL and 825 (89.4%) with non-decline ADL. Decline ADL exhibited a higher risk of hospitalization for HF and mortality compared with non-decline ADL. A multivariate analysis revealed that decline ADL emerged as an independent risk factor of hospitalization for HF [hazard ratio (HR), 1.42; 95% confidence interval (CI): 1.01-1.96; p=0.046] and mortality (HR, 1.95; 95% CI: 1.23-2.99; p<0.01). Although 66.3% of patients with decline ADL were registered for long-term care insurance, few received daycare services (32.7%) or home-visit medical services (8.2%). CONCLUSIONS: Decline in ADL is a predictor of hospitalization for HF and mortality in HF patients.

Peptide self-cleavage at a canaline residue: application to a solubilizing tag system for native chemical ligation.

Canaline (Can) is a non-proteinogenic amino acid containing an aminooxy group in its side chain. Can-containing peptides can be synthesized by standard Fmoc SPPS using Fmoc-Can(2-Cl-Trt). Here, for the first time, a Can residue within a peptide sequence was found to spontaneously cleave the main chain amide bond under slightly acidic conditions (pH 4-5). Contrastingly, Can-containing peptides are completely stable under the acidic conditions for HPLC purification (pH ca. 2) and under the neutral conditions for native chemical ligation (NCL). Taking advantage of these unique pH-dependent properties of Can, a novel solubilizing tag system for NCL-mediated protein synthesis using (Lys/Arg)n-Can was developed.

Easy-to-Attach/Detach Solubilizing-Tag-Aided Chemical Synthesis of an Aggregative Capsid Protein.

A solubilizing Trt-K10 tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt-K10 tag comprises a hydrophilic oligo-Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol-type introducing reagent Trt(OH)-K10 under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol-additive-free native chemical ligation can be modified directly in a one-pot manner to facilitate the isolation of the product by high-performance liquid chromatography. Finally, the Trt-K10 tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy-to-attach/detach tag-aided method, a hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.

1,2,4-Triazole-aided native chemical ligation between peptide-N-acyl-N'-methyl-benzimidazolinone and cysteinyl peptide.

1,2,4-Triazole facilitated native chemical ligation (NCL) between peptide-MeNbz (MeNbz: N-acyl-N'-methyl-benzimidazolinone) and a cysteinyl peptide in the absence of thiol additives. The method enabled one-pot desulfurization and iodine oxidation after NCL. Additionally, the direct isolation of the target peptide from the NCL reaction mixture with an activated thiopropyl-Sepharose resin was achieved.

N-Sulfanylethylaminooxybutyramide (SEAoxy): A Crypto-Thioester Compatible with Fmoc Solid-Phase Peptide Synthesis.

An N-sulfanylethylaminooxybutyramide (SEAoxy) has been developed as a novel thioester equivalent for native chemical ligation. SEAoxy peptide was straightforwardly synthesized by conventional Fmoc solid-phase peptide synthesis without a problem. Moreover, SEAoxy peptide could be directly applied to native chemical ligation owing to the intramolecular N-to-S acyl shift that releases the peptide-thioester in situ. This methodology was successfully applied to the synthesis of two bioactive peptides.

Imidazole-Aided Native Chemical Ligation: Imidazole as a One-Pot Desulfurization-Amenable Non-Thiol-Type Alternative to 4-Mercaptophenylacetic Acid.

Various bioactive proteins have been synthesized by native chemical ligation (NCL) and its combination with subsequent desulfurization (e.g., conversion from Cys to Ala). In NCL, excess 4-mercaptophenylacetic acid (MPAA) is generally added to facilitate the reaction. However, co-elution of MPAA with the ligation product during preparative high-performance liquid chromatography sometimes reduces its usefulness. In addition, contamination of MPAA disturbs subsequent desulfurization. Here, we report for the first time that imidazole can be adopted as an alternative to MPAA in NCL using a peptide-alkylthioester. The efficiency of the imidazole-aided NCL (Im-NCL) is similar to that of traditional MPAA-aided NCL. As model cases, we successfully synthesized adiponectin(19-107) and [Ser(PO3 H2 )65 ]-ubiquitin using Im-NCL with a one-pot desulfurization.

Combination of Thiol-Additive-Free Native Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine.

We report a novel strategy for native chemical ligation (NCL). Alanines not located at a ligation site are temporarily replaced with cysteines, and this enables efficient thiol-additive-free NCL, with subsequent desulfurization to regenerate the target peptide. We synthesized stresscopin-related peptide and neuroendocrine regulatory peptide-2 (NERP-2) by this method. We confirmed that both conventional alkyl thioester and thioester-equivalent N-acyl-N'-methyl-benzimidazolinone (MeNbz) can be adopted as thioester components for thiol-additive-free NCL of multi-Cys-containing peptides.

Development of a sufficiently reactive thioalkylester involving the side-chain thiol of cysteine applicable for kinetically controlled ligation.

N(α) -Trifluoroacetyl-Cys-Leu-NH2 (TfaC-Leu-NH2 ) was incorporated into thioesters through its side-chain thiol group to develop a more reactive peptide-thioester than the commonly used peptide-3-mercaptopropionic acid (MPA)-thioester. The TfaC-thioester could be readily synthesized by solid-phase peptide synthesis (SPPS) with Boc chemistry using in situ neutralization protocols in sufficient yield without any side reaction associated with the use of TfaC. This thioester proved to display a much higher reactivity in the thiol-free native chemical ligation (NCL) reaction than the MPA-thioester and to be comparable to the thioarylester, such as the 4-mercaptophenylacetic acid (MPAA)-thioester, in terms of the ligation rate. We were able to demonstrate the usefulness of the TfaC-thioester by using it to synthesize neuromedin S via a one-pot sequential NCL approach followed by desulfurization. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 503-511, 2016.

Successful treatment of primary malignant lymphoma of the penis by organ-preserving rituximab-containing chemotherapy.

Primary penile lymphoma is extremely rare. Here we report the case of a 67-year-old man with the chief complaints of difficulty in urination and priapism, who was eventually diagnosed with primary malignant lymphoma of the penis. Pathological examination of excision biopsy of the left inguinal lymph node revealed malignant CD20+ diffuse large B-cell lymphoma. We treated this patient with a systemic rituximab-chemotherapy regimen and obtained good results, in terms of both functional and cosmetic outcomes. Soluble interleukin-2 receptor was a useful tumor marker for evaluating the therapeutic effect. The patient has been in remission for 10 months after the discontinuation of chemotherapy.

Identification of the endogenous cysteine-rich peptide trissin, a ligand for an orphan G protein-coupled receptor in Drosophila.

There are many orphan G protein-coupled receptors (GPCRs), for which ligands have not yet been identified, in both vertebrates and invertebrates, such as Drosophila melanogaster. Identification of their cognate ligands is critical for understanding the function and regulation of such GPCRs. Indeed, the discovery of bioactive peptides that bind GPCRs has enhanced our understanding of mechanisms underlying many physiological processes. Here, we identified an endogenous ligand of the Drosophila orphan GPCR, CG34381. The purified ligand is a peptide comprised of 28 amino acids with three intrachain disulfide bonds. The preprotein is coded for by gene CG14871. We designated the cysteine-rich peptide "trissin" (it means for triple S-S bonds) and characterized the structure of intrachain disulfide bonds formation in a synthetic trissin peptide. Because the expression of trissin and its receptor is reported to predominantly localize to the brain and thoracicoabdominal ganglion, trissin is expected to behave as a neuropeptide. The discovery of trissin provides an important lead to aid our understanding of cysteine-rich peptides and their functional interaction with GPCRs.

Conversion chemotherapy using cetuximab plus FOLFIRI followed by bevacizumab plus mFOLFOX6 in patients with unresectable liver metastases from colorectal cancer.

Recently, in patients with unresectable colorectal liver metastasis, liver resection sometimes becomes possible by intensive systemic chemotherapy, i.e. conversion therapy. However, among cases that do not respond well to first-line chemotherapy, it is rare that second-line chemotherapy results in a marked response allowing liver resection. We consider that the liver resection rate may be increased by initiating second-line treatment at an earlier stage before progression subsequent to first-line chemotherapy. We are conducting a multicentre Phase II study to evaluate the efficacy and safety of sequential chemotherapy using six cycles of cetuximab plus FOLFIRI (5-fluorouracil, folinic acid and irinotecan) followed by six cycles of bevacizumab plus FOLFOX (5-fluorouracil, folinic acid and oxaliplatin) as conversion chemotherapy. The primary endpoint is the liver resection rate during the bevacizumab + FOLFOX phase. Fifty patients are required for this study.

Hepatolithiasis in the hepatic hilum mimicking hilar cholangiocarcinoma: report of a case.

We report a rare case of hepatolithiasis, which was diagnosed as hilar cholangiocarcinoma and treated with hepatectomy and extrahepatic bile duct resection. A 59-year-old woman presented to a local hospital with liver dysfunction. Diagnostic imaging revealed a biliary stricture at the hepatic hilum and middle bile duct. Hilar cholangiocarcinoma was diagnosed, and she was referred to our hospital for definitive surgical treatment. She underwent left hepatic trisectionectomy, total caudate lobectomy, and extrahepatic bile duct resection. Gross examination of the resected specimen revealed intrahepatic stones firmly adherent to the bile duct wall. Pathological examination revealed no malignant lesions. The epithelium of the bile duct was absent underneath the stone, and the boundary between the stone and bile duct wall was ill defined. To our knowledge, this is the first case report of hepatolithiasis with a biliary stricture caused by peculiar stone formation, mimicking hilar cholangiocarcinoma.

Optimal oxidative folding of the novel antimicrobial cyclotide from Hedyotis biflora requires high alcohol concentrations.

Hedyotide B1, a novel cyclotide isolated from the medicinal plant Hedyotis biflora, contains a cystine knot commonly found in toxins and plant defense peptides. The optimal oxidative folding of a cystine knot encased in the circular peptide backbone of a cyclotide poses a challenge. Here we report a systematic study of optimization of the oxidative folding of hedyotide B1, a 30-amino acid cyclic peptide with a net charge of +3. The linear precursor of hedyotide B1, synthesized as a thioester by solid phase synthesis, was cyclized quantitatively by a thia-zip cyclization to form the circular backbone and then subjected to oxidative folding in a thiol-disulfide redox system under 38 different conditions. Of the oxidative conditions examined, the nature of the organic cosolvent appeared to be critical, with the use of 70% 2-propanol affording the highest yield (48%). The disulfide connectivity of the folded hedyotide was identical to that of the native form as determined by partial acid hydrolysis. The use of such a high alcohol concentration suggests that a partial denaturation may be necessary for the oxidative folding of a cyclotide with the inverse orientation of hydrophobic side chains that are externalized to the solvent face to permit the formation of the interior cystine core in the circularized backbone. We also show that synthetic hedyotide B1 is an antimicrobial, exhibiting minimal inhibitory concentrations in the micromolar range against both Gram-positive and -negative bacteria.

Gallbladder cancer involving the extrahepatic bile duct is worthy of resection.

OBJECTIVE: To clarify the value of resection of gallbladder cancer involving the extrahepatic bile duct. BACKGROUND: : Several recent studies have proven that jaundice and extrahepatic biliary involvement are independent predictors of a poor outcome. Only a few authors recommend resection of such advanced disease. METHODS: One hundred patients with pT3/4, pN0/1, M0 disease were the subjects of this study. Mortality and long-term outcome were analyzed using a prospectively collected database. RESULTS: The only factor associated with mortality in univariate and multivariate analyses was intraoperative blood loss. The 5-year survival rate and median survival time were 23% and 1.5 years for patients with pathologic extrahepatic biliary invasion (pEBI), and 54% and 15.4 years for patients without pEBI. Twelve patients with pEBI survived beyond 5 years. Multivariate analysis revealed that R1/2 resection and combined resection of adjacent organs other than the liver and extrahepatic bile duct (CRAO) were independent predictors of poor outcome. Five-year survival rate and median survival time after R0 resection without CRAO were 36% and 3.8 years even in patients with pEBI. In contrast, after R0 resection with CRAO 5-year survival and median survival time were 16% and 0.8 years, respectively. CONCLUSIONS: Patients with advanced gallbladder cancer with pEBI are candidates for resection when distant metastases are absent and R0 resection is achievable. When CRAO is unnecessary, surgical resection should be aggressively planned.

Using the greater omental flap to cover the cut surface of the liver for prevention of delayed gastric emptying after left-sided hepatobiliary resection: a prospective randomized controlled trial.

OBJECTIVE: The aim of this randomized controlled trial is to evaluate the effect on delayed gastric emptying (DGE) of using the greater omental flap to cover the cut surface of the liver after left-sided hepatobiliary resection. METHODS: From June 2007 to December 2008, all eligible patients were randomly assigned to either the greater omental flap group (OF group) or the control group (non-OF group). RESULTS: A total of 40 patients remained for final analysis. The incidence of DGE after left-sided hepatobiliary resection was 25%. The incidence of DGE showed no statistically significant differences between the OF group (10%) and the non-OF group (40%) (p = 0.065). The assessment of DGE using radiopaque rings revealed that changes over time in the gastric emptying ratio (GER, percentage of rings excreted from stomach) did not differ in a significant manner between the two groups. There were significant differences in changes over time in GER (p = 0.044) between the patients with and without DGE. The patients with DGE also showed higher GER at 5 h (p = 0.042) and at 6 h (p = 0.034) than those without DGE. CONCLUSIONS: Using the greater omental flap to cover the cut surface of the liver may reduce the incidence of DGE after left-sided hepatobiliary resection. Assessment using radiopaque markers may be useful to evaluate DGE.

Intraoperative bacterial translocation detected by bacterium-specific ribosomal rna-targeted reverse-transcriptase polymerase chain reaction for the mesenteric lymph node strongly predicts postoperative infectious complications after major hepatectomy for biliary malignancies.

BACKGROUND: There is little evidence indicating a causal linkage between bacterial translocation and postoperative infectious complication (POIC) in human studies. OBJECTIVE: To investigate the correlation between the occurrence of bacterial translocation in the mesenteric lymph node (MLN) and POIC with a sensitive quantitative method using bacterium-specific ribosomal RNA (rRNA)-targeted reverse transcriptase-polymerase chain reaction (RT-qPCR). METHODS: Patients who underwent major hepatectomy for biliary malignancies involving hepatic hilus were included in this study (n = 65). Mesenteric lymph nodes were harvested from the jejunal mesentery 2 times during the operation (MLN-1 harvested at laparotomy and MLN-2 harvested after tumor resection). Microorganisms were detected by a bacterium-specific rRNA-targeted RT-qPCR method. Perioperative factors and POIC were recorded prospectively. RESULTS: Of 65 patients, 51 completed the study. Microorganisms were detected in MLN-1 and MLN-2 in 15 (29.4%) and 19 (37.3%) patients, respectively. The detection of microorganisms in MLN-1 was significantly correlated with the incidence of preoperative cholangitis (P = 0.04), whereas the detection of microorganisms in MLN-2 was significantly correlated with the incidence of POIC (P = 0.002). In multivariate analysis, a positive result for detection of microorganisms in MLN-2 was one of the independent predictive factors of POIC (odds ratio = 26.1). CONCLUSIONS: Intraoperative analysis of MLNs (especially MLN-2) by rRNA-targeted RT-qPCR can strongly predict the occurrence of POIC after hepatectomy for biliary malignancy. This method is more sensitive and faster at detection of microorganisms than the conventional culture method. Therefore, we can obtain the information of bacterial translocation immediately after the surgery and can select the group of patients with high risk for POIC.