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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hepatectomy with simultaneous resection of the portal vein and hepatic artery for advanced perihilar cholangiocarcinoma: an audit of 50 consecutive cases.

OBJECTIVE: To outline our experience with hepatectomy with simultaneous resection of the portal vein and hepatic artery for advanced perihilar cholangiocarcinoma, and to discuss the clinical significance of this challenging hepatectomy. SUMMARY BACKGROUND DATA: Only a few authors reported negative results for this surgery in a very limited number of patients. METHODS: We retrospectively reviewed medical records of 50 patients with advanced cholangiocarcinoma who underwent hepatectomy (left trisectionectomy in 26, left hepatectomy in 23, and right hepatectomy in 1) with simultaneous resection and reconstruction of the portal vein and hepatic artery, focusing on surgical outcome and survival. RESULTS: The operative time was 776 +/- 191 minutes, and blood loss was 2593 +/- 1890 mL. Time of vessel resection and reconstruction was 25 +/- 19 minutes for the portal vein and 119 +/- 56 minutes for the hepatic artery. A total of 27 (54.0%) patients developed several kinds of complications, including intra-abdominal abscess (n = 13), wound infection (n = 9), bile leakage from liver stump (n = 9), and liver failure (n = 7). Relaparotomy was necessary in 5 (10.0%) patients. One (2.0%) patient died of a postoperative complication. Microscopic cancer invasion of the resected portal vein was found in 44 (88.0%) patients, while that of the resected hepatic artery was found in 27 (54.0%). The distal bile duct margin, proximal bile duct margin, and radial margin were positive for cancer in 2 (4.0%), 4 (8.0%), and 17 (34.0%) patients, respectively. Consequently, R0 resection was achieved in 33 (66.0%) patients. The 1-, 3-, and 5-year survival rates were 78.9%, 36.3%, and 30.3%, respectively. Survival for 30 patients with pM0 disease who underwent R0 resection was better, being 40.7% at the 3- and 5-year time points. CONCLUSION: Major hepatectomy with simultaneous resection and reconstruction of the portal vein and hepatic artery is technically demanding. However, this surgery can be performed with acceptable mortality by an experienced surgeon and offers a better chance of long-term survival in selected patients.

Retroperitoneal solitary fibrous tumor-induced hypoglycemia associated with high molecular weight insulin-like growth factor II.

A man, aged 65 years, presented with frequent episodes of hypoglycemia and unconsciousness. Hypoglycemia was accompanied by undetectable serum insulin and C-peptide levels and a high serum insulin-like growth factor (IGF)-II level. He was found to have a retroperitoneal solitary fibrous tumor. He underwent successful resection of the tumor and had no hypoglycemic episodes after the operation. Immunohistochemical analysis revealed positive immunostaining for IGF-II in tumor cells. The presence of the high-molecular-weight form of IGF-II in the patient's serum was confirmed by immunoblotting, which suggests that his hypoglycemia was due to an increase in the plasma level of IGF-II secreted by the tumor.

[Pathologically complete response of multiple liver metastases from rectal cancer treated with mFOLFOX6 plus bevacizumab].

We report a case of pathologically complete response of multiple liver metastases from rectal cancer after neoadjuvant chemotherapy. The patient was a 74-year-old woman who had advanced rectal cancer with synchronous liver metastases (T4N1M1). Following resection of the primary tumor, she received biweekly mFOLFOX6 plus bevacizumab neoadjuvant chemotherapy. After 5 courses, the liver tumors were markedly reduced in size. Three weeks after the final treatment, she underwent partial hepatectomies. Histologically, no cancer cells were detected in any resected specimens. The postoperative course was uneventful, and she has been well without recurrence for one year at the time of writing. Regimens containing bevacizumab may result in good tumor response. Surgical resection is crucial for proof of pathologically complete response.

Clinicopathologic study of cholangiocarcinoma with superficial spread.

OBJECTIVE: To review our experience with cholangiocarcinoma with superficial spread, to clarify its clinical features, and to discuss treatment strategies. SUMMARY BACKGROUND DATA: Most of the previous reports on cholangiocarcinoma with superficial spread were case reports. Little is known about this type of cholangiocarcinoma. METHODS: The medical records of 471 patients with cholangiocarcinoma who underwent resection (351 perihilar and 120 distal cancers) were retrospectively reviewed, focusing on superficial spread, which was defined as noninvasive cancer extension of more than 20 mm. RESULTS: Superficial spread was found in 69 (14.6%) of 471 patients, and its length was 54 +/- 19 mm. Histologically, papillary and well differentiated adenocarcinomas were observed more frequently in cholangiocarcinomas with superficial spread (C(+SS)), compared with those without superficial spread (C(-SS)). Histologic indexes showing tumor aggressiveness, including lymphatic, venous, and perineural invasions, were lower in C(+SS), and all factors of tumor staging (pT, pN, and pM) were less advanced in C(+SS) than in C(-SS). Regarding surgical procedure, a combined hepatectomy and pancreatoduodenectomy was indicated in 26 (37.7%) of the 69 patients with C(+SS), but in only 25 (6.2%) of the 402 patients with C(-SS). Positivity of the proximal ductal margin was higher in C(+SS) than in C(-SS) (18.8% vs. 11.9%), although this was not statistically significant. All positive proximal ductal margins in C(+SS) were because of carcinoma in situ, whereas invasive cancer was the main reason for positivity in C(-SS). Survival (excluding 29 in-hospital deaths) was significantly better in the patients with C(+SS) than in those with C(-SS) (5- and 10-year survival rates; 48.8% and 19.6% vs. 26.8% and 16.6%, P = 0.0009). Survival was comparable between the patients with a negative ductal margin and those with a positive margin with carcinoma in situ. On multivariate analysis, the presence or absence of superficial spread was not identified as a prognostic factor. CONCLUSIONS: C(+SS) is associated with less advanced, slower growing tumors and better survival compared with C(-SS). In many cases of C(+SS), the survival does not depend on the complete resection of all the superficial spread but on the stage of the main lesion.

Value of multidetector row CT in the assessment of longitudinal extension of cholangiocarcinoma: correlation between MDCT and microscopic findings.

BACKGROUND: A few authors have reported the value of multidetector row CT (MDCT) for evaluating the longitudinal extent of cholangiocarcinoma. They have not focused on CT attenuation of a tumor and actual tumor extent along the bile ducts. We designed the present study to analyze attenuation. METHODS: Between January 2003 and July 2005, 113 consecutive patients with cholangiocarcinoma underwent a surgical resection following MDCT. Of these MDCT studies, 73 (perihilar cholangiocarcinoma, n = 62; middle and distal cholangiocarcinoma, n = 11) were suitable for analysis, and the patients were enrolled in the study. Patients were divided according to tumor hypoattenuation and hyperattenuation on MDCT. Histologic differentiation, desmoplastic reaction, and vascular density were microscopically compared with the tumor attenuation to differentiate the characteristics of the attenuation. The extent of cancer along the bile duct diagnosed by MDCT was compared with the actual extent determined by the microscopic findings. RESULTS: Hyperattenuated tumor was observed in 40 patients. There was no difference in histologic differentiation, desmoplastic reaction, or vascular density between the hyperattenuated and hypoattenuated cholangiocarcinomas. Neither the proximal nor the distal borders between the normal and thickened bile duct wall could be determined in the 33 patients with hypoattenuated tumor; in contrast, an accurate assessment of extent of tumor was obtained in 76% of the proximal borders and 82% of the distal borders in the 40 patients with hyperattenuated tumor. CONCLUSIONS: Although the cause of the difference between the hyperattenuated and hypoattenuated cholangiocarcinoma still is unclear, MDCT can be an alternative to direct cholangiography in selected patients with hyperattenuated cholangiocarcinoma.

Small pancreatic carcinoma misdiagnosed as superficially spreading cholangiocarcinoma.

We report a case of small pancreatic carcinoma misdiagnosed as superficially spreading cholangiocarcinoma using percutaneous transhepatic cholangioscopy (PTCS). The patient was a 72-year-old man admitted to a local hospital with obstructive jaundice. The patient underwent percutaneous transhepatic biliary drainage and PTCS. He was referred to our hospital with a diagnosis of superficially spreading cholangiocarcinoma. Cholangiography revealed a stenosis of the common bile duct, and also revealed some irregularities from the common hepatic duct to the left hepatic duct, suggesting a superficial spread of cancer. No pancreatic tumor was identified by endoscopic retrograde pancreatography or by enhanced computed tomography. Cholangioscopy disclosed an elevated tumor with torsional vessels and granular mucosal lesions, which were extended to the left hepatic duct. Repeated cholangioscopic biopsies of the bile duct mucosa revealed adenocarcinoma. The patient was diagnosed with superficially spreading cholangiocarcinoma extending to the left hepatic duct and the right anterior hepatic duct. Left trisectionectomy combined with pancreatoduodenectomy was performed. The cut surface of the resected specimen showed a pancreatic head tumor that was 8 mm in diameter. Histological findings of the resected specimen revealed adenocarcinoma arising from the pancreatic head with invasion in the common bile duct. Additionally, extensive inflammatory granulation tissue was observed along the surface of the bile duct, without any evidence of carcinoma. This case implies to us that the results of PTCS, even after repeated biopsies, should be interpreted with great caution.