Guidelines for the management of biliary tract and ampullary carcinomas: surgical treatment

The only curative treatment in biliary tract cancer is surgical treatment. Therefore, the suitability of curative resection should be investigated in the first place. In the presence of metastasis to the liver, lung, peritoneum, or distant lymph nodes, curative resection is not suitable. No definite consensus has been reached on local extension factors and curability. Measures of hepatic functional reserve in the jaundiced liver include future liver remnant volume and the indocyanine green (ICG) clearance test. Preoperative portal vein embolization may be considered in patients in whom right hepatectomy or more, or hepatectomy with a resection rate exceeding 50%–60% is planned. Postoperative complications and surgery-related mortality may be reduced with the use of portal vein embolization. Although hepatectomy and/or pancreaticoduodenectomy are preferable for the curative resection of bile duct cancer, extrahepatic bile duct resection alone is also considered in patients for whom it is judged that curative resection would be achieved after a strict diagnosis of its local extension. Also, combined caudate lobe resection is recommended for hilar cholangiocarcinoma. Because the prognosis of patients treated with combined portal vein resection is significantly better than that of unresected patients, combined portal vein resection may be carried out. Prognostic factors after resection for bile duct cancer include positive surgical margins, especially in the ductal stump; lymph node metastasis; perineural invasion; and combined vascular resection due to portal vein and/or hepatic artery invasion. For patients with suspected gallbladder cancer, laparoscopic cholecystectomy is not recommended, and open cholecystectomy should be performed as a rule. When gallbladder cancer invading the subserosal layer or deeper has been detected after simple cholecystectomy, additional resection should be considered. Prognostic factors after resection for gallbladder cancer include the depth of mural invasion; lymph node metastasis; extramural extension, especially into the hepatoduodenal ligament; perineural invasion; and the degree of curability. Pancreaticoduodenectomy is indicated for ampullary carcinoma, and limited operation is also indicated for carcinoma in adenoma. The prognostic factors after resection for ampullary carcinoma include lymph node metastasis, pancreatic invasion, and perineural invasion

Cysteine Residues in the Major Capsid Protein, Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

<div><p>The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted <i>in vitro</i> protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, <i>in vitro</i> translated C247A Vp1 formed pentamers, but <i>in vitro</i> translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.</p></div

Structural difference around C80 between JCV Vp1 and SV40 Vp1.

<p>Vp1 structures of JCV and SV40 are shown in green and red, respectively. The different residues within 4 Å of C80 of the Vp1s are shown as stick models. An arrow indicates C80 in JCV Vp1 and C87 in SV40 Vp1.</p

Stabilities of WT Vp1 and C80A and C247A mutant Vp1s expressed in HeLa cells.

<p>(A) HeLa cells transfected with an empty plasmid (Mock) or plasmid encoding WT, C80A, or C247A Vp1 were pulse-labeled for 5 min and then either harvested immediately (lanes 1 to 3) or chased for 12 h (lanes 4 to 6) or 24 h (lanes 7 to 10). The anti-Vp1 immunocomplexes (IP) prepared from the lysates were analyzed by SDS-PAGE and autoradiography. (B) The intensities of the bands in <i>A</i> were quantified with an image analyzer and plotted as relative values compared to those obtained at 0 h of chase. Data are the means ± S.D. of values from three independent experiments. The solid line, dotted line, and broken line represent WT, C80A, and C247A samples, respectively.</p

Oligomerization analysis of <i>in vitro</i> synthesized WT and mutant Vp1s.

<p>(A) Electron micrographs of VLPs (VLP; scale bar, 10 nm) and pentamers (EGTA + DTT; scale bar, 10 nm) formed by WT JCV Vp1. (B) The mixtures resulting from the <i>in vitro</i> transcription-coupled-translation of the empty pURE2 plasmid (Mock) and of the Vp1-encoding pURE2-Vp1 (WT) were supplemented with DTT, boiled, separated by SDS-PAGE, and immunoblotted with a rabbit anti-Vp1 antibody. An arrowhead marks the position of monomeric Vp1. An arrow indicates nonspecific bands. (C) Sedimentation profiles of <i>in vitro</i> translated single cysteine substitution mutants. Monomeric Vp1s, pentameric Vp1s, and the <i>in vitro</i> translation products for WT Vp1 or cysteine point mutant Vp1s (C42A, C80A, C97A, C200A, C247A, C260A, or C80T) were separated by 5–20% sucrose gradient sedimentation under denaturing conditions, and the resulting fractions were examined for the presence of Vp1 by SDS-PAGE and immunoblotting.</p

Structural environments of JCV Vp1 C80 and C247 in relation to Vp1 stability.

<p>(A) Alignment of amino acid sequences of JCV, SV40, BKV, and MPyV Vp1s in the vicinity of C80. Amino acids homologous to C80 are in boldface. (B) C80T mutant Vp1, but not C80A or C80S Vp1, is stable. Lysates of HeLa cells were analyzed by SDS-PAGE and immunoblotting for Vp1. These cells were prepared 48 h after transfection with the expression plasmid encoding eithe WT Vp1 or each C80 mutant Vp1 or after transfection with empty expression plasmid (Mock). (C) Comparison of the structures of JCV and MPyV Vp1s surrounding C80. The JCV structure is superimposed on the MPyV structure. The sulfur atom of JCV's C80 and the methyl group of MPyV's T97 are circled in red. (D) Alignment of amino acid sequences of JCV, SV40, BKV, and MPyV Vp1s in the vicinity of C247. Amino acids homologous to C247 are in boldface. (E) All C247 substitution mutant Vp1s expressed in HeLa cells are unstable. Lysates of HeLa cells transfected with the expression plasmid for WT Vp1 or for each C247 mutant Vp1 or after transfection with empty expression plasmid (Mock) were analyzed for Vp1 by SDS-PAGE and immunoblotting for Vp1.</p

Cysteine residues in JCV Vp1.

<p>Three-dimensional model of the JCV Vp1 pentamer was constructed using data from an X-ray crystal structure of JCV Vp1 (PDB code: 3NXG) using the Discovery Studio 3.0 software package (Accelrys, San Diego, CA). Structure of the JCV Vp1 pentamer viewed on its top (A) and side (B) are shown as transparent surface and ribbon representation. Red spheres indicate the positions of cysteine residues on Vp1.</p

HA titers and Vp1 expression levels of JCV Vp1 cysteine point mutants in mammalian cells.

<p>(A) Schematic diagram of cysteine point mutant Vp1s. Each of the six cysteine residues in the 354-amino-acid JCV Vp1 is marked with a “C” for the WT Vp1 (WT), and the cysteine residue mutated into alanine is designated as “A” for each point mutant Vp1. (B) HA titers of extracts from Vp1-expressing cells. HA titers were determined from the last dilution showing hemagglutination for two-fold serially diluted extracts prepared from HeLa cells transfected with the expression plasmids for WT or individual point mutant Vp1s. The corresponding dilutions of extract from JCV-infected cells served as a positive control, whereas those of PBS or of HeLa extract from transfection with the empty plasmid (Mock) were used as negative controls. (C) Levels of Vp1 expression. Quantitation of the mutant Vp1s and actin in HeLa cells transfected with the Vp1 expression plasmids or with an empty vector (Mock) were analyzed by SDS-PAGE and immunoblotting for Vp1 (Vp1) and for actin (act).</p

Cysteine point-mutant Vp1s synthesized <i>in vitro</i> are stable.

<p>The empty pURE2 plasmid (Mock) or pURE2-Vp1 plasmids encoding either WT Vp1 or cysteine point mutant Vp1s were subjected to cell-free transcription-coupled translation, and the translation products were examined for Vp1 by SDS-PAGE and immunoblotting.</p