These results suggest that the intraductal growth type of tumour should be treated as a distinct entity compared with other types of ICC. Multiple tumours and high serum CA19-9 level were signs of dismal prognosis, whereas not all patients with lymph node involvement had a poor prognosis.

Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. When observed by immunofluorescence in HeLa S3 and HepG2 cells, endogenous BIG1 and coexpressed BIG2 were distributed in a punctate pattern throughout the cytosol, and also concentrated in the perinuclear region, where endogenous BIG1 and BIG2 each partially colocalized with Golgispecific 58K protein and ␥-adaptin. On Western blot analysis, both BIG1 and BIG2 were clearly more abundant in the cytosol than in the microsomal fractions. After density gradient centrifugation of a microsomal fraction, BIG1 and BIG2 were recovered in the same fraction as ␤-COP, a marker for Golgi membranes. When cytosol from HeLa S3 cells was subjected to gel filtration and fractions were analyzed by Western blotting, the largest percentages of both BIG1 and BIG2 were detected in fractions containing proteins with a molecular mass of >670 kDa. Western blotting using anti-peptide antibodies specific for BIG1 or BIG2 demonstrated that Ϸ70% of BIG2 was immunoprecipitated along with 100% of BIG1 by the anti-BIG1 IgG, and Ϸ75% of BIG1 was coprecipitated with 100% of BIG2 by the anti-BIG2 IgG. All observations were consistent with the conclusion that significant fractions of BIG1 and BIG2 exist as components of the same macromolecular complexes in bovine brain cytosol and are similarly localized in cultured cells.

Forty-four patients with advanced gallbladder carcinoma (18 with stage pT3 and 26 with stage pT4 of the Union Internacional Contra la Cancrum classification) were aggressively managed by extended heptatic resection in 33 patients, bile duct resection in 28, pancreaticoduodenectomy in seven, gastrointestinal resection in eleven and portal vein resection and reconstruction in seven. Adjacent organ involvement was classified as follows: type I, hepatic involvement with or without gastrointestinal invasion (Ia, Ib); type II, bile duct involvement with or without gastrointestinal invasion (IIa, IIb); type III, hepatic and bile duct involvement with or without gastrointestinal invasion (IIIa, IIIb); type IV, gastrointestinal involvement without hepatic or bile duct invasion. Fourteen of 15 patients with type I tumours had a curative resection compared with seven of 26 with type III lesions (P < 0.0001). The surgical mortality rate was two of 15 patients with type I tumours, seven of 26 with type III tumours and nine of 44 for the whole group. The long-term survival rate after curative resection was four and two of 23 at 3 and 5 years respectively, significantly better than two and none of 21 at 1 and 2 years after non-curative resection (P < 0.01). The survival rate after curative resection for patients with type I tumours was four and two of 14 at 3 and 5 years respectively, significantly better than for other types (P < 0.05). This classification of advanced gallbladder carcinoma according to involvement of adjacent organs might be helpful in planning surgery for this condition; in particular, type I tumours should be treated by a radical surgical procedure to achieve a favourable outcome.

Activation of ADP-ribosylation factors (ARFs), ϳ20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: ϳ200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and ϳ50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ϳ209-kDa protein 99.5% identical in deduced amino acid sequence (1,849 residues) to a BFAinhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is ϳ74% identical (1,785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a ϳ202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His 6 fusion protein in Sf9 cells, accelerated guanosine 5-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for ϳ50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ϳ200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFAsensitive ARF function in Golgi transport.