Oncocytic biliary cystadenocarcinoma is a form of intraductal oncocytic papillary neoplasm of the liver.

Biliary cystadenocarcinoma with oncocytic differentiation was first reported in 1992. This is a report of a second case. The patient (a 71-year-old man) was admitted to our hospital complaining of abdominal fullness. Multicystic lesions were identified in the left hepatic lobe radiologically. The patient died of peritoneal dissemination of carcinoma 20 months later. At autopsy, the tumor of the left hepatic lobe was found to be composed of adjoining multiple cystic lesions and a solid lesion with infiltration of the hepatic hilus and peritoneal dissemination. Histologically, the multicystic lesions were covered by papillary neoplastic epithelial cells with an eosinophilic granular cytoplasm resembling that of oncocytes and a fine fibrovascular core. The cyst wall was fibrous, but there was no mesenchymal stroma. In the solid lesion and infiltrated areas, acidophilic and granular carcinoma cells formed small glandular or solid cord patterns with much mucin secretion (mucinous carcinoma). Immunohistochemically, carcinoma cells of both components were found to contain many mitochondria and showed the phenotypes of hepatocytes and cholangiocytes. Interestingly, the intrahepatic biliary tree also was invaded by carcinoma cells. This may be a case of intraductal oncocytic papillary neoplasm of the left hepatic lobe followed by secondary cystic dilatation of the affected bile duct.

Spontaneous occurrence of chronic non-suppurative destructive cholangitis and antimitochondrial autoantibodies in MRL/lpr mice: possible animal model for primary biliary cirrhosis.

MRL/MP mice bearing the lymphoproliferative gene lpr (known as MRL/MP-lpr/lpr or MRL/Ipr mice) are known to spontaneously develop severe autoimmune diseases such as glomerulonephritis, arteritis and arthritis at an early stage of their life. They have also been reported to develop severe sialadenitis, suggesting that this mouse could be a model for Sjögren's syndrome. Primary biliary cirrhosis, an autoimmune disease characterized by chronic non-suppurative destructive cholangitis and the occurrence of antimitochondrial antibodies, is frequently associated with Sjögren's syndrome. In this study, we examined whether cholangitis and/or antimitochondrial antibodies occur in this mouse model, using more than 100 young and old MRL/Ipr mice. We frequently found portal inflammation associated with cholangitis of small intrahepatic bile ducts, especially in older mice. There was also infiltration of inflammatory cells (monocytes) as well as CD4-positive T cells. Epithelioid granuloma and bile-duct loss were also occasionally found. These histological features resemble primary biliary cirrhosis. In addition, antimitochondrial antibodies were shown by immunocytochemistry to be present in the sera of MRL/Ipr mice. There is currently no established animal model for primary biliary cirrhosis. Therefore, further studies on MRL/Ipr mice, with respect to pathogenesis of primary biliary cirrhosis, are warranted.

Pathology and pathogenesis of idiopathic portal hypertension with an emphasis on the liver.

Idiopathic portal hypertension (IPH) is characterized by a long-standing presinusoidal portal hypertension of unknown etiology in adults. Some unidentified agent(s) affect(s) the intrahepatic small portal veins or portal tracts. Immunological disturbance, thromboembolism, infectious etiology and/or increased fibrogenesis in portal tracts are suspected as being candidates for the primary agent(s). During the long clinical course of IPH, several pathological changes may occur, including subcapsular parenchymal atrophy, atrophy of the liver, portal and parenchymal fibrosis, and portal venous phlebosclerosis and thrombosis. The last-named of these lesions is mostly found in patients with a history of splenectomy. Subcapsular parenchymal and hepatic atrophy may result from a hepatocellular dropout via apoptosis or necrosis because of intrahepatic hemodynamic disturbances, particularly chronic portal venous blood insufficiency. Pericellular fibrosis and thin fibrous septa are also frequently found and associated with activated perisinusoidal cells positive for smooth muscle actin. At the same time, vague nodular hyperplasia of hepatocytes not surrounded by fibrous septa is not infrequently seen. It may resemble nodular regenerative hyperplasia, partial nodular transformation, or focal nodular hyperplasia. However, liver cirrhosis does not occur even at the terminal stage. Taking these findings into consideration, a new staging of IPH with a combination of hepatic parenchymal atrophy and portal venous thrombosis was proposed: non-atrophic liver without subcapsular parenchymal atrophy (stage I), non-atrophic liver with subcapsular parenchymal atrophy (stage II), atrophic liver with subcapsular parenchymal atrophy (stage III), and portal venous occlusive thrombosis (stage IV). IPH livers are likely to progress from stage I to stage III. Stage IV, which occurs relatively late, has a poor prognosis. This staging is applicable to clinical and autopsy cases without any histological data.

Frequent molecular identification of Campylobacter but not Helicobacter genus in bile and biliary epithelium in hepatolithiasis.

Bacterial infection of the biliary tree and bile stasis may be causally related to hepatolithiasis, but which bacterial species are involved and their roles in the pathogenesis of hepatolithiasis have not been ascertained. Recently, the Helicobacter genus was detected in human bile and biliary mucosal samples by molecular techniques, and its association with several biliary diseases has been suggested. The Campylobacter genus, which is closely related to the Helicobacter genus, has also recently been identified as causative of human gastrointestinal diseases. This study attempted to elucidate whether Helicobacter and/or Campylobacter bacteria are present in bile samples and biliary mucosal specimens from hepatolithiasis patients and whether they are involved in the pathogenesis of hepatolithiasis. The 16S rRNA gene of the Helicobacter and of the Campylobacter genus was examined by polymerase chain reaction in DNA samples extracted from bile and/or microdissected biliary epithelium from 69 patients with hepatolithiasis and control patients with choledocholithiasis, cholecystolithiasis, and normal gall bladders. The Helicobacter genus was detected in 1 of 8 (13%) biliary epithelial samples in hepatolithiasis and 1 of 10 (10%) bile samples in choledocholithiasis. The Campylobacter genus was detected in 3 of 14 (21%) bile samples and 5 of 8 (63%) epithelial samples in hepatolithiasis, and in 2 of 15 (13%) bile samples and 1 of 8 (13%) epithelial samples in cholecystolithiasis. The detection rate for Campylobacter in biliary epithelium of hepatolithiasis was significantly higher than in the bile or biliary epithelium of control groups (p<0.05). By a phylogenetic analysis based on nucleotide sequences, the Campylobacter genuses detected in hepatolithiasis were clustered with C. rectus or C. showae. The frequent detection of the Campylobacter 16S rRNA gene in bile, and especially in biliary epithelium of hepatolithiasis, suggests a pathogenetic relationship with Campylobacter infection.

Hepatocellular carcinoma arising in non-alcoholic steatohepatitis.

The incidence and significance of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH) has not been previously evaluated in detail. We recently experienced a case of NASH with multicentric HCC in a female patient. At the age of 58 years, the patient was diagnosed with non-insulin-dependent diabetes mellitus, treated by insulin therapy. The patient did not drink alcohol. She was negative for all serological markers of hepatitis B and C virus infection. Because of liver dysfunction, a needle biopsy was performed at the age of 62 years, and pathological findings, such as fatty change, Mallory's body, nuclear glycogen and pericellular fibrosis, suggested a diagnosis of NASH. Subsequently, four nodules were detected in the liver by imaging. Liver biopsies were performed from each nodule. One nodule was pathologically diagnosed as a pseudolymphoma, while three other nodules were moderately differentiated HCC (10 years after the diagnosis of non-alcoholic steatohepatitis), well-differentiated HCC (11 years later) and dysplastic nodule (11 years later), suggesting multicentric occurrence of HCC. This case suggests that HCC could be a late complication of NASH.

Single amino acid substitutions in flexible loops can induce large compressibility changes in dihydrofolate reductase.

To address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (v(o)) and adiabatic compressibility (beta(s)(o)) were determined for a series of mutants with amino acid replacements at Gly67 (7 mutants), Gly121 (6 mutants), and Ala145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15 degrees C. These mutations induced large changes in v(o) (0.710-0.733 cm(3). g(-1)) and beta(s)(o) (-1.8 x 10(-6)-5.5 x 10(-6) bar(-1)) from the corresponding values for the wild-type enzyme (v(o)=0.723 cm(3). g(-1), beta(s)(o) = 1.7 x 10(-6) bar(-1)), probably due to modifications of internal cavities. The beta(s)(o) value increased with increasing v(o), but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between beta(s)(o) and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large beta(s)(o) value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (k(cat)) and in part due to increased affinity for the substrate (K(m)), despite the fact that the mutation sites are far from the catalytic site. These results demonstrate that the flexibility of the DHFR molecule is dramatically influenced by a single amino acid substitution in one of these loops and that the flexible loops of this protein play important roles in determining the enzyme function.

Protein expression and genetic alterations of p53 and ras in intrahepatic cholangiocarcinoma.

AIMS: The significance of molecular and genetic alterations of p53 and ras in the development and progression as well as the histological differentiation of intrahepatic cholangiocarcinoma (ICC) was evaluated. METHODS AND RESULTS: We examined immunohistochemically ras p21 protein and p53-related products (p53 protein, WAF-1 and mdm-2) in 43 cases of ICC. In addition, point mutations of ras and p53 were examined genetically in selected ICC cases by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequence analysis. Point mutation of K-ras gene codon 12 was detected in three of 14 cases and one of 15 cases by PCR-RFLP and direct sequence analysis, respectively. Immunoreactivity of ras p21 protein was not detected. Point mutation of p53 was detected in three of 15 cases. p53 protein was immunohistochemically detectable in 33 of 43 cases. Immunoreactivity of p53 was more frequent in well-differentiated and less frequent in poorly differentiated cases. Immunoreactivity of WAF-1 and mdm-2 was seen in 16 and eight of 43 cases, respectively. Both proteins were frequently detected in the cases positive for p53 protein. CONCLUSION: These results suggest that dysregulation of ras is involved in at least 20% of ICC and expression of p53 protein is more significantly involved in ICC, particularly in the well and moderately differentiated cases. While some cases of p53 expression may be explainable by point mutation of p53, there may be some epigenetic phenomena that stabilize p53 protein in ICC. That is, wild type p53 may be stabilized and then detectable by forming complexes with other molecules of p53 downstream effector genes, such as WAF-1 and mdm-2.

Generation of monoclonal antibodies to murine bile duct epithelial cells: identification of annexin V as a new marker of small intrahepatic bile ducts.

Biliary epithelial cells (BECs) are distributed along the length of both the extrahepatic and intrahepatic biliary tree, but have distinctly different phenotypes and functions according to their anatomical location. It has been reasoned that the distinct appearance of pathogenic lesions in different biliary diseases may be associated with the expression of distinct proteins. These data prompted us to immunize rats with cultured murine BECs with the objective of determining if there are unique antigens on BECs. Of the 45 monoclonal antibodies (mAbs) produced, 12 mAbs (MBEC 1-12) were selected for detailed study based on their classification into three major groups. These groups included four antibodies (MBEC 1-4) that reacted in a staining pattern typical of mucin. A second group of mAbs, MBECs 5 to 8, reacted strongly along the biliary tract and by immunoblot analysis, reacted with several bands ranging from 44 kd to 64 kd. These antibodies were considered as markers of pan BECs and their staining pattern proved similar to that of a control polyclonal pan-cytokeratin. The final group of mAbs, MBECs 9 to 12, recognized a 36-kd antigen using lysates of murine BECs. These antibodies also predominantly stained small peripheral bile ducts. The reactive antigen was purified by immunoprecipitation and microsequenced; the peptides sequenced showed 100% homology with murine annexin V. The identification of annexin V with predominantly intrahepatic bile ducts, is of significant interest because of the multiple roles of annexin V, including that of membrane cytoskeletal interactions during transport and apoptosis.

Definition and pathology of primary sclerosing cholangitis.

Although primary sclerosing cholangitis (PSC) is not a common disease, it is important in the differential diagnosis of hepatobiliary tract diseases in clinical practice. A diagnosis of PSC should be made only after the exclusion of similar diseases with well known etiologies or pathogeneses. In this review, the pathology of classical PSC and its variants or related diseases is highlighted. PSC is histologically characterized by progressive periductal fibrosis with luminal stenosis or obliteration, along with the formation of a fibrous core, as well as dilatation (cholangiectasis). Its etiology is unknown. Bacterial ascending cholangitis is superimposed on its long clinical course. Such a heterogeneous distribution of biliary lesions with biliary obliteration and cholangiectasis is responsible for the radiological demonstration of biliary abnormalities, particularly the beaded appearance. Sampling variability is common in needle or wedge biopsied specimens. As a result of biliary damage, the liver shows progressive cholestatic change followed by biliary fibrosis and cirrhosis, and this hepatic progression is divisible into four stages. There are several variants of PSC or related diseases, such as localized biliary sclerosis and stenosis, sclerosing cholangitis associated with inflammatory pseudotumor, and PSC-autoimmune hepatitis overlapping syndrome. Cholelithiasis, including secondary hepatolithiasis and, to a lesser degree, biliary carcinoma and dysplasia, are also known to develop at the perihilar bile ducts as a late complication of PSC.

Relationship between interleukin-6 and proliferation and differentiation in cholangiocarcinoma.

AIMS: Interleukin-6 (IL-6) has been implicated as a mediator of growth control in several human neoplasms. The significance of IL-6 expression in human cholangiocarcinoma was examined in this study. METHODS AND RESULTS: IL-6 expression was examined in 43 surgically resected cholangiocarcinomas and a cholangiocarcinoma cell line CCKS1, derived from abdominal metastasis of moderately differentiated adenocarcinoma, by immunohistochemical and in-situ hybridization techniques. In non-neoplastic bile ducts, IL-6 was constitutively but weakly expressed. In surgical cases of cholangiocarcinoma, IL-6 was frequently and strongly expressed in the cytoplasm of well-differentiated cholangiocarcinoma, while its expression was decreased, and less intense or absent in moderately and poorly differentiated areas, respectively. IL-6 mRNA was detected in the cytoplasm of carcinoma cells of two cases of cholangiocarcinomas positive for IL-6. IL-6 was detected in hepatic bile from two cholangiocarcinoma cases studied. The proliferation antigen Ki67 was found to be more frequently expressed in IL-6 negative carcinoma cells than in IL-6 positive carcinoma cells (P < 0.01). In cultured carcinoma cells line CCKS1, IL-6, IL-6 mRNA and IL-6 receptor alpha chain were detected in the cytoplasm of carcinoma cells, suggesting an autocrine effect of IL-6 on carcinoma cells. CONCLUSION: IL-6 expression is inversely related to cell proliferation and positively related to differentiation in cholangiocarcinoma.

aly/aly mice: a unique model of biliary disease.

An autosomal recessive murine mutation, coined "aly/aly" or "alymphoplasia," was recently reported. Homozygotes for aly are defective in both humoral and cell-mediated immune function and have diffuse lymphoid cell infiltration of various tissues, particularly around the conduit ducts of the pancreas and salivary glands. In pilot studies in our laboratories, aly/aly mice were found to have peculiar biliary tract lesions, which were analyzed histologically and immunohistochemically in the present study. The livers of aly/aly mice older than 8 weeks consistently showed a variable lymphoid cell infiltration with lymph follicle formation in portal tracts; intrahepatic biliary epithelial cells showed various types of damage including pseudopyloric gland metaplasia and proliferative changes. In addition, the extrahepatic bile duct and intrahepatic large bile duct were found to contain an acidophilic substance in their epithelial cytoplasm. In the lumen and occasionally in the cytoplasm of these bile ducts, acidophilic crystals were also seen. Ultrastructurally, the intracytoplasmic acidophilic substances consisted of membrane-bound intracytoplasmic inclusions with homogeneous electron density, likely derived from rough endoplasmic reticulum (ER). Immunohistochemically, the cytoplasmic acidophilic substances were simultaneously positive for cystatin C, gastrin, serotonin, and somatostatin. In contrast, the acidophilic crystals did not react with any of these antibodies. These findings suggest that the intracytoplasmic acidophilic substances may contain a precursor of the peptide hormones, possibly because of defective secretion or intracellular transport. We believe that the aly/aly mouse is a useful model for the analysis of biliary metabolic events, and for studies of the interaction of the immune system and biliary destruction.

Isolation, culture and characterization of biliary epithelial cells from different anatomical levels of the intrahepatic and extrahepatic biliary tree from a mouse.

We developed methods to isolate biliary epithelial cells (BECs) from the gallbladder (GB), common bile duct (CBD), intrahepatic large bile duct (ILBD) and small bile duct (ISBD) of a mouse, simultaneously. ILBD and ISBD were cut from the biliary tree after collagenase perfusion of the liver. BECs from all of these biliary segments were cultured as explants on collagen gel. BECs spread from the explants and formed cellular sheets. Areas of these sheets composed entirely of BECs were cut and placed on other gels as subculture, and this continued for 10 passages. Primary and passage cultured BECs on gel were composed of a monolayer of epithelial cells. Passaged cultured BECs in gel formed a spherical cyst lined by a single epithelial layer. Ultrastructurally, microvilli were dense on the luminal surface, and junctional complex and interdigitation was identifiable on the lateral surfaces. These features were similar in both primary and passaged cultured BECs, irrespective of their anatomical origin. Major histocompatibility complex antigens and intercellular adhesion molecule-1 were induced on the basolateral cell membranes of primary and passaged cultured BECs, by interferon-gamma. Although several phenotypic, structural and probable biological features of BECs inherent to each anatomical level may be lost after culture on gel, a combination of this method, several immunological modifications in experimental animals, and addition of immunologically active substances to the culture medium will make the immunopathologic analysis of biliary diseases possible.

Increased expression of interleukin-6 and tumor necrosis factor-alpha in pathologic biliary epithelial cells: in situ and culture study.

We examined the pathologic significance of the expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), both proinflammatory cytokines, on intrahepatic biliary epithelial cells, using immunohistochemical and in situ hybridization techniques as well as culture study. IL-6 and TNF-alpha were expressed in the cytoplasm of biliary epithelial cells of damaged small bile ducts and bile ductules, particularly in primary biliary cirrhosis. Their expression on the bile ducts was mild to moderate in other hepatobiliary diseases and mild or absent in normal livers. Signals of IL-6 mRNA and TNF-alpha mRNA were detected in the cytoplasm of biliary epithelial cells, especially in primary biliary cirrhosis. Immunoelectron microscopic study supported this. TNF receptor and to a lesser degree IL-6 receptor alpha-chain were detected on these damaged bile ducts, suggesting an autocrine effect. By Western blotting and enzyme-linked immunosorbent assay, IL-6 and TNF-alpha were frequently detected in gallbladder bile from primary biliary cirrhosis, and their titers were higher compared with other hepatobiliary diseases. Culture of intrahepatic biliary epithelial cells revealed that they expressed IL-6 and secreted IL-6 in the culture media. These results suggest that the intrahepatic biliary epithelial cells are able to synthesize IL-6 and probably TNF-alpha and are involved in the production of bile duct lesions by means of receptor-mediated processes, particularly biliary epithelial proliferation and destruction and autoimmune augmentation, in primary biliary cirrhosis.

Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium.

Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied. In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained. Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis.

The translin ring specifically recognizes DNA ends at recombination hot spots in the human genome.

We previously showed that consensus sequences exist at the chromosomal breakpoints in lymphoid malignancies and that these sequences are specifically recognized by a novel DNA binding protein, Translin. In the present study, the native form of Translin was established to be a ring-shaped structure by electron microscopy and crystallographic studies. It was also determined that this multimeric Translin formed by the subunits is responsible for its binding to target sequences situated only at single-stranded DNA ends. Furthermore, DNA-damaging reagents were found to initiate a signaling pathway for the active nuclear transport of Translin. The results support the hypothesis that staggered breaks occur at recombination hot spots and Translin has a pivotal function in recognition of the generated single-stranded DNA ends.

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI.

Three mutants of Escherichia coli ribonuclease HI, in which an invariant acidic residue Asp134 was replaced, were crystallized, and their three-dimensional structures were determined by X-ray crystallography. The D134A mutant is completely inactive, whereas the other two mutants, D134H and D134N, retain 59 and 90% activities relative to the wild-type, respectively. The overall structures of these three mutant proteins are identical with that of the wild-type enzyme, except for local conformational changes of the flexible loops. The ribonuclease H family has a common active site, which is composed of four invariant acidic residues (Asp10, Glu48, Asp70 and Asp134 in E.coli ribonuclease HI), and their relative positions in the mutants, even including the side-chain atoms, are almost the same as those in the wild-type. The positions of the delta-polar atoms at residue 134 in the wild-type, as well as D134H and D134N, coincide well with each other. They are located near the imidazole side chain of His124, which is assumed to participate in the catalytic reaction, in addition to the four invariant acidic residues. Combined with the pH profiles of the enzymatic activities of the two other mutants, H124A and H124A/D134N, the crystallographic results allow us to propose a new catalytic mechanism of ribonuclease H, which includes the roles for Asp134 and His124.

Reestablishment of rabbit gallbladder epithelial cells in collagen gel culture and their alterations by cytochalasin B and transforming growth factor beta-1. A morphologic study.

We previously developed a model in which rabbit gall bladder epithelial cells in collagen gels proliferated and formed multicellular spherical cysts after 2 to 4 days. In the present study, we examined in depth the dynamic processes of loss and reestablishment of cell polarity of rabbit gallbladder epithelial cells isolated and cultured in collagen gel. Six hours after being place in culture, the isolated epithelial cells had lost the morphologic features and phenotypic markers inherent in the in vivo gallbladder mucosa, and autophagic vacuoles appeared transiently, reflecting epithelial cell injury, or remodelling, or both. After 12 hours, mucin dots appeared in clumps of epithelial cells and gradually became larger, and the epithelial cell clumps were transformed into multicellular cysts after 1 to 2 days. The luminal surfaces of the mucin dots (intracytoplasmic inclusions or small lumens sealed by several epithelial cells) and multicellular cysts were covered by microvilli and presented profiles of mucus glycoprotein and carbohydrate residues shared with the in vivo gallbladder mucosa. The presence of cellular adhesion structures and the distribution of cellular organelles toward the luminal surface implied the reestablishment of epithelial cell polarity. The addition of cytochalasin B induced many mucin-positive cytoplasmic inclusions covered by microvilli in the epithelial cells of the multicellular cysts, while the addition of transforming growth factor beta 1 promoted maturation of the multicellular cysts. This short term culture is useful for the analysis of the polarity of biliary epithelial cells and for examining disorders in this polarity.

Histopathology of the liver in non-cirrhotic portal hypertension of unknown aetiology.

Non-cirrhotic, long-standing portal hypertension of unknown aetiology is being re-evaluated histopathologically and clinically. In this study, we examined 107 livers with this condition (92 wedge biopsy and 15 autopsy specimens) from five institutions in Japan. These cases were histologically categorized into four groups: idiopathic portal hypertension (66 cases), nodular regenerative hyperplasia (14 cases), partial nodular transformation (two cases), and incomplete septal cirrhosis (25 cases). These four groups shared several histological features: dense portal fibrosis with portal venous obliteration and intralobular slender fibrosis. In addition, the histopathological features characteristic of one group were also found to a mild degree in other groups. The histopathological lesions preceding portal venous obliteration remain speculative. However, the portal venous obliteration may be responsible for the occurrence of sustained portal hypertension and several of the pathological changes in these livers. It seems likely that idiopathic portal hypertension, nodular regenerative hyperplasia, partial nodular transformation and incomplete septal cirrhosis comprise a family of non-cirrhotic, long-standing portal hypertension in Japan, and the histological differences between them may reflect chronological progression of a single disease.

Crystal structure of a pyrimidine dimer-specific excision repair enzyme from bacteriophage T4: refinement at 1.45 A and X-ray analysis of the three active site mutants.

Crystallographic study of bacteriophage T4 endonuclease V, which is involved in the initial step of the pyrimidine dimer-specific excision repair pathway, has been carried out with respect to the wild-type and three different mutant enzymes. This enzyme catalyzes the cleavage of the N-glycosyl bond at the 5'-side of the pyrimidine dimer, and subsequently incises the phosphodiester bond at the apyrimidinic site through a beta-elimination reaction. The structure of the wild-type enzyme refined at 1.45 A resolution reveals the detailed molecular architecture. The enzyme is composed of a single compact domain classified as an all-alpha structure. The molecule is stabilized mainly by three hydrophobic cores, two of which include many aromatic side-chain interactions. The structure has a unique folding motif, where the amino-terminal segment penetrates between two major alpha-helices and prevents their direct contact, and it is incompatible with the close-packing category of helices for protein folding. The concave surface, covered with many positive charges, implies an interface for DNA binding. The glycosylase catalytic center, which comprises Glu23 and the surrounding basic residues Arg3, Arg22 and Arg26, lie in this basic surface. The crystal structures of the three active-site mutants, in which Glu23 was replaced by Gln(E23Q) and Asp (E23D), respectively, and Arg3 by Gln (R3Q), have been determined at atomic resolution. The backbone structures of the E23Q and R3Q mutants were almost identical with that of the wild-type, while the E23D mutation induces a small, but significant, change in the backbone structure, such as an increase of the central kink of the H1 helix at Pro25. In the catalytic center of the glycosylase, however, these three mutations do not generate notable movements of protein atoms, except for significant shifts of some bound water molecules. Thus, the structural differences between the wild-type and each mutant are confined to the remarkably small region around their replaced chemical groups. Combined with the biochemical studies and the difference circular dichroism measurements, these results allow us to conclude that the negatively charged carboxyl group of Glu23 is essential for the cleavage of the N-glycosyl bond, and that the positively charged guanidino group of Arg3 is crucial to bind the substrate, a DNA duplex containing a pyrimidine dimer. The amino terminal alpha-amino group is located at a position approximately 4.4 A away from the carboxyl group of Glu23. These structural features are generally consistent with the reaction scheme proposed by Dodson and co-workers.

Architectures of class-defining and specific domains of glutamyl-tRNA synthetase.

The crystal structure of a class I aminoacyl-transfer RNA synthetase, glutamyl-tRNA synthetase (GluRS) from Thermus thermophilus, was solved and refined at 2.5 A resolution. The amino-terminal half of GluRS shows a geometrical similarity with that of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of the same subclass in class I, comprising the class I-specific Rossmann fold domain and the intervening subclass-specific alpha/beta domain. These domains were found to have two GluRS-specific, secondary-structure insertions, which then participated in the specific recognition of the D and acceptor stems of tRNA(Glu) as indicated by mutagenesis analyses based on the docking properties of GluRS and tRNA. In striking contrast to the beta-barrel structure of the GlnRS carboxyl-terminal half, the GluRS carboxyl-terminal half displayed an all-alpha-helix architecture, an alpha-helix cage, and mutagenesis analyses indicated that it had a role in the anticodon recognition.