Undifferentiated (embryonal) sarcoma of the liver in middle-aged adults: smooth muscle differentiation determined by immunohistochemistry and electron microscopy

Undifferentiated (embryonal) sarcoma of the liver (UESL) is a rare pediatric liver malignancy that is extremely uncommon in middle-aged individuals. We studied 2 cases of UESL in middle-aged adults (1 case in a 49-year-old woman and the other in a 62-year-old man) by histology, immunohistochemistry, and electron microscopy to clarify the cellular characteristics of this peculiar tumor. One tumor showed a mixture of spindle cells, polygonal cells, and multinucleated giant cells within a myxoid matrix and also revealed focal areas of a storiform pattern in a metastatic lesion. The other tumor was composed mainly of anaplastic large cells admixed with few fibrous or spindle-shaped components and many multinucleated giant cells. In both cases, some tumor cells contained eosinophilic hyaline globules that were diastase resistant and periodic acid-Schiff positive. Immunohistochemically, the tumor cells showed positive staining for smooth muscle markers, such as desmin, alpha-smooth muscle actin, and muscle-specific actin, and also for histiocytic markers, such as alpha-1-antitrypsin, alpha-1-antichymotrypsin, and CD68. Electron microscope examination revealed thin myofilaments with focal densities and intermediate filaments in the cytoplasm of tumor cells. Our studies suggest that UESL exhibits at least a partial smooth muscle phenotype in middle-aged adults, and this specific differentiation may be more common in this age group than in children. Tumor cells of UESL with smooth muscle differentiation in middle-aged adults show phenotypic diversity comparable to those of malignant fibrous histiocytoma with myofibroblastic differentiation.     

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5

Background: Eukaryotic RNA polymerase II is composed of more than 10 polypeptide chains. The minimum and essential subunits for RNA synthesis have not yet been identified. Toward this ultimate goal, we analysed the topological arrangement of the putative subunits. Here we report a subunit–subunit contact network involving subunit 5 of the fission yeast Schizosaccharomyces pombe RNA polymerase II.
Results: The rpb5 ⁺ gene encoding subunit 5 of RNA polymerase II was cloned from the fission yeast Schizosaccharomyces pombe . The polypeptide predicted from DNA sequence of the rpb5 ⁺ gene consists of  210 amino acids with a calculated molecular weight of 23 914. The homology of the amino acid sequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human hRPB25, respectively. Far-Western blot analysis of S. pombe RNA polymerase II using ³²P-labelled recombinant Rpb5 fused to glutathione S-transferase (GST) as a probe, indicated that Rpb5 binds strongly to membrane-immobilized Rpb1, Rpb2 and Rpb3 and weakly to Rpb5 and a 15-kDa subunit (Rpb8 or Rpb11). In agreement with this result, the ³²P-labelled Rpb3 probe showed a strong binding signal against Rpb5 in addition to Rpb1 and Rpb2. The existence of Rpb5–Rpb3 contact was supported by detection of complexes formed between these two proteins synthesized in vitro using protein-immobilized beads.
Conclusion: Rpb3 and Rpb5, the putative subunits of RNA polymerase II, associate each other to form binary complexes. These two subunits also bind to the two large subunits, Rpb1 and Rpb2, independently.     

Aberrant B1 cell migration into the thymus results in activation of CD4 T cells through its potent antigen-presenting activity in the development of murine lupus

B1 cells have different origin and function from conventional B (B2) cells and are considered to be involved in autoantibody production in the development of autoimmune disease. We found that B1 cells preferentially accumulated in the target organs including thymus in aged BWF1 mice, a murine model for systemic lupus erythematosus, and that B lymphocyte chemoattractant (BLC/CXCL13) expression was increased in the thymus before the onset of lupus nephritis, while stromal cell-derived factor-1 (SDF-1/CXCL12) and secondary lymphoid tissue chemokine (SLC/CCL21) expression remained unchanged. Adhesion molecules such as peripheral node addressin (PNAd), ICAM-1, and VCAM-1 were also expressed on endothelial cells in the enlarged thymic perivascular space (PVS) in aged BWF1 mice. BLC protein and PNAd were co-localized on these high-endothelial-venules-like vessels in enlarged PVS. B1 cells expressed higher level of costimulatory molecules and showed a potent antigen-presenting activity in allogeneic mixed lymphocyte reaction comparable to splenic dendritic cells. Interestingly, B1 cells stimulated proliferation of autologous thymic CD4 T cells in the presence of IL-2. These results indicate that aberrant B1 cell trafficking into the thymus due to ectopic high expression of BLC may result in an activation of self-reactive T cells in the development of murine lupus.     

Development of a rapid PCR method using the insertion sequence IS1203 for genotyping Shiga toxin-producing Escherichia coli O157

We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing). DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203. To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within 1 or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981). This method can be used for rapid and simplified genotyping.     

Identification, characterization, and expression pattern of the chicken EKLF gene.

EKLF/KLF1 was the first of the Krüppel-like factors (KLFs) to be identified in mammals and plays an important role in primitive and definitive erythropoiesis. Here, we identify and characterize EKLF in the chicken (cEKLF). The predicted amino acid sequence of the zinc finger region of cEKLF is at least 87.7% similar to mammalian EKLF proteins and is 98.8% and 95% similar to the EKLF orthologues in Xenopus and zebrafish, respectively. During early embryonic development, cEKLF expression is seen in the posterior primitive streak, which gives rise to hematopoietic cells, and then in the blood islands and in circulating blood cells. cEKLF mRNA is expressed in blood cells but not in brain later in chicken embryonic development. cEKLF mRNA is increased in definitive compared with primitive erythropoiesis. The conserved sequence and expression pattern of cEKLF suggests that its function is similar to its orthologues in mammals, Xenopus, and zebrafish.     

A comparative study of gut‐associated lymphoid tissue in calf and chicken

The calf contains two types of Peyer's patches (PPs): jejunal and ileal. The ileal PP has been thought to be equivalent to the bursa of Fabricius (BF) as a central lymphoid organ. The morphologies of ileal and jejunal PPs in the calf were compared with those of the BF and the caecal tonsil (CT) in the chicken. Immunoglobulin G–positive (IgG⁺) cells appear in the follicles of them all and exhibited a dendritic appearance after birth. We investigated whether the IgG in these follicles was produced in situ. IgG‐producing cells were detected in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. CD4⁺ cells were distributed in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. The data suggest that Ig class switching occurs in both jejunal PP follicles and CT follicles, but does not occur in either the ileal PP follicles or the bursal follicles. Because CD4⁺ T cells would be prerequisite for Ig class switching in these follicles, IgG⁺ cells of the follicular medullas in the ileal PP and the BF would trap immune complexes from the gut lumen. The primary B‐cell repertoire might be selected by gut‐derived antigens in the ileal PP and the BF before seeding the periphery. Anat Rec 266:207–217, 2002. © 2002 Wiley‐Liss, Inc.     

The effects of 5-bromodeoxyuridine and isoproterenol on the postnatal differentiation of rat submandibular gland

The effects of 5-bromodeoxyuridine (BrdU) on the postnatal differentiation of rat submandibular gland and on the isoproterenol-induced changes of differentiation were studied. The rats were injected with BrdU for three consecutive days, beginning at two days of age. The total dose of BrdU was 0.9 mg/g body weight. BrdU caused a severe retardation of growth up to 15 days of age. A rapid growth of the animals between 15 and 22 days indicated a recovery from the effect of BrdU. The growth of the submandibular gland was retarded similarly with a corresponding decrease in DNA, RNA and protein content. Incorporation of tritiated thymidine into the submandibular gland was not altered in the BrdU-treated animals at one and three days after the last injection of the analog. At days 15 and 22 the rate of thymidine incorporation was greater in the submandibular gland of BrdU-treated rats as compared to littermate controls. Isoproterenol stimulated thymidine incorporation into the submandibular glands of two-week-old rats. This stimulation was not observed in rats which received BrdU at age 7–9 days, prior to the administration of isoproterenol. Electron microscopic observations, including a quantitative analysis of the frequency distribution of the various cell types in the terminal tubules and developing acini, indicated a retardation of acinar cell differentiation in the glands of BrdU-treated rats. In addition, there was an increase in the number and size of the secretory granules in the terminal tubule cells. BrdU treatment, however, caused no obvious pathologic alterations in the submandibular gland. Administration of isoproterenol for five days, beginning at five days of age, caused an apparent acceleration of the differentiation of acinar cells. In the glands of isoproterenol-treated rats the acinar cells were enlarged and were filled with homogenous secretory granules. Pretreatment with BrdU partially inhibited the effects of isoproterenol on differentiation and hypertrophy of the submandibular gland. With the given dose of BrdU, approximately 5% of thymine was replaced by bromouracil in the DNA of the submandibular gland. Such a replacement would be consistent with a direct effect of BrdU on the cytodifferentiation in the submandibular gland. However, because of the severe retardation of growth of the BrdU-treated rats, indirect effects can not be excluded.     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test

In order to evaluate the utility of the mouse lymphoma assay(MLA) for detecting in vitro clastogens and spindle poisonsand to compare it with the in vitro chromosomal aberration test(CA), we conducted an international collaborative study of theMLA that included 45 Japanese laboratories and seven overseaslaboratories under the cooperation of the Ministry of Healthand Welfare of Japan and the Japanese Pharmaceutical Manufacturer'sAssociation. We examined 40 chemicals; 33 were reportedly positivein the CA but negative in the bacterial reverse mutation assay,six were negative in both assays and one was positive in both.We assayed mutations of the thymidine kinase (TK) locus (tk)of L5178Y tk^+/– mouse lymphoma cells using the microwellmethod. According to our standard protocol, cells were exposedto the chemical for 3 h, cultured for 2 days and TK-deficientmutants were expressed in 96-well plates under trifluorothymidine.Each chemical was coded and tested by two or three laboratories.Among the 34 CA-positive chemicals, positive MLA results wereobtained for 20 and negative results were obtained for nine.The remaining five chemicals were inconclusive or equivocalbecause of discrepant inter-laboratory results or reproduceddiscrepant results, respectively. Among the six CA-negativechemicals, one was negative in the MLA, two were positive andthree were inconclusive. Thus, the MLA could detect only 59%(20/34) of CA-positive chemicals. We concluded that the MLAwas not as sensitive as the CA. Some MLA-negative chemicalsevoked positive responses in the CA only after long continuoustreatment. These might also be genotoxic in the MLA with longcontinuous treatment. Improvement of the MLA protocol, includingalteration of the duration of the treatment, might render theMLA as sensitive as the CA. ⁸ To whom correspondence should be addressed. Tel: +81 3 37009847; Fax: +81 3 3700 2348; Email: sofuni{at}nihs.go.jp