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To clarify the mode of secretion of specific atrial granules, rat atrial muscle cells were examined by transmission electron microscopy. Atrial granule formation and exocytotic features of granules were clearly seen. Abrupt breaks in the unit membrane structure of mature granules were observed in thin sections, but these breaks were not detected in freeze-fracture replicas. These findings support the concept that the granule contents are released to the extracellular space by exocytosis.

The cytoskeletons of two established chick embryo cell (CEC) lines were examined by fluorescence and electron microscopy and compared with those of control cells and cells transformed by Rous sarcoma virus (RSV). In normal CEC, many stress fibers were observed. On the other hand, stress fibers were disorganized in nontransformed spontaneously established CEC, non-tumorigenic CEC partially transformed with a chemical carcinogen, and tumorigenic RSV-transformed CEC. In the normal CEC, actin filaments formed several bundles along the processes of the cell. Stereo-images of the peripheral region revealed bundles of filaments which were located along the attached side to the substrate. A fine well preserved network of filaments was also observed. On the other hand, in spontaneously established, partially transformed and RSV-transformed CEC, a fine network of filaments, but no actin cables, was found. These results support previous evidence that the cytoskeletal changes themselves are not directly related to the transformation or tumorigenicity of cells.

Dry pleural dissemination in non-small cell lung cancer, defined as solid pleural metastasis of lung cancer without pleural eff usion, is a condition occurring in T4 lung cancer. Positron emission tomography (PET) has been reported to be useful for the diagnosis and staging of lung cancer. It has been reported that positive findings on PET scans of indeterminate pleural abnormalities at computed tomography (CT) are sensitive to malignancy. We encountered two cases of dry small pleural dissemination of adenocarcinoma of the lung preoperatively detected by PET/CT. A 75-year-old man and a 66-year-old man underwent CT scan, which demonstrated solitary tumor in the lung, an enlarged mediastinal lymph node, and a small pleural nodule less than 10 mm in size, all of which were positive findings on the fluorine 18 fluorodeoxyglucose (FDG) PET portion of an integrated PET/CT. Both patients underwent thoracoscopic biopsy of the dry pleural nodule revealing dissemination of adenocarcinoma of the lung (T4). Whereas histological thoracoscopic diagnosis remains mandatory before planning treatment, our cases may suggest that PET/CT will be useful as a screening modality for dry pleural dissemination of lung cancer.

Oxidative stress, including the reactive oxygen or nitrogen species generated in the enzymatical oxidationor auto-oxidation of an excess amount of dopamine, is thought to play an important role in dopaminergic neurotoxicity. Dopamine and its metabolites containing 2 hydroxyl residues exert cytotoxicityin dopaminergic neuronal cells, primarily due to the generation of highly reactive dopamine and DOPA quinones. Dopamine and DOPA quinones may irreversibly alter protein function through the formation of 5-cysteinyl-catechols on the proteins. Furthermore, the quinone formation is closely linked to other representative hypotheses such as mitochondrial dysfunction, inflammation, oxidative stress, and dysfunction of the ubiquitin-proteasome system, in the pathogenesis of neurodegenerative diseases. Therefore, pathogenic effects of the dopamine quinone have recently focused on dopaminergicneuron-specific oxidative stress. In this article, we primarily review recent studies on the pathogenicity of quinone formation, in addition to several neuroprotective approaches against dopaminequinone-induced dysfunction of dopaminergic neurons.

Cherubism is a rare developmental lesion of the jaw that is generally inherited as an autosomal dominant trait. Recent studies have revealed point mutations in the SH3BP2 gene in cherubism patients. In this study, we examined a 6-year-old Korean boy and his family. We found a Pro418Arg mutation in the SH3BP2 gene of the patient and his mother. A father and his 30-month-old younger brother had no mutations. Immunohistochemically, the multinucleated giant cells proved positive for CD68 and tartrate-resistant acid phosphatase (TRAP). Numerous spindle-shaped stromal cells expressed a ligand for receptor activator of nuclear factor kB (RANKL), but not in multinucleated giant cells. These results provide evidence that RANKL plays a critical role in the differentiation of osteoclast precursor cells to multinucleated giant cells in cherubism. Additionally, genetic analysis may be a useful method for differentiation of cherubism.

The aim of this study was to determine the optimal temperature of graft preservation after ex vivo gene transfer to rat lung isografts. Left lungs were harvested and infused with cationic lipid/LacZ-DNA complex via the pulmonary artery, and the grafts were stored for 4h. The grafts (n＝7) were allocated into groups IﾝIV according to the storage temperature:4℃, 10℃, 16℃, and 23℃, respectively. Forty-eight h after orthotopic transplantation, the arterial blood gas was analyzed and the peak airway pressure (PAP) and the level of LacZ protein production in the grafts were measured by reverse transcription polymerase chain reaction. After reperfusion, the grafts were stained with hematoxylin and eosin. The grafts in groups III and IV showed more deterioration as evidenced by decreased arterial oxygen tension, increased PAP, and predominant infiltration of inflammatory cells compared with groups I and II. The level of LacZ production was significantly lower in group I than in groups IIﾝIV. The optimal temperature of lung graft preservation after ex vivo gene transfer was determined to be 10℃, balancing considerations of lung injury and efficiency of transgene expression.

Hepatic and pancreatic differentiation from ES cells is of great interest for the impact that this knowledge could have on the treatment of hepatic and diabetic patients. The liver and pancreas initially develop by budding from the embryonic endoderm. Thus, the development of the endoderm represents an important step and has an integral common role in initiating the early stages of pancreatic and liver development. We know that the development of hepatocytes and insulin-producing pancreatic beta-cells from ES cells represents the culmination of a complex developmental program. However, there has been recent progress in directing ES cells to endoderm and early-stage hepatic and pancreatic progenitor cells. We here discuss the role of the microenvironment, transcriptional factors and cytokines, which have been recognized as important molecules during the major steps of the development of the liver and pancreas. We also present the most recent advances and efforts taken to produce definitive endoderm-committed ES cells for the further differentiation of hepatocyte-like and insulinproducing cells. Recent progress in the search for new sources of hepatocytes and beta-cells has opened up several possibilities for the future of new perspectives for future of new prophylactic and therapeutic possibilities for liver diseases and diabetes.

Testicular Sertoli cells highly express dynamin 2 and amphiphysin 1. Here we demonstrate that dynamin 2 is implicated in phosphatidylserine (PS)-dependent phagocytosis in Sertoli cells. Immunofluorescence and dual-live imaging revealed that dynamin 2 and amphiphysin 1 accumulate simultaneously at ruffles. These proteins are specifically bound in vitro. Over-expression of dominant negative dynamin 2 (K44A) inhibits liposome-uptake and leads to the mis-localization of amphiphysin 1. Thus, the cooperative function of dynamin 2 and amphiphysin 1 in PS-dependent phagocytosis is strongly suggested.

The molecular defects in the catalase gene, levels of m-RNA and properties of the residual catalase studied by scientists are reviewed in human (Japanese, Swiss and Hungarian) and non-human (mouse and beagle dog) acatalasemia with reference to the bioinformatics. Japanese acatalasemia-I, the G to A transition at the fifth position of intron 4 of the catalase gene, limited the correct splicing of the mRNA and synthesized trace catalase with normal properties. Hungarian acatalasemia type C showed a splicing mutation. In the Japanese acatalasemia II and the type A and B of Hungarian acatalasemia, the deletion or insertion of nucleotides was observed in the coding regions, and the frame shift altered downstream amino acid sequences and formed truncated proteins. In the Hungarian acatalasemia D, the substitution of a nucleotide in the exon was found. In mouse and beagle dog acatalasemia, the substitution of nucleotides in the coding regions was also observed. Studies of residual catalase in Swiss, mouse and beagle dog acatalasemia showed that aberrant catalase protein degrades more quickly than normal catalase in cells. The experimental research in genetic toxicology concerning the effect of oxidative stressors (nitrogen monoxide, nitrogen dioxide and so on) on Japanese acatalasemic blood and acatalasemic mice is described. The clinical features of Japanese and Hungarian acatalasemic subjects are also described.

We have recently shown that a new therapeutic modality using the REIC/Dkk-3 gene (Ad-REIC) is effective against various human cancers, including those of prostate, testis and breast origins. The aim of the present study was to examine the sensitivity of bladder cancers to Ad-REIC and to clarify the molecular mechanisms that determine sensitivity/resistance. We found that 2 human bladder cancer cell lines, T24 and J82, are resistant to Ad-REIC. In T24 and J82 cells, the ER stress response and activation of JNK were observed in a manner similar to that in the sensitive PC3 cells. Translocation of Bax to mitochondria occurred in PC3 cells but not in T24 and J82 cells. Bcl-2 was remarkably overexpressed in T24 and J82 compared with the expression levels in sensitive cell lines. Treatment of T24 and J82 cells with a Bcl-2 inhibitor sensitized the cells to Ad-REIC-induced apoptosis. The results indicate that some human bladder cancers are resistant to apoptosis induced by overexpression of REIC/Dkk-3, which is at least in part due to up-regulation of Bcl-2. These results provide a basis for possible use of Bcl-2 as a marker of sensitive cancers and to try to sensitize resistant cancers to Ad-REIC by down-regulation of Bcl-2.

The insulin-like growth factor I receptor (IGF-IR) is exceptionally overexpressed in many cervicalcancer-derived cell lines. It is postulated that a decrease of p53 protein levels due to human papillomavirus (HPV) infection may contribute to the up-regulation of IGF-IR expression in cervical cancer cells because transcription of IGF-IR is strictly down-regulated by p53. To evaluate this fact in clinical cervical cancer specimens, we checked the expression levels and activated status of IGF-IR by immunohistochemistry. Formalin-fixed and paraffin-embedded specimens obtained by conization or hysterectomy were stained with anti-IGF-IR and with an antibody recognizing phosphorylated tyrosine at its c-terminus. The expression levels of IGF-IR were significantly high in cervical intraepithelial neoplasia (CIN) III and invasive cancer specimens. Phosphorylation of IGF-IR was promoted in all CIN and invasive cancer specimens, and its intensity was related to the promotion of lesions. Interestingly, IGF-IR overexpression was missing in the basal layer of CIN I and II lesions, whereas it was evenly distributed in CIN III and invasive cancer lesions. This IGF-IR overexpression pattern may be utilized in the diagnosis of HPV infection status in CIN lesions.

Autoimmune hepatitis (AIH) is a chronic and progressive disease characterized by histological interface hepatitis, hypergammaglobulinemia, and circulating autoantibodies. Multiple factors, including molecular mimicry, a genetic background including major histocompatibility complex class II, and defective function of regulatory T-cells, are involved in the pathogenesis. The diagnosis is made based on the scoring system of the International Autoimmune Hepatitis Group, the sensitivity and specificity of which are90%, respectively. AIH is classified into 3 sub-types based on the profiles of circulating autoantibodies: anti-nuclear antibody and/or smooth muscle antibody-positive (type 1), anti-liver-kidney microsomal antibody-positive (type 2), and anti-soluble liver antigen/liver-pancreas antigen antibody- positive (type 3). Recently, however, the number of atypical cases lacking the usual features has increased-for example, patients with acute-onset or fulminant-type AIH, autoantibody-negative patients, male patients, and patients with bile duct injury-and thus the clinical features of AIH have been diversified. AIH is responsive to immunosuppressive treatment in most cases; however, relapse occurs in more than 80% of patients within 1 year after immunosuppressive treatment withdrawal. The 10-year survival rate and the 10-year hepatocellular carcinoma-free rate are90%, respectively, indicating that some patients reach liver failure or develop hepatocellular carcinoma. To improve the prognosis of these patients, persistent normalization of transaminase is required.

The establishment of permanent cell line that can produce an alpha-fetoprotein has made tissue culture a powerful tool for the study of alpha-fetoprotein. For this reason, the hepatoma cells of rat ascites hepatoma AH70B were cultured in vitro and some biological characters of the isolated six clones examined. The cultured cells were morphologically epithelial and the mode of chromosome number in hypotetraploid range, and possessed tumorigenicity. The cells secreted alpha-fetoprotein at the high level and a few components of serum proteins in the culture medium for more than one year. Alpha-Fetoprotein was also detected in cytoplasm by fluorescent antibody technique. The examined character was little different among the six colonial clones. From the present cloning procedure, it was suggested that the cultured cells derived from a single cell were secreting alpha-fetoprotein and several components of serum proteins together.

The mechanism of action of the drug was investigated from various points of view. The findings may be summarized as follows: 1. In the experiments of the degranulation of mesenteric mast cells of rats, menaquinone proved to significantly inhibit the degranulation either in active or passive sensitization with the reagin-like antibody. 2. Menaquinone did not inhibit the formation of the reagin-like antibody. 3. In the experiements of the degranulation of basophilic granulocytes from patients of bronchial asthma, the rate of appearance of A form basophilic cells upon addition of the antihuman IgE goat serum was not markedly but significantly inhibited in the patients treated with menaquinone for long periods, as compared with that in the control, whereas the in vitro addition of menaquinone did not exert a significant inhibitory action.

A study was carried out to clarify the mechanism of nuclear extrusion of mammalian erythroid cells by observing erythroblasts of rabbit under various conditions in vitro. The animals were made anemic by phenylhydrazine injection and erythroblasts were obtained from the peripheral blood and observed morphologically after a certain time of incubation. After two hour incubation at 37 degrees C, about 50% of erythroblasts were denucleated. The nuclear extrusion was remarkably suppressed by the inhibitor for electron transport system or by uncouplers for oxidative phosphorylation. It was also arrested by the inhibitor of cell movement, like cytochalasin B. In contrast, monoiodo-acetic acid, ouabain and colchicine hardly inhibited the nuclear extrusion. The observations indicated that the nuclear extrusion of mammalian erythroblast is an energy-dependent process in connection with the function of contractile microfilamentous system susceptible to cytochalasin B.

Anemia or polycythemia was induced in male rats. These males were conjugated with healthy, untreated female litter mates by FANG'S method of aortic parabiosis that resulted in complete cross circulation of blood between the two animals. The sex chromosomes of cells in erythropoiesis in various hemopoietic organs were examined in the treated male animals. The anemic parabionts indicated sharp increases in chimerical rates with erythroid marrows being evident. Polycythemic parabionts indicated marked decreases in chimerical rates with evidence of myeloid marrows. These findings suggested that the so-called stem cells in peripheral blood of the female parabiont migrated to the bone marrow of the male partner and that these migrating cells differentiated to erythroblast. The possible relationships between erythropoiesis and other cell proliferations in the hemopoietic organs are discussed.

In mouse bearing progressive cancer a decrease was present in the allogeneic inhibitory activity of T-lymphocytes, which constitutes the core of immunological surveillance system in mammalians. For tests, methylcholanthrene-induced tumor (MC-tumor) was isografted subcutaneously on the back between scapulae of C3H mice, and the lymphocytes were prepared from the regional axillary lymph nodes removed from these mice at 1, 2, 3, or 4 weeks after grafting. These lymph nodes cells were cultured together with 40-fold numbers of allogeneic JTC-11 cells derived from Ehrlich cancer cells in a culture medium containing 2.0% (v/v) PHA for 24 or 48 hours. The proliferation rate of JTC-11 cells (increased numbers) at weekly interval was considered the allogeneic inhibitory activity of lymph node cells. As a result it was demonstrated that in the early stage after tumor transplantation, i.e., in the first or second week, regional lymph node cells showed a strong allogeneic inhibitory activity, as in the case with lymph-node cells from normal mice, but at progressive stage of cancer, i.e., the third or fourth week when tumors were larger, such activity was completely lost. It seems that mice with progressive cancer showed a decrease of allogeneic inhibitory activity, i.e., a disruption of homeostasis was present.

It has previously been shown that the barrier system for high environmental salinity is closely related to the salt-resistance of Staphyloccus aureus. The present investigation was undertaken to clarify the energy dependency for the maintenance of intracellular univalent cation contents in cells grown on high concentration of salt containing medium. The results are summarized as follows: (1) The growth of 10% NaCl-Staph which was grown in the 10% NaCl containing nutrient broth was more sensitive to NaN3 than Normal-Staph which was grown only on nutrient broth. The anaerobic conditions in both media demonstrated a more powerful effect on growth inhibition of 10% NaCl-Staph than Normal-Staph. Therefore, 10% NaCl-Staph must have a higher energy dependency than Normal-Staph. (2) The high sensitivity to uncouplers, such as DNP and FCCP in 10% NaCl-Staph, also suggested an energy dependency which was probably related to respiration and not to anaerobic glycolysis. (3) The intracellular Na+ contents of Normal-Staph and 10% NaCl-Staph were 12.0 and 152.9 mmoles per Kg wet weight of cells respectively, and the content of K+ in 10% NaCl-Staph (90.2 mmoles per Kg wet weight) was lower than that of Normal-Staph (215.8 mmoles per Kg wet weight). These intracellular Na+ and K+ contents were strongly affected by the addition of various inhibitors to the medium. The measurements of intracellular univalent cation contents indicated the existance of an adaptively developed barrier system in 10% NaCl-Staph and the existence of energy-dependent transport mechanisms for efflux of Na+ in Normal-Staph and for the influx of K+ in 10% NaCl-Staph.

In vitro quantitative biosynthetic studies were carried out on bone marrow cells obtained from an eleventh case with gamma heavy chain disease. The findings indicate that neither cytoplasmic nor extracellular degradation was responsible for the presence of the gamma heavy chain fragment in serum. The absence of a covalent-bound light chain was also confirmed.

In vitro transformations of brain cells of hamsters of various ages were examined after the administration of human adenovirus type 12 (Ad 12) to determine the type and origin of the target cell. Hamster brain cells at all examined ages were transformed by Ad12. Although the virus was not isolated, virus specific tumor antigen was demonstrated in the transformed cells. The histological features of tumors that developed by transplantation of transformed cells closely resembled Ad12-induced brain tumors. The transformed cell focus tended to appear near the embryonic brain cell (EB cell) or glioblastic cell (GB cell). The transformed cells were morphologically similar to the EB or GB cell. Some subcultured transformed cells showed a rosette-like pattern, and the surrounding space arrangement was similar to that of the ventricular wall. The incidence of brain cell transformations decreased with increased hamster age. This decreased incidence with age corresponded to the decreased numbers of EB or GB cells present in progressively older hamsters. From these results, it is concluded that the target cells of AD12 in hamster brain cell cultures are probably the EB or GB cells.