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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.

We studied the effects of advanced glycation end products (AGEs), which are known to accumulate in patients with diabetes, autoimmune diseases, or those who smoke, on embryonal development. Pronuclear (PN) embryos were obtained by flushing the fallopian tubes of rats after superovulation and mating. The cleavage rate and blastocyst yield were evaluated at 24, 72, 96, and 120 h of culture. Glyoxal, an AGE-forming aldehyde, suppressed embryonal development at every stage from PN to blastocyst in a concentration-dependent manner. The cleavage rate of the embryo was also signifi cantly decreased by treatment with glyoxal at concentrations of 1 mM or higher. The blastocyst yield was significantly decreased by treatment with glyoxal at concentrations of 0.5 mM or higher. N-acetyl-L-cysteine (L-NAC) at 1 mM significantly suppressed the glyoxal-induced embryonal toxicity. BSA-AGEs at 5 microg/ml or higher concentration signifi cantly reduced the cleavage rate and blastocyst yield compared to those for BSA-treated embryos. L-NAC at 1 mM significantly suppressed BSAAGE-induced embryonal toxicity. Because AGEs are embryo-toxic, AGE contamination may influence the pregnancy rate of in vitro fertilization and embryo transfer. AGEs, which are increased in women under pathological conditions, may also be involved in their infertility.

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.

To evaluate morbidity in Hybrid Radical Prostatectomy (HRP, hybridized laparoscopic and open retropubic radical prostatectomy). The operative and pathological outcomes obtained in 25 consecutive patients who underwent HRP were reviewed. The median operating time was 220min, median blood loss was 550ml, and no patient required an allogenic blood transfusion. No severe postoperative complications were observed. The surgical margin was positive in 12% of all patients, and in 1 patient with pT2 or less (4.5%). These results indicate that HRP is safe and may be able to combine the benefits of both laparoscopic and open procedures.

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.

Percutaneous cardiopulmonary support (PCPS) has been applied for cardiopulmonary arrest (CPA). We have developed a novel method of cardiopulmonary resuscitation using PCPS combined with liposome-encapsulated hemoglobin (TRM645) to improve oxygen delivery to vital organs. Ventricular fibrillation was electrically induced to an adult goat for 10 min. Next, PCPS (30 ml/kg/min, V/Q: 1) was performed for 20 min. Then, external defibrillation was attempted and observed for 120 min. The TRM group (n5) was filled with 300 mL of TRM645 for the PCPS circuit. The control group (n5) was filled with the same volume of saline. The delivery of oxygen (DO2) and oxygen consumption (VO2) decreased markedly by PCPS after CPA, compared to the preoperative values. DO2 was kept at a constant level during PCPS in both groups, but VO2 slowly decreased at 5, 10, and 15 min of PCPS in the control groups, demonstrating that systemic oxygen metabolism decreased with time. In contrast, the decreases in VO2 were small in the TRM group at 5, 10, and 15 min of PCPS, demonstrating that TRM645 continuously maintained systemic oxygen consumption even at a low flow rate. AST and LDH in the TRM group were lower than the control. There were significant differences at 120 min after the restoration of spontaneous circulation (p<0.05).

We evaluated the infection risks in the neonatal intensive care unit (NICU) using data of NICU infection surveillance data. The subjects were 871 NICU babies, consisting of 465 boys and 406 girls, who were cared for between June 2002 and January 2003 in 7 medical institutions that employed NICU infection surveillance. Infections were defined according to the National Nosocomial Infection Surveillance (NNIS) System. Of the 58 babies with nosocomial infections, 15 had methicillin-resistant Staphylococcus aureus (MRSA) infection. Multiple logistic regression analysis demonstrated that the odds ratio for nosocomial infections was significantly related to gender, birth weight and the insertion of a central venous catheter (CVC). When the birth weight group of more than 1, 500g was regarded as the reference, the odds ratio was 2.35 in the birth weight group of 1,000-1,499g and 8.82 in the birth weight group of less than 1,000g. The odds ratio of the CVC () for nosocomial infection was 2.27. However, other devices including artificial ventilation, umbilical artery catheter, umbilical venous catheter, and urinary catheter were not significant risk factors. The incidence of MRSA infection rapidly increased from 0.3% in the birth weight group of more than 1,500g to 2.1% in the birth weight group of 1,000-1,499g, and to 11.1% in the birth weight group of less than 1,000g. When the birth weight group of more than 1,500g was regarded as the reference, multiple logistic regression analysis demonstrated that the odds ratio was 7.25 in the birth weight group of 1,000-1,499g and 42.88 in the birth weight group of less than 1,000g. These odds ratios were significantly higher than that in the reference group. However, the application of devices did not cause any significant differences in the odds ratio for MRSA infection.

In order to make the mechanism concerned with the sound-susceptibility in the organ of Corti clear, we observed the organ of Corti with the phase-contrast microscope, after the microdissection of the cochleae in human, dogs, guinea pigs and hamsters by Engstroöm's surface preparation technique. As a result, we have formulated a hypothesis for the mechanism of the sound-susceptibility in the organ of Corti. Further, we have inferred the contrary recruitment phenomenon (hypothesis), by explaining theoretically such a clinical fact as the recruitment phenomenon or the cochlear hearing loss by applying our first formula of hypothesis. Finally, we described the application of the contrary recruitment phenomenon (hypothesis) to the early discovery or diagnosis of the false normal ear or cochlea, in other words, latent hearing loss.

For the purpose to study the citrate metabolism in liver diseases, blood citrate, blood glucose and serum non-esterified fatty acids (NEFA) in fasting state were measured in the subjects with chronic hepatitis and with liver cirrhosis. Citrate and glucose were measured by the enzymatic methods. NEFA was measured colorimetrically. Fasting blood citrate level was investigated in relation to the type and extent of these liver diseases.Results revealed the following: 1. Fasting blood citrate level rose with the severity of liver diseases, especially in decompensated liver cirrhosis. 2. No significant difference in fasting blood citrate level was found between the subjects with and without glucose intolerance. 3. Fasting blood citrate level had a closer correlation with serum NEFA level than with blood glucose level. From these results, it has been concluded that the increase in blood citrate level in liver diseases is due to the impaired uptake of citrate by the liver and the increased release of citrate from peripheral tissues.

A purified and homogeneous preparation of rat alpha-fetoprotein (AFP) was separated into two components, AFPa and AFPb, by polyacrylamide gel electrophoresis. These two components had a definite difference in electrostatic net charge and gave only a single band on sodium dodecyl sulfate-electrophoresis. Neuraminidase-treated AFP gave clearly separable, slower moving four to six and finally two components depending on the time of incubation with neuraminidase. The time-dependent conversion of each AFPa and AFPb into slower migrating components upon neuraminidase treatment was confirmed by re-electrophoresis of separated and similarly treated AFPa and AFPb.

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 effect of glucose load on the levels of blood glucose, serum non-esterified fatty acids (NEFA) and liver citrate was investigated in carbontetrachloride-intoxicated (injured) rats and compared with non-intoxicated controls. The citrate level in the liver from injured animals showed 15-fold of the value of the control. Glucose load on these animals caused gradual decrease in the citrate level, whereas similar administration to the control caused inverse results. The serum NEFA levels were lowered by glucose load in both of injured and control animals. The pattern of changes in the citrate level after glucose load in the liver from injured animals was similar to that in the muscle from the control, suggesting a similarity on citrate metabolism between the injured liver and the muscle.The possible mechanisms for these results were discussed in relation to the difference in citrate metabolism between the liver and the muscle.

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.

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 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.

Native and heat-treated RNAs from the purified Schmidt-Ruppin strain of Rous sarcoma virus (RSV) were fractionated by sucrose density gradients in the presence of ribonuclease inhibitor diethyl-pyrocarbonate and observed by electron microscopy. The structure of native 60-70S RNA was classified into two forms: tanglefolded type and linear type. In the tangle-folded type double stranded portions were observed in several sites. A high frequency of 60-70S RNA were 1.0 mum and 3-3.5 mum in length. Molecules with length about 9mum were of the tangle-folded type while molecules shorter than 6 mum were of the linear form. The structure of heat-treated RNA(30-40S) was linear with the most frequent length being 1-1.5 mum. These results indicate that native 60-70S RNA is folded with the total molecular length being in the order of 6 to 9 mum. Molecules about 3mum long are likely to be the main subunits of 60-70S RNA, and they are fragmented further into smaller subunits of about 1 mum length.

As a step in the elucidation of the mutual relationship between the degree of cancer progress and the antitumor activity of lymphocytes from different sites in cancer-bearing body, we isografted methylcholanthrene-induced tumor (MC-tumor) subcutaneously on the back of mice. The regional axillary lymph nodes, spleen and distant mesenteric lymph nodes were removed from these animals one, two, three, and four weeks later. We mixed lymphocytes prepared from these lymphatic tissues with primary MC-tumor culture cells and cultured together to estimate antitumor acitivity of lymphocytes from different sites. It has been found that a strong antitumor activity can be seen only in those regional axillary lymph node cells taken out one or two weeks after tumor transplatation and such an activity is weakened by three or four weeks. On the other hand, distant mesenteric lymph node cells one or two weeks after the transplantation have no antitumor activity as yet, while at the terminal cancer stage of four weeks there appears a stronger antitumor activity than that of regional lymph nodes. In the spleen, a strong antitumor activity can be observed in the third week after tumor transplantation, but the activity disappears by the fourth week. These findings support our previous findings in that for the tumor onset after the transplantation the antitumor activity seems to appear first in the regional lymph nodes, and when the tumor grows beyond a certain size, such an activity diminishes while it appears in further distant lymphatic tissues.