Lack of Bcl11b tumor suppressor results in vulnerability to DNA replication stress and damages.

Bcl11b/Rit1 is involved in T-cell development and undergoes chromosomal rearrangements in human T-cell leukemias. Thymocytes of Bcl11b(-/-) newborn mice exhibit apoptosis at a certain developmental stage when thymocytes re-enter into the cell-cycle. Here, we show that Bcl11b-knockdown T-cell lines, when exposed to growth stimuli, exhibited apoptosis at the S phase with concomitant decreases in a cell-cycle inhibitor, p27 and an antiapoptotic protein, Bcl-xL, owing to transcriptional repression. This repression was a likely consequence of the impairment of Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase associating with Bcl11b. Activation of the apoptotic process cleaved the mediator protein, Claspin, and inhibited phosphorylation of cell-cycle checkpoint kinase 1 (Chk1) that plays a central role in sensing and responding to incomplete replication. Bcl11b(-/-) thymocytes also failed to phosphorylate Chk1 when UV irradiated. These results implicate Bcl11b in the remedy for DNA replication stress and maintenance of genomic integrity.

Increased susceptibility to apoptosis in CD45(+) myeloma cells accompanied by the increased expression of VDAC1.

Expression of CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45(+) U266 proliferates in response to a growth factor, interleukin-6. Here, we show that CD45(+) myeloma cell lines were more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress, than CD45(-) cells. Reactive oxygen species and calcium ion seemed to be involved in the susceptibility to apoptosis of CD45(+) U266. The activation of the src family kinases associated with CD45 phosphatase played an important role in the augmented apoptosis in CD45(+) U266 by oxidative stress. These results indicate that the CD45-expression renders myeloma cells competent for not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45(+) myeloma cells dependently upon the circumstantial stimuli. Furthermore, voltage-dependent anion channel (VDAC) 1 was identified as a gene highly expressed in CD45(+) U266 by cDNA subtraction. The increased expression of VDAC1 seemed to augment the sensitivity to the ER-stress because the VDAC1-transfected U266 was more susceptible to the thapsigargin-induced apoptosis. Thus, CD45 expression accompanied by the increased VDAC1 expression sensitizes myeloma cells to the various extracellular stimuli that trigger apoptosis via the mitochondrial pathways.

The effect of basic fibroblast growth factor on the regeneration of guinea pig olfactory epithelium.

Olfactory receptor cells are widely thought to regenerate after degeneration and also thought to show turnover in normal circumstances in animal olfactory epithelium. The identity of the factor that controls proliferation and differentiation of olfactory receptor cells is a very important problem that has yet to be resolved. In this study, the mitogenic effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on olfactory receptor cells in guinea pig olfactory epithelium was examined. The intraperitoneal injection of 1,000 ng bFGF/day for 14 days increased the cells in proliferation detected by immunostaining with proliferating cell nuclear antigen (PCNA), while neither EGF nor low-dose bFGF had any effect. These results support the idea that an adequate dose of bFGF plays an important role in the neurogenesis in the olfactory epithelium. Further study is needed to clarify the efficacy of bFGF in the damaged olfactory epithelium, but bFGF may provide a therapeutic option for olfactory disturbances caused by complete or partial loss of olfactory receptor cells.

Resistance of primary cultured mouse hepatic tumor cells to cellular senescence despite expression of p16(Ink4a), p19(Arf), p53, and p21(Waf1/Cip1).

Primary cultured mouse hepatic cells become senescent within a short period, although rare cells form colonies from which continuously proliferating cell lines can be established. In contrast, hepatic tumor (HT) cells show little senescence and higher colony-forming capacity. To assess this difference, we investigated p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression in primary normal and HT cells, together with cell lines established from both. In primary normal cells, p16(Ink4a)/p19(Arf) were expressed only in association with senescence and disappeared at later stages of colony formation. In contrast, primary HT cells showed sustained p16(Ink4a)/p19(Arf) expression from the beginning. No p16(Ink4a)/p19(Arf) alterations, such as deletion, mutations, or hypermethylation, were detected in the primary HT cells, although most cell lines derived from either normal or HT cell colonies lost p16(Ink4a) or p19(Arf) expression owing to hypermethylation or homozygous deletion of p16(Ink4a)/p19(Arf). On the other hand, primary normal and HT cells and most cell lines showed constitutively elevated expression of p53/p21(Waf1/Cip1), with a further increment after ultraviolet ir-radiation, indicating a functionally normal p53 pathway. These results indicate that primary HT cells are resistant to senescence despite retaining p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression and that loss of p16(Ink4a)/p19(Arf) function is associated only with establishment of the cell lines.

Investigation on diabetic autonomic neuropathy assessed by power spectral analysis of heart rate variability in WBN/Kob rats.

This article investigates the development of cardiovascular autonomic dysfunction caused by diabetes mellitus. We performed power spectral analysis of heart rate variability in WBN/Kob rats as a model of spontaneous diabetes. The heart rate of the rats was measured continuously for 24 hours with an implanted telemetric transmitter, and power spectral analysis of heart rate variability was performed on continuous electrocardiograms. At 4 to 5 months of age, the rats indicated a tendency toward a decrease in plasma insulin concentration without hyperglycemia. At 8 to 9 months of age, they showed remarkable hyperglycemia, loss of the circadian rhythm of the heart rate, and reversion or loss of the circadian rhythm of the blood pressure. By the power spectral analysis of heart rate variability, it became apparent that the circadian rhythm of the low frequency/high frequency ratio was absent even in prediabetic WBN/Kob rats. In addition, the circadian rhythms of the high-frequency power level and low frequency/high frequency ratio were absent in diabetic WBN/Kob rats. These findings indicate that the autonomic nervous system in WBN/Kob rats is progressively damaged from the prediabetic to diabetic state. In conclusion, diabetic autonomic neuropathy may be characterized by the appearance of sympathetic overactivity that precedes the impairment of parasympathetic activity.

Heat transfer and flow characteristics on a gas turbine shroud.

The work described in this paper is an experimental investigation of the heat transfer from the main flow to a turbine shroud surface, which may be applicable to ceramic gas turbines. Three kinds of turbine shrouds are considered with a flat surface, a taper surface and a spiral groove surface opposite to the blades in an axial flow turbine of actual turbo-charger. Heat transfer measurements were performed for the experimental conditions of a uniform heat flux or a uniform wall temperature. The effects of the inlet flow angle, rotational speed, and tip clearance on the heat transfer coefficient were clarified under on- and off-design flow conditions. The mean heat transfer coefficient was correlated to the blade Reynolds number and tip clearance, and compared with an experimental correlation and measurements of a flat surface. A comparison was also made for the measurement of static pressure distributions.

Long-term administration of highly purified eicosapentaenoic acid ethyl ester prevents diabetes and abnormalities of blood coagulation in male WBN/Kob rats.

We investigated the effect of long-term administration of highly purified eicosapentaenoic acid ethyl ester (EPA-E), an n-3 polyunsaturated fatty acid, on the development of diabetes, insulin resistance, and abnormalities of blood coagulation in male WBN/Kob rats, a model of spontaneous diabetes mellitus. After 8-month oral EPA-E treatment, the incidence of diabetes at a dose of 0.1, 0.3, and 1.0 g/kg was 92%, 50%, and 17%, respectively. Its incidence was suppressed significantly and dose-dependently at a dose of 0.3 g/kg or higher compared with the rate (100%) for the vehicle control. Additionally, EPA-E significantly and dose-dependently decreased the elevation of plasma glucose after an oral glucose load and increased the glucose infusion rate (GIR) during the euglycemic insulin-glucose clamp test at a dose of 0.1 g/kg or higher compared with the vehicle control. Furthermore, EPA-E significantly and dose-dependently ameliorated coagulation-related parameters, including the prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen level, and factor II, V, VII, VIII, IX, X, XI, and XII and antithrombin III (AT III) activities, and fibrinolysis-related parameters, including plasminogen, tissue-type plasminogen activator (t-PA), alpha2-plasmin inhibitor (alpha2-PI), and plasminogen activator inhibitor (PAI), and also suppressed ADP- or collagen-induced platelet aggregation and the cholesterol to phospholipid (C/P) molar ratio in platelet membranes at a dose of 0.1 g/kg or higher. These data demonstrate multiple actions of the product in these laboratory animals. These include changes in platelet function, coagulation/fibrinolysis factors, plasma immunoreactive insulin secretion, and plasma glucose/insulin resistance.

Age-related changes in coagulation, fibrinolysis, and platelet aggregation in male WBN/Kob rats.

We investigated the age-related changes in blood coagulation, fibrinolysis, and platelet aggregation in male WBN/Kob rats, animals that exhibit spontaneously diabetes mellitus at more than 6 months of age. The rats aged 6 months or more showed significant hyperglycemia, hypoinsulinemia, and hyperlipidemia. As changes in coagulation parameters, the data indicated significant increases in factors II, V, VII, VIII, IX, X, and XII activities; a significant decrease in antithrombin III activity in rats more than 6 months of age; significant increases in fibrinogen level and factor XI activity; and significant decreases in prothrombin time and activated partial thromboplastin time in those more than 9 months of age. As changes in fibrinolytic parameters, the animals showed significant decreases in plasminogen and tissue-type plasminogen activator, and significant increases in alpha2-plasmin inhibitor and plasminogen activator inhibitor at more than 6 months of age. In addition, there were significant correlations between the plasma levels of coagulation/fibrinolytic markers and the 4-hour fasting glucose or lipids. Furthermore, they displayed significant increases in ADP- or collagen-induced platelet aggregation and in cholesterol/phospholipid molar ratio in platelets at more than 9 months of age. The increase in cholesterol/phospholipid ratio may be responsible for hyperaggregation of platelets in diabetic animals. These findings suggest that WBN/Kob rats are suitable for research on blood coagulation abnormalities in diabetes. However, further studies are needed to clarify the details of the mechanisms involved.

Long-term administration of highly purified eicosapentaenoic acid ethyl ester improves the dysfunction of vascular endothelial and smooth muscle cells in male WBN/Kob rats.

We investigated the effect of long-term administration of highly purified eicosapentaenoic acid ethyl ester (EPA-E), an n-3 polyunsaturated fatty acid, on the dysfunction of the endothelium and smooth muscle cells in male WBN/Kob rats, a model of spontaneous diabetes mellitus. After oral 8-month treatment with EPA-E, the agent significantly and dose-dependently increased the migration activity of vascular endothelial cells and also decreased 5-bromodeoxyuridine (BrdU) uptake by vascular smooth muscle cells at a dose of 0.1 g/kg or higher. In addition, there were significant correlations between the endothelial cell migration or smooth muscle cell proliferation and the 4-hour fasting glucose level. These findings suggest that EPA-E has a suppressive effect on thrombosis and atherosclerosis.

Gain of chromosomes 15 and 19 is frequent in both mouse hepatocellular carcinoma cell lines and primary tumors, but loss of chromosomes 4 and 12 is detected only in the cell lines.

Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial cell lines derived from normal livers of C57BL/6JxC3H/HeJ F(1) and C3H/HeJxC57BL/6J F(1) mice. In the primary tumors, non-random gain of chromosomes 15 and 19 was found in seven and five of 14 hepatocellular carcinomas, respectively. On the other hand, in the cases of both liver epithelial and hepatocellular carcinoma cell lines, frequent changes were loss of chromosomes 4 (4/9 cell lines) and 12 (3/9) as well as gain of chromosomes 15 (5/9) and 19 (4/9). These results indicate that the chromosomal gain is associated with both in vivo carcinogenesis and establishment of cell lines, while the loss is specific for the latter. PCR analysis using polymorphic microsatellite DNA markers revealed that the loss of chromosome 12 as well as chromosome 4 was much more frequent for the C57BL/6J hepatocarcinogenesis-resistant rather than the susceptible C3H/HeJ strain.

Self-renewal and differentiation of a basic fibroblast growth factor-dependent multipotent hematopoietic cell line derived from embryonic stem cells.

Despite the accumulation of informat on on the origin of hematopoietic stem cells, it is still unclear how these cells are generated in ontogeny. Isolation of cell lines equivalent to early embryonic hematopoietic progenitor cells can be helpful. A multipotent hematopoietic progenitor cell line, A-6, was isolated from H-1 embryonic stem (ES) cells. The self-renewal of A-6 cells was supported by basic-fibroblast growth factor (b-FGF) and their differentiation into definitive erythroid cells, granulocytes and macrophages was induced after co-culture with ST-2 stromal cells. A-6 cells were positive for the surface markers of hematopoietic stem cell, c-kit, CD31, CD34, Flt3/Flk2, PgP-1, and HSA, but were negative for that of the differentiated cells. Reverse transcription-polymerase chain reaction analysis showed that A-6 cells produced mRNA from SCL/tal-1 and GATA-2 genes. Among various cytokines examined, on y stem cell factor (SCF) and Flt3/Flk2 ligand (FL) supported the proliferation of A-6 cells instead of b-FGF. The FL, as well as b-FGF, supported the self-renewal of A-6 cells, whereas SCF induced differentiation into myeloid cells. A-6 cells will be useful for the characterization of hematopoietic progenitor cells derived from ES cells and provide a model system to realize the control mechanisms between self-renewal and different ation of hematopoietic stem cells.

Functional pulmonary surfactant deficiency and neonatal respiratory disorders.

The pathophysiology of functional deficiency of pulmonary surfactant in the neonatal respiratory disorders represented by MAS, hemorrhagic lung edema and ARDS was discussed. The removal of inhibitor(s) is the cardinal procedure for MAS and the lavage with surfactant solution seems to be promising. In case of replacement therapy, we should consider using a different dose compared to the one used in RDS due to lung immaturity, in order to optimize results.

Morphological characteristics of the dermal papillae in the development of pressure sores.

Mechanisms of skin break down in the development of human pressure sores are still unclear. This study was undertaken to clarify the morphological characteristics of the dermal papillae in the skin associated with pressure sores. Skin tissues were excised from the sacrum of a Japanese subject post mortem, where a superficial pressure sore had developed. Light microscopic and transmission and scanning electron microscopic examinations were performed. It was found that the atrophic, irregular contour and alignment of the dermal papillae were characteristic of the boundary area between healthy and damaged areas. In addition, a relatively dense network of collagen fibres in the papillary layer of the boundary area was observed when compared with the healthy area. These findings suggest that the morphological changes of the papillae observed in the boundary area affect microcirculation, impairing tissue viability by inhibiting nutritive blood supply and by accumulating metabolic byproducts which predispose to tissue damage.

Inhibition of gene expression from the human c-erbB gene promoter by a retroviral vector expressing anti-gene RNA.

Anti-gene is a potent inhibitor of transcriptional promoter activity and subsequent gene expression. This property has been exploited to suppress the expression of a variety of oncogenes for regulating tumor proliferation or viral activities. In this paper, we describe a novel retroviral vector designed to express human c-erbB anti-gene RNA and to reduce the promoter activity in the cells. Mouse fibroblast NIH3T3 cells were stably transfected with an expression construct containing a truncated human c-erbB gene promoter fused to the firefly luciferase reporter gene. Infection into these cells of the c-erbB anti-gene retroviral vector targeted to the 26 bp pyrimidine-rich element in the human c-erbB gene promoter resulted in a dose-dependent decrease in the luciferase activity of the cells. Retroviral vector expressing anti-gene RNA may be useful as an alternative program of gene regulation in the cells.

Roles of the Pas1 and Par2 genes in determination of the unique, intermediate susceptibility of BALB/cByJ mice to urethane-induction of lung carcinogenesis: differential effects on tumor multiplicity, size and Kras2 mutations.

The C3H/HeJ (C3H), A/J and BALB/cByJ (BALB) mouse strains are respectively resistant, sensitive and intermediate regarding the induction of lung tumors by urethane. The phenotypic difference between C3H and A/J is largely determined by the Pas1 (Pulmonary adenoma susceptibility 1) gene on chromosome 6, the A/J allele of which dominantly increases the tumor burden. We recently found that BALB mice possess a unique lung tumor resistance gene on chromosome 18, designated Par2 (Pulmonary adenoma resistance 2), which partially, but dominantly suppresses the sensitive phenotype of A/J mice (Oncogene 13: 1599-1604, 1996). It has, however, remained unclear why BALB mice carrying the Par2 gene are significantly more sensitive to urethane-induced lung carcinogenesis than C3H mice that have no dominant lung tumor resistance genes. In the present study, using (C3H x BALB)F1 x C3H backcross mice treated with urethane, we demonstrated that BALB mice possess the disease allele of the Pas1 gene despite their 15-fold more resistance relative to A/J mice (LOD = 22.6). The BALB Par2 allele only significantly reduced the mean lung tumor multiplicity (LOD = 4.4) in the backcross population carrying the BALB allele of Pas1, indicating that the intermediate BALB phenotype may at least in part be the result of interactions between these two dominant genes. While the BALB Pas1 allele increased both the mean multiplicity and size of lung tumors, the BALB Par2 allele affected only the mean tumor multiplicity, implying that they are involved in different stages of multi-step lung carcinogenesis. In addition, we found that 68% of lung tumors from the BALB Pas1-positive backcross mice contained activating point mutations of the Kras2 oncogene, tightly linked to the Pas1 locus, whereas these genetic alterations were absent in tumors from BALB Pas1-negative mice. The Par2 genotype exhibited no effect on this parameter. Since the activating point mutations were observed exclusively in the BALB allele as already reported with lung tumors in (C57BL/6J x BALB/cJ)F1 mice, BALB Pas1 or possibly Kras2 itself may confer selective growth advantage on the affected urethane-initiated lung lesions.

Loss of heterozygosity at loci on chromosome 4, a common genetic event during the spontaneous immortalization of mouse embryonic fibroblasts.

Spontaneously immortalized fibroblast cell lines derived from embryonic tissues of C3D2F1, mice were analyzed for loss of heterozygosity (LOH) at multiple chromosomal loci to identify candidate suppressor loci for immortalization. Among 47 simple sequence repeat (SSR) loci selected for screening, those on chromosome 4 exhibited an exceptionally high LDH incidence of up to 89%. Only four other chromosomes (8, 11, 12, and 18) showed LOH, with the highest incidence being 33%. To further localize candidate suppressor genes on mouse chromosome 4, detailed deletion mapping was performed with 18 cell lines and 14 SSR markers. The greatest LOH incidence (94%) was observed at the D4Mit14 locus located on distal chromosome 4, indicating that a major suppressor gene resides in this region. On the other hand, at the D4Mit77 locus, 30 cM proximal to the D4Mit14 locus, we found the SSR to be homozygously lost in 39% of the cell lines. Because the D4Mit77 is tightly linked to the tumor suppressor gene p16, for which homozygous deletion has been reported in various human tumor cell lines, we also examined our fibroblast cell lines for gross aberrations of the p16 gene by using the Southern blot method. The p16 gene was found to be homozygously deleted in 56% of the cell lines. Although this result implies that the p16 gene plays a role as a suppressor gene for immortalization, the combined incidence of LOH and homozygous deletion at the D4Mit77 locus was 72%, which is significantly lower than the observed incidence at the D4Mit14 locus. Consequently, we concluded that immortalization of mouse embryonic fibroblasts may involve more than one suppressor gene on chromosome 4.

Genetic dissection of susceptibility to murine ovarian teratomas that originate from parthenogenetic oocytes.

The LT/Sv mouse strain is characterized by its abnormally high incidence of spontaneous ovarian teratomas. These tumors have been shown to originate from parthenogenetic oocytes, which are spontaneously induced to divide. Both spontaneous parthenogenesis and ovarian teratomas are extremely rare for other mouse strains, including C57BL/6J. To identify the genes responsible for this unique phenotype of female LT/Sv mice, we performed linkage analysis of female (C57BL/6J x LT/Sv)F2 mice. A locus on chromosome 6 designated Ots1 (ovarian teratoma susceptibility) was identified as the single major locus that increases the frequency of teratomas in a semidominant manner.

Successful hematopoietic reconstitution by transplantation of umbilical cord blood cells in a transfusion-dependent child with Diamond-Blackfan anemia.

A 4-year-old boy with Diamond-Blackfan anemia and a history of multiple transfusions underwent umbilical cord blood transplantation from his HLA-identical female sibling born by vaginal delivery at 38 weeks. The patient was prepared with busulfan, cyclophosphamide and antilymphocyte globulin. Methotrexate and cyclosporin A were given for the prophylaxis of GVHD. Regimen-related toxicity was not observed and successful engraftment occurred, including the erythroid series. No evidence of acute or chronic GVHD has been observed for 14 months after transplantation. This is the first case of successful umbilical cord blood transplantation to a patient with Diamond-Blackfan anemia.