Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.     

Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication

The Rad17-replication factor C (Rad17-RFC) and Rad9-Rad1-Hus1 complexes are thought to function in the early phase of cell-cycle checkpoint control as sensors for genome damage and genome replication errors. However, genetic analysis of the functions of these complexes in vertebrates is complicated by the lethality of these gene disruptions in embryonic mouse cells. We disrupted the Rad17 and Rad9 loci by gene targeting in the chicken B lymphocyte line DT40. Rad17-/- and Rad9-/- DT40 cells are viable, and are highly sensitive to UV irradiation, alkylating agents, and DNA replication inhibitors, such as hydroxyurea. We further found that Rad17-/- and Rad9-/- but not ATM-/- cells are defective in S-phase DNA damage checkpoint controls and in the cellular response to stalled DNA replication. These results indicate a critical role for chicken Rad17 and Rad9 in the cellular response to stalled DNA replication and DNA damage.     

Angiogenic endothelium-specific nestin expression is enhanced by the first intron of the nestin gene

Nestin is a member of intermediate filaments abundantly expressed in neural stem cells and glioblastomas. The nestin gene has four exons and three introns, and neural cell-specific expression is regulated by the second intron. We previously reported that nestin was invariably detected in the tumor endothelium in gliomas even though tumor cells were negative for nestin. In the present study, we further confirmed nestin immunostaining in tumor endothelium of a variety of common cancers, including lung, stomach, colon, and cervical carcinomas. We examined an endothelium-specific regulator using human umbilical vein endothelial cells (HUVECs) and human glioblastoma-derived U251 cells. In a luciferase reporter assay, the first intron plus 5' upstream promoter (5'UP) gave the highest activity, followed by 5'UP, and the second intron plus 5'UP. However, the assay values were much lower by HUVEC extracts than by U251 cell extracts. Although green fluorescent protein expression was positive over all U251 cells under either the first intron, second intron, or ubiquitously active CAG promoter, the fluorescence in HUVECs was limited to a few cells even under the first intron. This difference came from the growth feature of HUVECs which exhibit growth arrest by contact inhibition. We found that the nestin expression was specific to proliferative endothelium, by using proliferation markers in hemangioblastomas and in situ hybridization. Using an endothelial tube formation assay, tyrosine kinase domain-deleted VEGF receptor KDR effectively abolished the tube formation under the first intron. We suggest that the nestin expression in tumor endothelium is enhanced by the first intron.     

Myopathy phenotype in transgenic mice expressing mutated PABPN1 as a model of oculopharyngeal muscular dystrophy

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD)is a late-onset disorder characterized clinically by progressiveptosis, dysphagia and limb weakness, and by unique intranuclearinclusions in the skeletal muscle fibers. The disease is causedby the expansion of a 10-alanine stretch to 12–17 alanineresidues in the poly(A)-binding protein, nuclear 1 (PABPN1;PABP2). While PABPN1 is a major component of the inclusionsin OPMD, the exact cause of the disease is unknown. To elucidatethe molecular mechanism and to construct a useful model fortherapeutic trials, we have generated transgenic mice expressingthe hPABPN1. Transgenic mice lines expressing a normal hPABPN1with 10-alanine stretch did not reveal myopathic changes, whereaslines expressing high levels of expanded hPABPN1 with a 13-alaninestretch showed an apparent myopathy phenotype, especially inold age. Pathological studies in the latter mice disclosed intranuclearinclusions consisting of aggregated mutant hPABPN1 product.Furthermore, some TUNEL positive nuclei were shown around degeneratingfibers and a cluster of it in the lesion in necrotic musclefibers. Interestingly, the degree of myopathic changes was moreprominent in the eyelid and pharyngeal muscles. Further, muscleweakness in the limbs was apparent as shown by the fatigabilitytest. Nuclear inclusions seemed to develop gradually with aging,at least after 1 week of age, in model mouse muscles. We establishedthe first transgenic mouse model of OPMD by expressing mutatedPABPN1, and our model mice appear to have more dramatic alternationsin myofiber viability. ^* To whom correspondence should be addressed. Tel: +81 963736083; Fax: +81 963736599; Email: yamamura{at}gpo.kumamoto-u.ac.jp     

Sensorless estimation of pressure head and flow of a continuous flow artificial heart based on input power and rotational speed

The present study has proposed a new method for estimating the pressure head (P(t)[mm Hg]) and flow (Q(t)[L/min]) of a centrifugal pump on the basis of voltage (V(t)[V]), current (I(t)[A]), and rotational speed (N(t)[k(rpm)]) of the DC motor for a pump without any additional sensors. In the proposed estimation method, two auto-regressive exogenous (ARX) models are employed. One ARX model has an output, P(t) or Q(t), and three inputs, VI(t) = V(t)I(t) and N(t) and the steady state gain (K) of the system from VI(t) to N(t). It can be assumed that K may include the information on viscosity of blood. The coefficient parameters of this ARX model are identified in an off-line fashion before implantation of the pump. After implantation, P(t) or Q(t) is estimated by the same ARX model with the already identified parameters. The other ARX model is used to identify Kon the basis of VI(t) and N(t) in an on-line fashion every time the viscosity of blood may change. In the experiment, a mock circulatory system consisting of a centrifugal pump and a reservoir with 37% glycerin or water was employed. The root mean square error between measured Q(t) and its estimate obtained from the proposed method was 1.66L/min. On the other hand, a different method based on a single ARX model with inputs of VI(t) and N(t), but without the additional input of K, yielded the corresponding estimation error of 2.22L/min. This means that the proposed method can reduce its estimation error by about 25% in comparison with a method that cannot cope with the change in blood viscosity.     

Mechanisms of inactivation of the p16INK4a gene in leiomyosarcoma of soft tissue: decreased p16 expression correlates with promoter methylation and poor prognosis

The p16INK4a tumour suppressor gene, encoding p16 protein, plays a crucial role in regulation of the G1 cell-cycle phase. To investigate the potential role of p16 in soft tissue leiomyosarcoma (LMS), an immunohistochemical analysis was performed of 77 LMSs for p16 expression. Decreased expression of the p16 protein was identified in 25 of 77 LMSs (32%). Decreased expression of p16 correlated significantly with large tumour size (p=0.0038). In a univariate analysis, large tumour size and decreased expression of p16 were statistically significant adverse prognostic factors (p=0.025 and p=0.0021, respectively). In a multivariate analysis including conventional clinicopathological parameters, decreased expression of p16 protein was revealed as the only independent unfavourable prognostic factor (p=0.012). To elucidate the mechanisms of inactivation of the p16INK4a gene, 49 LMSs for which genomic DNA was available were examined; analysis for homozygous deletion, mutation, and promoter hypermethylation was conducted using differential PCR, PCR-SSCP, and methylation-specific PCR, respectively. Promoter hypermethylation was detected in 11 of 49 LMS cases (22%); homozygous deletion was detected in 3 of 49 cases (6%); and mutation was not recognized in any of the cases studied. Eight of 15 cases (53%) with decreased expression of p16 protein revealed methylation of the p16INK4a gene promoter. Promoter hypermethylation correlated closely with decreased expression and poor prognosis (p=0.0014 and p=0.0088, respectively). These results suggest that decreased expression of p16 protein can be considered as an independent reliable prognostic parameter in patients with soft tissue LMS. Furthermore, promoter methylation was more frequent than either homozygous deletion or mutation in this tumour, and promoter methylation was also shown to have a strong association with inactivation of the p16INK4a gene.     

Genetic and epigenetic alterations of the PTEN gene in soft tissue sarcomas

The PTEN/MMAC1 ( PTEN ) gene was identified as a tumor suppressor gene encoding a cytoplasmic protein that controls cellular processes. To investigate the potential role and the alteration of the PTEN gene in soft tissue sarcomas (STSs), we searched for homozygous deletion and promoter hypermethylation in a series of 48 STSs that was composed of malignant fibrous histiocytoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, including 2 cases with a mutation that we previously reported; differential polymerase chain reaction and methylation-specific polymerase chain reaction, respectively, were used for the analyses. Furthermore, to determine whether PTEN gene alterations are involved in the down-regulation of PTEN expression, we examined the expression of PTEN protein in 38 cases in which paraffin-embedded tissues were available for immunohistochemical analysis. In addition to our previous results showing that 2 (4%) of 51 cases had a PTEN mutation, promoter methylation was recognized in 6 (13%) of 48 cases, and homozygous deletion was detected in 1 (2%) of 48 cases in the current study. Of 6 cases with promoter methylation of PTEN gene, 5 were malignant peripheral nerve sheath tumor. Decreased expression of PTEN protein was recognized in 11 (29%) of 38 STS cases. Of 9 cases with PTEN alterations (6 cases with promoter methylation, 2 with mutation, and 1 with homozygous deletion), 3 (33%) showed decreased expression of PTEN protein. Furthermore, decreased expression of the PTEN gene showed a statistically significant correlation with high MIB-1 labeling index in 38 STS cases examined ( P = .0441). In conclusion, promoter methylation and homozygous deletion of the PTEN gene were found to be relatively rare events in cases of STS, as is mutation of the gene. Of 9 cases with a PTEN alteration, 3 (33%) showed a decrease in PTEN expression, indicating that PTEN gene alterations seem to play a minor role in the inactivation of PTEN in these tumors. Furthermore, although a further detailed analysis of a larger number of cases is still necessary, the present results suggest that PTEN expression may be a useful indicator of cell proliferation in patients with STS.     

Electron-dense deposition patterns and the outcomes of idiopathic membranous nephropathy in Japanese

A considerable diversity in prognosis is seen with membranous nephropathy (MN). In terms of pathological findings, the presence of tubulointerstitial lesions was emphasized as a poor prognostic factor. However, the glomerular factors affecting the long-term outcome of idiopathic human MN have remained unclear. We reviewed the initial clinicopathological factors affecting the primary and secondary outcomes in 105 patients with primary MN, as well as reviewing previous reports. Based on electron microscopic (EM) findings, we could divide patients into two subtypes and one subgroup; i.e., homogeneous type with a synchronous phase of electron-dense deposits, with large dense deposits (deep subgroup) and heterogeneous type with various phases of dense deposits. The homogeneous type showed no endstage renal failure, and had earlier remission as compared with the heterogeneous type. For the secondary outcome, heterogeneous type and deep subgroup were also independent risk factors. However, there was no significant difference in the final primary or secondary outcome for any treatment subgroups. These results indicated that our category of EM findings was a beneficial marker of the primary and secondary outcomes in MN; the homogeneous type of MN with synchronous phase of electron-dense deposits (except for the "deep" subgroup) had a good outcome.     

Difference in the expression of three tight junction proteins, barmotin, occludin, and ZO-1, in phenotypically different human colon cancer cell lines

Epithelioid disorganization is a hallmark of gastrointestinal cancers and is believed to be associated with malignant phenotypes such as invasiveness and the potentiality for metastasis. Although tight junctions (TJs) are known to be crucial for the maintenance of polarized organization of the gastrointestinal epithelium, changes in the TJ proteins in human cancers have not yet been fully elucidated. In this report, we investigated the expression and localization of three TJ proteins-barmotin (7H6 antigen), occludin, and ZO-1-in three phenotypically different human colon cancer cell lines exhibiting differnt grades of epithelioid organization. All three proteins were localized at the most apical part of the cell border corresponding to the site of TJs in T₈₄ cells, in which epithelioid organization was well preserved. In contrast, in COLO320DM cells, which showed no epithelioid phenotypes, occludin was not detectable at either the protein or mRNA level, although barmotin and ZO-1 were present in the cytoplasm. In the third cell line, DLD-1, which showed an epithelioid phenotype intermediate between T₈₄ and COLO320DM, aberrant expression of occludin was found in the basolateral cell membrane. On the other hand, barmotin was present in the cytoplasm, whereas ZO-1 was localized at the cell border. These observations showed that changes in the expression of TJ proteins occur in close correlation with epithelioid disorganization in human colon cancers.