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.     

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.     

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.     

Morphological changes in mouse sublingual gland parenchyma subjected to chorda tympani resection

Morphological changes in the mouse sublingual gland parenchyma subjected to parasympathetic nerve block were investigated. Mice were subjected to unilateral resection of the chorda tympani, near its point of joining with the lingual nerve. After 1, 2, 3, 5, 10 or 20 weeks, the mice were killed and their sublingual glands were removed and processed for light and electron microscopy. Two weeks after resection, the space between the adjoining lobules of the glands on the treated side began to be expanded, and by 10 weeks were 10 times the size of the spaces in the glands of the untreated mice. Three weeks after resection, the lobule area decreased to about 72% of the area of glands in the untreated mice and the acinus area to about 52%. However, no significant difference was seen between the numbers of acini in each group. Electron microscopy showed that the glands on the treated side contained fewer secretory granules than the glands in the untreated mice, though there was no difference in size. Neither the lobules of the glands on the treated side nor those of the glands of the untreated mice contained many TUNEL-positive cells. These findings suggest that following parasympathetic nerve resection, mouse sublingual gland acinar cells undergo atrophy with a reduction size rather than cell death.     

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.     

Gain-of-function polymorphism in mouse and human Ltk: implications for the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE), a complex multigenic disease, is a typical antibody-mediated autoimmune disease characterized by production of autoantibodies against a variety of autoantigens and immune complex-type tissue inflammation, most prominently in the kidney. Evidence suggests that genetic factors predisposing to aberrant proliferation/maturation of self-reactive B cells initiate and propagate the disease. In SLE-prone New Zealand Black (NZB) mice and their F1 cross with New Zealand White (NZW) mice, B cell abnormalities can be ascribed mainly to self-reactive CD5+ B1 cells. Our genome-wide scans to search for susceptibility genes for aberrant activation of B1 cells in these mice showed evidence that the gene, Ltk, encoding leukocyte tyrosine kinase (LTK), is a possible candidate. LTK is a receptor-type protein tyrosine kinase, belonging to the insulin receptor superfamily, and is mainly expressed in B lymphocyte precursors and neuronal tissues. Sequence and functional analyses of the gene revealed that NZB has a gain-of-function polymorphism in the LTK kinase domain near YXXM, a binding motif of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). SLE patients also had this type of Ltk polymorphism with a significantly higher frequency compared with the healthy controls. Our findings suggest that these polymorphic LTKs cause up-regulation of the PI3K pathway and possibly form one genetic component of susceptibility to abnormal proliferation of self-reactive B cells in SLE.     

Immunohistochemical analysis of thymidylate synthase, p16(INK4a), cyclin-dependent kinase 4 and cyclin D1 in colorectal cancers receiving preoperative chemotherapy: significance of p16(INK4a)-mediated cellular arrest as an indicator of chemosensitivity to 5-fluorouracil

High expression of thymidylate synthase (TS) is allegedly associated with the chemoresistance to 5-fluorouracil (5-FU) in colorectal cancers. However, low TS expression does not necessarily imply chemosensitivity. Inactivation of p16(INK4a) correlates with poor prognosis in various cancers. We immunohistochemically evaluated the relationship between the expression of TS, p16(INK4a), CDK4 and cyclin D1 and the effect of 5-FU-based chemotherapy in colorectal cancers. After antigen retrieval, immunoperoxidase staining was performed on the paraffin-embedded, biopsy and surgical specimens of 37 advanced colorectal cancers preoperatively treated with peroral administration of 5-FU derivatives. As a control group, 31 colorectal cancers without preoperative treatment were analyzed. High TS expression was found in 23 (74%) of 31 tumors resected from histological non-responders and in 19 (61%) of 31 controls but in none of six responders. High p16(INK4a) expression was seen in 83% of the responders, 52% of the non-responders and 32% of the controls. The TS-low/p16(INK4a)-high phenotype was noted in 83% of the responders, but only in 3% of the non-responders (P = 0.0001). Induction of p16(INK4a) expression after chemotherapy was predominantly seen in the responders. Neither CDK4 nor cyclin D1 expression was related to the chemotherapeutic effects. In conclusion, the combination of low expression of TS and induction of p16(INK4a) after chemotherapy can be important indicators of the sensitivity to 5-FU-based chemotherapy in colorectal cancers.     

Possible role of hepatocyte growth factor in regeneration of human peritoneal mesothelial cells

Human peritoneal mesothelial cells (HPMCs) play an important role in peritoneal functions. During long term peritoneal dialysis, it has been reported that HPMCs are damaged by high glucose solution via the signal of transforming growth factor (TGF)-beta1 produced by HPMCs. In this study, we focused on the effect of hepatocyte growth factor (HGF), known as an anti-fibrotic and anti-TGF-beta1 agent, on HPMCs damaged by high glucose solution. HPMCs were isolated from specimens of the omentum from nonuremic patients after informed consent had been obtained. After confirming adhesion for 6 hours, 100 microL of DMEM with 0.5%FCS were added at different concentrations (D-glucose; 6, 30 mM) with or without HGF (10, 30, 100 ng/mL) for 48 hours. We examined the effects of a high concentration of glucose and then focused on following four critical points: 1) the production of HGF from HPMCs exposed to a high concentration of glucose, 2) the expression of c-Met on HPMCs, 3) the viability of those cells, and 4) matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-2 (TIMP-2). The following significant changes are described herein: high glucose solution and TGF-beta1 i) decreased HGF production from HPMCs and ii) up-regulated expression of c-Met on HPMCs, and addition of HGF iii) restored viability of HPMCs damaged by glucose, iv) suppressed TGF-beta1 production by HGF, and v) induced up-regulation of MMP-2 and decreased TIMP-2 production by HPMCs. Levels of HGF decreased by high concentrations of glucose in the peritoneal cavity may induce the loss of HPMCs and thereby result in peritoneal fibrosis. These results suggest that HGF is an effective agent in the regeneration of peritoneal membrane damaged by high glucose solution.