Development of an optimized 5-stage protocol for the in vitro preparation of insulin-secreting cells from mouse ES cells

In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage. GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 10⁶ cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.     

Lower extremity muscle activity during different types and speeds of underwater movement

To compare the activity of lower extremity muscles during land walking (LW), water walking (WW), and deep-water running (DWR), 9 healthy young subjects were tested at self-selected low, moderate, and high intensities for 8 sec with two repetitions. Surface EMG electrodes were placed on the tibialis anterior (TA), soleus (SOL), medial gastrocnemius (GAS), rectus femoris (RF), and biceps femoris (BF). During DWR, the SOL and GAS activities were lower than LW and WW. The BF activities were higher during DWR than LW and WW. It was considered that the lower activity of SOL and GAS depended on water depth, and higher activity of BF occurred by greater flexion of the knee joint or extension of the hip joint during exercise.     

Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.     

ROCK inhibitor Y-27632 attenuates stellate cell contraction and portal pressure increase induced by endothelin-1

By using a selective ROCK inhibitor Y-27632, the role of Rho-ROCK signaling in the function of hepatic stellate cells in culture was studied. Stellate cells maintained the "star-like" configuration of the quiescent stage in the presence of Y-27632, while the expression of smooth muscle alpha-actin and PDGF receptor beta was not affected by the agent. Serum-stimulated migration of the cells was significantly suppressed by Y-27632. The contraction of stellate cells induced by 5 nM endothelin-1 was attenuated by the agent in a dose-dependent manner. Formation of F-actin stress fibers and phosphorylation of myosin light chain was apparently reduced by Y-27632 even under the stimulation with endothelin-1. On the other hand, ex vivo liver perfusion experiment revealed that endothelin-1 (2 nM)-induced increase of portal vein constriction was almost completely inhibited by 20 microM Y-27632 with a concomitant improvement of hepatocyte degeneration. These results suggest that ROCK is one of the key regulators of stellate cell motility and that the clinical application of ROCK inhibitors such as Y-27632 should be considered in the reduction of portal hypertension in liver fibrosis and cirrhosis.     

In vivo gene transfer of an extracellular domain of platelet-derived growth factor beta receptor by the HVJ-liposome method ameliorates bleomycin-induced pulmonary fibrosis

A number of investigators have reported augmented expression of PDGF in lungs with idiopathic pulmonary fibrosis (IPF) or with other types of pulmonary fibrosis. To accomplish such a regulation of PDGF activity, we constructed an expression plasmid of the extracellular domain of PDGF receptor beta chain (XR), which lacks intracellular tyrosine kinase domain and transmembrane portions, and estimated the therapeutic effects of XR gene transfer through the trachea on bleomycin-induced lung fibrosis of C57BL/6 mice using the hemagglutinating virus of Japan(HVJ)-liposome method. The XR gene transfer ameliorated the increases in the wet weight and hydroxyproline content and the histopathologic changes of the lung induced by bleomycin. These findings suggest that PDGF plays a crucial role in the pathogenesis of pulmonary fibrosis, and that XR gene transfer using the HVJ-liposome method may limit the progression of pulmonary fibrosis.     

HVJ-envelope vector for gene transfer into central nervous system

To overcome some problems of virus vectors, we developed a novel non-viral vector system, the HVJ-envelope vector (HVJ-E). In this study, we investigated the feasibility of gene transfer into the CNS using the HVJ-E both in vitro and in vivo. Using the Venus reporter gene, fluorescence could be detected in cultured rat cerebral cortex neurons and glial cells. In vivo, the reporter gene (Venus) was successfully transfected into the rat brain by direct injection into the thalamus, intraventricular injection, or intrathecal injection, without inducing immunological change. When the vector was injected after transient occlusion of the middle cerebral artery, fluorescence due to EGFP gene or luciferase activity could be detected only in the injured hemisphere. Finally, luciferase activity was markedly enhanced by the addition of 50 U/ml heparin (P<0.01). Development of efficient HVJ-E for gene transfer into the CNS will be useful for research and clinical gene therapy.     

Fasting-induced suppression of pulsatile luteinizing hormone secretion is related to body energy status in ovariectomized goats

The effect of energy status on the response of luteinizing hormone (LH) pulse frequency to acute short-term energy deficiency created by fasting in estradiol-treated ovariectomized Shiba goats was studied in two experiments. In experiment 1, eight goats whose mean body weight (BW) was 25.6 +/- 5.8 (mean +/- S.D.)kg were fed 500 g hay cubes daily for 1 week. Then they were fasted for 3 days. Blood samples were collected for 4 h at 6 min intervals on the last day of feeding, first, second and third day of fasting for LH analysis. The goats were divided into light (<24 kg, n = 4) and heavy (> or =24 kg, n = 4) groups for data analysis. There was no difference in LH pulse frequency between the last day of feeding and each day of fasting in the heavy group. LH pulse frequency was significantly (P < 0.05) suppressed on the second day (3.3 +/- 1.3 pulses/4 h) and on the third day (2.3 +/- 1.9 pulses/4 h) relative to the day prior to fasting (4.8 +/- 1.5 pulses/4 h) in the light group. In experiment 2, BW plus a body mass index (gBMI: (body weight (kg)/withers height (m)/body length (m)) x 10) were measured to define energy status. Nine goats (BW, 25.6 +/- 5.8 kg) were fed 500 g hay cubes daily for a week and then fasted for 3 days. Then they were divided into two groups offered either a maintenance (n = 4) or a restricted (n = 5) level of feeding for 4 weeks. The restricted level of feeding was 30% of maintenance requirement based on the BW recorded weekly. The feeding level was then adjusted to maintain BW for a further week followed by 3 day fasting for restricted animals. Blood samples were collected for 6 h at 10 min intervals on the day prior to fasting and on third day of fasting before and after the dietary manipulation. BW (26.6 +/- 2.2 to 26.8 +/- 3.8 kg) and gBMI (8.4 +/- 0.4 to 7.8 +/- 0.3) remained constant over the period prior to fasting for the maintenance animals but were significantly lower (P < 0.05) after 4 weeks for the restricted goats (BW, 26.3 +/- 2.1 to 21.5 +/- 2.4 kg; gBMI, 8.4 +/- 0.9 to 6.9 +/- 0.7). There was no significant difference in the LH pulse frequency between feeding and fasting day in both sampling periods in the maintenance group. In the restricted group, LH pulse frequency was not suppressed by fasting in the first sampling period (6.8 +/- 2.9 to 5.2 +/- 2.5 pulses/6 h), whereas it tended to be suppressed (4.8 +/- 3.1 to 1.6 +/- 2.3 pulses/6 h; P < 0.06) and was significantly (P < 0.05) correlated to body weight (r = 0.70) and gBMI (r = 0.81) after the dietary manipulation. These results suggest that the suppressive effect of short-term energy restriction (fasting) on pulsatile LH secretion is related to body energy status.     

Causal relationship between the loss of RUNX3 expression and gastric cancer

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.     

TNF combined with IFN-alpha accelerates NF-kappaB-mediated apoptosis through enhancement of Fas expression in colon cancer cells

Immunostaining and EMSA revealed that NF-kappaB was activated strongly by TNF/IFN-alpha compared to TNF alone in a human colon adenocarcinoma cell line, RPMI4788. Although inhibition of activated NF-kappaB, by using an NF-kappaB decoy, reduced cell viability after treatment with TNF only, NF-kappaB decoy resulted in recovery of cell viability after TNF/IFN-alpha treatment. Caspase-3 activity was increased in cells induced by TNF/IFN-alpha, while suppression of caspase-3 activity was observed in cells transfected with NF-kappaB decoy and then treated by TNF/IFN-alpha. On the other hand, Fas expression was strongly enhanced by TNF/IFN-alpha, and inhibition of TNF/IFN-alpha-induced NF-kappaB activation, by using NF-kappaB decoy, decreased Fas expression. Cell viability and caspase-3 activity decreased in cells treated with TNF/IFN-alpha and anti-FasL antibody. Taken together, our findings suggest that activated NF-kappaB induced by the crosstalk between TNF and IFN-alpha is a novel pro-apoptotic signal acting via enhancement of Fas expression.