Biomedical Research

Online ISSN: 1880-313X
Publications
Identifying the TCR Vα and Vβ repertoires for the 37F8 cells purified with A24-modified WT1 tetramer. TCR α and β genes were amplified with a thermal cycler with PCR coding region-specific primer pairs for TRAC and various TCR α chains, or TRBV5-1, and TCRBC1/2. (A) TCR α and β PCR products were separated on agarose gels and visualized by UV illumination. TRAV12-2, TRAV12-3, and TRAV41 genes were detected using PCR with a TCR α primer-set. (B) Illustration of TCR α and β chains of the WT1-specific 37F8 cells. The 37F8 cells had two types of α chains (A12-3, A41) and one β chain (B5-1).  
Establishing WT1-specific CTL lines from PBMCs by MLPC. Upper panel (A) shows the CTL epitopes of A24- modified and natural WT1 tetramers used in this study. Lower panel (B) shows representative results for WT1-specific CTLs (37F8) induced by MLPC with A24-modified or natural WT1 peptides. The 37F8 cells were stained with A24-modified or natural WT1 tetramers. HIV tetramer was used as a negative control. The percentages of tetramer + cells among CD8 + T cells are indicated.  
Article
Wilms' tumor gene 1 (WT1) has been proposed as an attractive target for cancer immunotherapy. A natural 9-mer peptide (CYTWNQMNL), which bound to human leukocyte antigen (HLA)-A∗24:02, was identified from among WT1-specific cytotoxic T lymphocyte (CTL) epitopes. This natural WT1 CTL epitope peptide was further modified (CMTWNQMNL) to enhance its binding affinity to HLA-A∗24:02. This modified WT1 CTL epitope peptide was superior to the natural peptide for inducing HLA-A∗24:02-restricted WT1-specific CTLs. Here we induced several WT1 CTLs that reacted with both modified and natural WT1 tetramers from peripheral blood mononuclear cells. Then, T-cell receptor (TCR) genes were isolated from these WT1 CTLs to determine their Vα and Vβ usage. These TCR genes were transduced into human T lymphoma cells to establish a stable cell line, SK37, which expressed a WT1-specific TCR. We confirmed that SK37 cells reacted with both modified and natural WT1 tetramers, which indicated that SK37 cells could be a useful tool for WT1 tetramer reagent quality assurance. One the basis of these findings, we propose that this WT1 tetramer, which was quality-assured using established SK37 cells, will contribute to reliable immunomonitoring of tumor-specific CTL responses of cancer patients who receive WT1-targeted cancer vaccine therapy or TCR-gene therapy.
 
Article
OSU03012, a novel celecoxib derivative, has been shown to inhibit proliferation and induce apoptosis in numerous cancer cell lines. However, not much is known about its influence on cell volume regulation and cardiac function in the mammalian heart. We examined the effects of OSU-03012 on cell volume and action potentials in mouse ventricular cells. Video image analysis showed that cell volume increased on application of OSU-03012 in a dose-dependent manner. The action potential duration (APD) at 50% and 90% repolarization (APD(50) and APD(90) respectively) as well as the resting membrane potential (RMP) were measured in current-clamp experiments. OSU-03012 had little effect on APD(50) and RMP but induced approximately 30% shortening of APD(90). These results for cell volume and AP are similar to those in cells under ischaemia/hypoxia, and we confirmed that the shortening of APD(90) was almost completely recovered by glibenclamide, a potent inhibitor of ATP-sensitive potassium channels.We concluded that OSU-03012 may lead to cell swelling and shortening of AP via reduced ATP production in mouse ventricular cells.
 
Article
The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 (DVD), is a potent inducer of cell differentiation and inhibits proliferation of cells such as human promyelocytic leukemia HL-60 cells. In the present study, we examined the effects of DVD on the expression of exportin-1 and exportin-t, which play essential roles in the transport of mRNA and tRNA, respectively. The results of reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction indicated that DVD down-regulated the gene expression of these exportins. Western blotting revealed the decreased production of these proteins in DVD-treated cells. Thus, the present findings of decreased expression of exportin-1 and exportin-t induced by DVD can be correlated to inhibition of the proliferation of HL-60 cells.
 
Article
Using a DNA microarray, we analyzed about 16,600 genes for changes in expression associated with the differentiation of human promyelocytic leukemia HL-60 cells induced by 1alpha,25-dihydroxyvitamin D3 (DVD). Many of the up-regulated genes could be correlated to differentiation-associated changes toward a monocyte/macrophage lineage, and many down-regulated genes could be correlated to repressed cell growth. The present study revealed the down-regulated gene expression of importins and exportins 1, 5, 7, and exportin-tRNA. Thus, the present results confirmed our previous findings of down-regulation of exportin 1 and exportin-tRNA by DVD. Gene expression of exportin 6 is suggested to be regulated differently from that of exportins 1, 5, 7, and exportin-tRNA. The down-regulation of nuclear transport factors may be deeply associated with the differentiation of HL-60 cells induced by DVD.
 
Distribution of miR-219 and miR-124 in the central nervous system. The expression profiles of miR-219 and miR-124 in various human brain regions and cultured cells are shown. EC, endothelial cells.
Plasma miR-124 was increased in the rat MCAO model. A: Plasma levels of miR-124 in rats that underwent MCAO (n = 5, open circles) or sham-operated rats (n = 5, closed circles) were measured by real-time RT-PCR. The copy number per 100 μL of plasma was calculated, and plotted against the time course. Data below the detection limit (C T , approximately 31.5) are represented as 60 copies per 100 μL of plasma. Data are presented as the mean ± SD, and analyzed by multiple analyses of variance (MANOVA). *, P < 0.05; *** < 0.0001. B and C: Plasma levels of miR-451 and 5S rRNA were measured by real-time RT-PCR. Relative expression level (2 35-CT ) was plotted against the time course.
Analysis of correlation between the cerebral infarct size and plasma miR-124 level. A : A typical TTC staining pat- tern of the brain of an MCAO rat. B : Linear regression analysis. Size of the cerebral infarction was plotted against plasma miR-124 concentration. R 2 and P values are shown in the figures (n = 13). TTC, 2,3,5-triphenyltetrazolium chloride; MCAO, 
Article
MicroRNAs (miRNAs) are endogenous small RNAs that play an important role in various physiological processes by downregulating target genes. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In this study, miRNA array analysis in various tissues showed that miR-124 is almost exclusively expressed in the central nervous system and neuronal cells, suggesting that it might be useful as a potential biomarker for neurological diseases. We examined whether plasma concentrations of brain-specific miRNA can serve as a biomarker for cerebral infarction, where the cerebral infarction was modeled by middle cerebral artery occlusion (MCAO) in the rat. Plasma concentrations of miR-124 were significantly elevated at 6 h, and remained elevated at 48 h after MCAO introduction. Thus, plasma concentration of miR-124 provides a promising candidate biomarker for early detection of cerebral infarction.
 
Article
The ICGN mouse strain is a glomerulosclerosis (GS) model that shows significant proteinuria, podocyte morphological abnormalities and increased extracellular matrix accumulation in the glomeruli, which represent the final common pathology associated with a variety of kidney diseases leading to end-stage renal failure. Previously, we demonstrated that GS in ICGN mice can be attributed to the deletion mutation of the tensin2 (Tns2) gene (Tns2(nep)). Further, the C57BL/6J (B6) mouse is resistant to GS caused by this mutation. 129/Sv is also a popular strain; however, its susceptibility to GS has not been defined. Thus, to determine whether 129/Sv is resistant or susceptible to GS, we produced a congenic strain carrying Tns2(nep) on the 129(+Ter)/Sv (129T) background. 129T congenic mice (129T-Tns2(nep)) did not exhibit albuminuria, renal anemia, increases in BUN, or any severe pathological changes until at least 16 weeks of age. These results indicate that 129T is resistant to GS. Although their usage in biomedical studies is already widespread, 129/Sv mice may afford a late-onset and unique strain applicable to kidney disease research.
 
Effects of MU and TPA on O-GlcNAcylation. Human skin fibroblasts were cultured for 48 h with 0.1 or 1.0 mM MU, or 0.1 or 1.0 μg/mL TPA, and the cell extracts were analyzed by western blotting with anti-O-GlcNAc antibody.  
Effect of MU and TPA on the phosphorylation of HAS2. (A) Human skin fibroblasts were cultured in the presence of 0.1 μg/mL TPA, 1 mM MU, and their combination for 48 h, and the cell extracts were divided into a phosphorylated protein fraction (IMAC+) and a non-phosphorylated protein fraction (IMAC−) by IMAC column. Each fraction was analyzed by western blotting with anti-HAS2 antibody. (B) The ratios of HAS2 in phosphorylated and non-phosphorylated protein fractions in (A) were quantified by Image Lab Software (Bio-Rad). Values are expressed as the total of dual bands in each fraction. Open bar, HAS2 in nonphosphorylated protein fraction (IMAC−); closed bar, HAS2 in phosphorylated protein fraction (IMAC+).  
Effect of MU and TPA on the synthesis of hyaluronan and the gene expression of HAS2. (A) Human skin fibroblasts were cultured in the presence of TPA, MU, and the combination of both as described in the figure for 48 h, and hyaluronan in the media was determined. Values are expressed as means of two separate experiments, each of them determined in duplicates. (B) After cultivating the cells in the presence of TPA, MU, and their combination for 24 h, total RNA was extracted from the cell layers and RT-PCR for HAS1, HAS2, and HAS3 was performed. (C) Real-time PCR was performed for HAS2 using the RNA as described in (B). Values are from three independent experiments, each of them determined in triplicate. Differences among values obtained from experiments of the treatment groups were compared with the untreated control group by Student t test (* indicates P < 0.05 vs TPA− and MU−).  
Article
The effect of 4-methylumbelliferone (MU), a hyaluronan synthase-suppressor, on O-linked β-Nacetylglucosaminylation (O-GlcNAcylation) was investigated in cultured human skin fibroblasts, and we found that MU stimulated O-GlcNAcylation of the cellular proteins. Since O-GlcNAcylation affects protein phosphorylation via Ser/Thr kinases, we examined the effect of MU on both the phosphorylation of hyaluronan synthase 2 (HAS2) and hyaluronan production. The cells were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and MU independently or in combination. The protein fraction of each cell culture was extracted and divided into 2 parts-phosphorylated and non-phosphorylated fractions-by immobilized metal-affinity chromatography. The hyaluronan level in the medium was determined by an ELISA-like assay. Addition of MU decreased the level of hyaluronan in the medium and that of HAS2 in the phosphorylated protein fraction. On the contrary, the addition of TPA increased the levels of both of them. Interestingly, the combination of TPA and MU lowered the levels of them in treated cells as compared to those in untreated control cells. These results suggest that TPA activated protein kinase C (PKC), which stimulates the phosphorylation of HAS2, and increased hyaluronan production. Further, MU may inhibit the phosphorylation of HAS2 by PKC through the stimulation of O-GlcNAcylation.
 
Article
Geldanamycin, a heat-shock protein 90 (Hsp90)-binding agent, modulates various cellular activities. The present study found that, although geldanamycin by itself had no effect on thymocyte viability, it induced apoptosis in thymocytes with a reduction of the mitochondrial transmembrane potential (DeltaPsim) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC). This apoptosis depended on transcription and translation, and on activation of caspase-8 and -3. Geldanamycin treatment in the presence of TPA also enhanced destabilization of Lck. This destabilization was independent of transcription and translation. It was inhibited, however, by conventional PKC inhibitors, preventing apoptosis. Proteasome inhibitor affected neither the degradation of Lck nor DNA fragmentation, although they inhibited reduction of DeltaPsim. These results suggest that the ubiquitin-proteasome system is not involved in Lck destabilization, and that DeltaPsim reduction is not directly related to the progression of apoptosis. Furthermore, inhibition of Lck in the presence of TPA induced apoptosis in thymocytes. Our findings suggest that Hsp90 modulates thymocyte apoptosis in concert with PKC through the destabilization of Lck and in a caspase-8- and -3-dependent manner.
 
Article
Several studies on alcohol and gastric emptying using the (13)C breath test showed that alcohol consumption delayed gastric emptying of meals in healthy male subjects. However, they did not employ female subjects, and the retention time of alcoholic beverages in the stomach has not been examined, yet. We examined the retention time (= gastric emptying rate) of alcoholic beverages in the stomach in healthy male and female subjects. We also examined whether the congeners (nonalcoholic components) of red wine have any effect on gastric emptying. The retention time of 60 mL of red wine, vodka, congeners of red wine, or mineral water, was measured using a (13)C labeled acetic acid breath test. In male subjects, the retention time of wine and vodka was significantly longer than that of congeners and mineral water. In female subjects, although the (13)C content in the breath was slightly but significantly decreased by wine and congeners, but not by vodka, and the parameters for gastric emptying did not differ significantly among the 4 drinks. That is, alcohol hardly influenced the retention time in female subjects. In conclusion, there are sex differences in the gastric emptying rate of alcohol.
 
Article
Growth hormone (GH) replacement therapy has been shown to have beneficial effects on linear growth enhancement in GH-deficient children over the past few decades. SMP-140 is a sterile liquid formation containing rhGH that is expected to improve patient compliance and accuracy of dosing, compared with the commercially available lyophilized form of GH. However, since there are no data showing that SMP-140 influences body elongation in animal models, we studied the effects of SMP-140 on body length in hypophysectomized (HPX) rats, which are used as animal models of GH deficiency. Consistent with the main feature of GH-deficient children, the body length of HPX rats was significantly shorter than that of sham-operated rats at the start of the study. SMP-140 (0.2, 1 and 5 mg/kg) was administered once daily to HPX rats for seven days, and resulted in a dose-dependent increase in body length and in the width of the growth plate cartilage. These results show that SMP-140 administration increases body length in an animal model of GH deficiency, and suggest that SMP-140 will be a useful agent for the treatment of growth-retarded children.
 
Article
The purpose of this study was to clarify the cell growth inhibitory mechanism of human breast cancer cells caused by selenium (Se) compounds. In the presence of 17β-estradiol (E(2)) at physiological concentrations, growth of estrogen receptor α (ERα)-positive T47D cells was markedly inhibited by 1 × 10(-6) mol/L methylseleninic acid (MSA) with no Se related toxicity.Under conditions where cell growth was inhibited, MSA decreased ERα mRNA levels and subsequent protein levels; further decreasing expression of estrogen-responsive finger protein (Efp) which is a target gene product of ERα and promotes G2/M progression of the cell cycle. Therefore, the decline in Efp expression is presumed to be involved in G2 arrest. Coincidentally, the antioxidative thioredoxin/ thioredoxin reductase (Trx/TrxR) system in cells was enhanced by the synergistic action of E(2) and MSA. It has been reported that ROS-induced oxidative stress enhanced ERα expression. E(2) increased production of intracellular ROS in T47D cells. Meanwhile, MSA significantly decreased E(2)-induced ROS accumulation. From these results, activation of the Trx/TrxR system induced by the coexistence of MSA and E(2) suppresses oxidative stress and decreases expression of ERα, and finally induces the growth arrest of T47D cells through disruption of ERα signaling.
 
Article
Paclitaxel and carboplatin (TC) chemotherapy is an effective and well-tolerated regimen against advanced endometrial cancer. Organic anion transporting polypeptide 1B3 (OATP1B3) and copper transporter 1 (CTR1) are critical for the uptake of paclitaxel and carboplatin, respectively. This study aimed to address the prognostic impact of OATP1B3 and CTR1 in endometrial cancer patients treated with adjuvant TC chemotherapy. We immunohistochemically evaluated the expressions of OATP1B3 and CTR1 in 47 stage III endometrial cancers. The high expression levels of OATP1B3 were significantly correlated with type I tumor (P = 0.0005). In univariate analysis, high expression levels of OATP1B3 (P = 0.047) and CTR1 (P = 0.009) were significantly associated with longer disease-free survival (DFS) and longer overall survival (OS), respectively. The patients with tumors showing high expression levels of at least one of OATP1B3 and CTR1 had potentially longer DFS (P = 0.058) and significantly longer OS (P = 0.003) sin the univariate analysis. Combined OATP1B3/CTR1 expression was the sole independent prognostic factor for longer OS in the multivariate analysis (P = 0.013). Our findings suggest that combined OATP1B3/CTR1 expression is a possible predictive/prognostic factor for a good outcome in stage III endometrial cancer patients treated with adjuvant TC chemotherapy.
 
Article
Clinical trials involving in patients with osteoporosis have reported that activated vitamin D3 (1α,25(OH)2D3, calcitriol) can prevent falling by acting on the skeletal muscles. However, pharmacological mechanisms of 1α,25(OH)2D3 with respect to skeletal muscle hypertrophy or atrophy are still poorly understood. Therefore, we examined changes in the expression of several related genes in human myotubes to test whether 1α,25(OH)2D3 influences hypertrophy and atrophy of skeletal muscle. Myotubes treated with 1α,25(OH)2D3 increased interleukin-6 (IL-6) expression and inhibited expression of tumor necrosis factor alpha (TNF-α), whereas the expression of insulin-like growth factor-1 (IGF-1) that is involved in muscle hypertrophy was not affected. However, 1α,25(OH)2D3 treatment significantly inhibited the expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), ubiquitin ligases involved in muscle atrophy. The analysis of pathways using microarray data suggested that 1α,25(OH)2D3 upregulates AKT-1 by inhibiting the expression of protein phosphatase 2 (PP2A), a phosphatase acting on AKT-1, in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, thereby inhibiting the expression of ubiquitin ligases. Thus, this study showed that 1α,25(OH)2D3 might have an inhibitory effect on the expression of MAFbx and MuRF1 in skeletal muscle and a suppressive effect on muscle degradation in patients with osteoporosis.
 
Article
This study investigated whether the suppression of hypoxia-inducible factor (HIF)-1α up-regulation prevents high-dose and long-term UVB-induced wrinkle formation and angiogenesis associated with increased matrix metalloproteinase (MMP)-2 and MMP-9 activities. Twenty-four hairless mice were assigned to three groups: 1) control, 2) UVB-irradiation (UVB), and 3) UVB-irradiation followed by hyperoxia (UVB+HO). The backs of the mice were exposed to UVB irradiation three times a week for 10 weeks. To suppress UVB-induced cutaneous HIF-1α up-regulation, the mice were exposed to hyperoxia (90% oxygen) for 2 h immediately after each UVB irradiation. The UVB and UVB+HO groups had significantly increased degrees of wrinkle formation, dermal blood vessel density, and MMP-2 and MMP-9 activities compared with the control group. HIF-1α expression levels were significantly higher in the UVB group than in the control group whereas these levels remained unchanged in the UVB+HO groups. The activity of type 1 collagenase which digests collagen type 1 (a main component of the dermis), was similar in all groups; furthermore, the dermal soluble collagen content was similar in all groups. These results suggest that the suppression of increases in HIF-1α levels alone is insufficient to restrain wrinkle formation caused by higher doses and longer periods of the UVB irradiation that led to the up-regulation of MMP-2 and MMP-9 activities.
 
Article
Plasminogen activator (PA) is the enzyme that converts plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated the effect of the inflammatory cytokine interleukin-1beta (IL-1beta) on PA secretion in human dental pulp cells. When the cells were stimulated by IL-1beta, PA activity in the medium was clearly increased in a time- and dose-dependent manner. This PA activity in the medium was reduced after immunoprecipitation with anti-uPA antibody, and uPA protein was detected in the immunoprecipitated fraction by Western blotting. However, no such effect was observed with anti-tPA antibody. In the IL-1beta-stimulated cells, expression of uPA mRNA was enhanced whereas expression of tPA mRNA was less. The IL-1beta-stimulated uPA mRNA expression and PA activities in the cell lysate and medium were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFkappaB inhibitor pyrolidinedithiocarbamate, and were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. These observations suggest that IL-1beta stimulates uPA production via activation of NFkappaB and tyrosine phosphorylation, and also secretion of the enzyme, and that the uPA/plasmin system appears to be involved in inflammation in human dental pulp.
 
Article
Internleukin-1 (IL-1) and IL-6 are the most potent proinflammatory cytokines being involved in inflammatory diseases such as periodontitis. The objective of this study was to examine the synergistic effects of IL-1β and IL-6 on gingival inflammation by targeting cultured human gingival fibroblasts (HGFs). HGFs were treated with IL-1β or IL-6/soluble IL-6R (sIL-6R), and total RNA and total cell lysate were collected to examine expression of gp130 known as a signal transducer of IL-6 using qRT-PCR and Western blotting. IL-1β-mediated IL-6 productivity in HGFs was examined using ELISA method. Likewise, after HGFs and THP-1 macrophages were treated with IL-1β, TNF-α and IL-6, sIL-6R productivity was examined. Next, HGFs were treated with IL-6/ sIL-6R after pretreatment of IL-1β, and the intracellular signals were examined using Western blotting. Finally, various mRNA/protein expressions in HGFs treated with IL-6/sIL-6R after pretreatment of IL-1β were examined using qRT-PCR and ELISA method. IL-1β increased significantly both gp130 and IL-6 expression in HGFs. IL-6 increased significantly sIL-6R production in THP-1 macrophages but not HGFs. Co-stimulation with IL-1β and IL-6/sIL-6R induced dramatically the phosphorylation of Stat3, ERK and JNK in HGFs. Interestingly, expression of various inflammation- related molecules such as MMP-1, MCP-1, IL-1ra, bFGF and VEGF were enhanced by co-stimulation with IL-1β and IL-6/sIL-6R in HGFs. Gingival inflammation is regulated by HGFs affected by both IL-1β and IL-6/sIL-6R synergistically through induction of gp130 expression, resulting in progression of periodontitis.
 
Heat map of microRNA (miRNA) expression in human tissues. The expression profile of 375 miRNAs in human tissues was examined by miRNA array analysis. Various tissue-specific miRNAs were identified, i.e., miR124 and miR-219-2-3p in the brain and cerebellum, miR449b in the lung, miR-122 in the liver, miR-499-5p in the heart, miR-1 in the skeletal muscle, and miR-216a and miR-216b in the pancreas. In the placenta, the miRNA profile showed a peculiar pattern and many miRNAs were almost exclusively expressed. B: brain, Cr: cerebellum, Lu: lung, AG: adrenal gland, K: kidney, Pl, placenta, Li: liver, H: heart, U: uterus, Te: testis, Tr: trachea, Pr: prostate, Co: colon, TG: thyroid gland, SM: smooth muscle, SG: salivary gland, Sp: spleen, Pa: pancreas 
Validation of tissue-specific microRNAs (miRNAs) in rat tissues. The expression levels of miR-216a, 216b, and 375 in rat tissues were examined by real-time reverse transcription-polymerase chain reaction. As result, miR-216a (A) and miR216b (B) were almost exclusively expressed in the pancreas. However, tissue-specific expression of miR-375 (C) was not confirmed. 
Validation of pancreatic microRNAs (miRNAs) in rat cells dissected by laser microdissection. The expressions of (A) miR-216a and (B) miR-216b were measured in Langerhans' islets and acinar cells. Both miRNAs were highly expressed in acinar cells. (C) The expression of miR-375 was examined in Langerhans' islets, acinar cells, and pituitary gland. MiR-375 expression was higher in Langerhans' islets than in acinar cells. However, miR-375 expression appeared to be very high in the pituitary gland. 
Article
MicroRNAs (miRNAs) are endogenous small RNAs (length, 18-ss23 nucleotides) that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In the present study, we explored cell- or tissue-specific miRNAs and assessed the applicability of miRNA profiling for identifying biomarkers of tissue injuries. miRNA analyses in various human and rat tissues identified several candidate miRNAs with possible tissue-specific expression, some of which have already been reported. In the present study, we focused on pancreas-specific miRNAs, miR-216a and miR-216b. Laser microdissection revealed that miR-216a and 216b were predominantly expressed in acinar cells of the pancreas as compared to Langerhans' islet. Plasma concentrations of miR-216a and miR-216b considerably increased in a rat model of L-arginineinduced acute pancreatitis. The current results have confirmed that miRNA expression profiling in various cells is useful for providing biomarkers for cell- or tissue-specific injuries.
 
Article
We previously demonstrated that c-Jun N-terminal kinase (JNK) phosphorylates serine 62 of Bcl-xL to induce the degradation of Bcl-xL and apoptosis in WEHI-231 cells upon BCR crosslinking. In order to elucidate the regulatory mechanisms underlying the phosphorylation of Bcl-xL, we prepared an assay system in which JNK phosphorylated Bcl-xL in HEK293T cells. Consequently, we found that a signal transduction molecule, alpha4, enhanced the phosphorylation of Bcl-xL by JNK, while the co-expression of C-terminal alpha4 (220-340) diminished the phosphorylation of Bcl-xL induced by JNK. Furthermore, full-length alpha4 associated with both JNK and Bcl-xL, whereas C-terminal alpha4 (220-340) associated only with Bcl-xL, not JNK. In addition, WEHI-231 cells transfected with the cDNA of C-terminal alpha4 (220-340) exhibited decreased phosphorylation of Bcl-xL and stronger resistance to apoptosis induced by BCR crosslinking. These results indicate that alpha4 is an important regulatory molecule of apoptosis induced by BCR crosslinking in WEHI-231 cells and that C-terminal alpha4 (220-340) functions as a dominant negative form.
 
Article
Acute renal failure (ARF) occurs in septic patients and is histologically characterized by tubular apical damages, including brush border breakdown. Nevertheless, little information is available to identify the apical injury at a molecular level. Type 2a Na-phosphate (Pi) co-transporter (NaPiT2a) is constitutively expressed by brush borders of proximal tubules under a healthy condition. Therefore, we investigated if NaPiT2a could be used as a negative marker to predict the renal dysfunction, using an animal model of septic ARF. After the treatment of lipopolysaccharide (LPS), mice manifested the tubular apical injury and renal dysfunction, as evidenced by the increase in blood urea nitrogen (BUN) levels. Immunohistochemical examination revealed that the expression of NaPiT2a by renal proximal tubules became faint, being reciprocal to the development of tubular hypoxia during sepsis. Inversely, the loss in apical NaPiT2a was restored in a regenerating stage, associated with the recovery from renal hypoxia. Overall, there was a negative correlation between the NaPiT2a expression and BUN levels or tubular injury scores in septic mice. Our data indicate that the loss of NaPiT2a is a reliable marker for predicting the progression of septic ARF, while local hypoxia might be involved in the decrease of NaPiT2a expression.
 
Article
Volume-regulated outwardly rectifying anion channel (VRAC) plays an important role in cell-volume regulation in many types of cells. Little is known about the regulation of VRAC by phosphatidylinositides (PIs), which include phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2). We examined the effect of PIs on the VRAC current activated in hypotonic solution in mouse ventricular cells. VRAC current was inhibited strongly by intracellular application of LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or anti-PIP3 antibody (PIP3-Ab), and less strongly by anti-PIP2 antibody (PIP2-Ab). LY294002 inhibited regulatory volume decrease in hypotonically swollen cells, which was in parallel with the VRAC inhibition. Intracellular PIP3 or PIP2 influenced neither the basal background current in isotonic solution nor the VRAC current in hypotonic solution. However, PIP3, but not PIP2, restored the VRAC current suppressed by LY294002 or PIP2-Ab. These results suggest that the activation of VRAC current requires the presence of intracellular PIP3, that PI3K-mediated increase in PIP3 level is sufficient to fully activate VRAC current, and that PIP3 alone without osmotic stimulation cannot induce VRAC current. We propose that VRAC in mouse ventricular cells is regulated by PIP3 and/or its down stream signaling pathways.
 
Article
Esophageal squamous cell carcinoma (ESCC) is considered one of the most aggressive cancers with poor prognosis. The high molecular weight cytokeratin 34βE12 (CK34βE12) is recognized by the antibody, that is expressed in the cytoplasm of epithelial basal cells, and has been considered as a potential marker for prostate cancer, breast cancer, and basaloid carcinoma of the lung. However, there are no clinicopathological studies investigating CK34βE12 expression at the invasive front of ESCC. In this study, we examined 170 surgically resected cases of ESCC to clarify the clinicopathological significance of CK34βE12 expression. CK34βE12 expression was found in 85.3% (145/170) of ESCC cases and was significantly correlated with lymph node metastasis (66.2% [96/145], P = 0.034), depth of tumor invasion (57.9% [84/145], P = 0.042), and differentiation (82.1% [119/145], P = 0.013). These results indicated that CK34βE12 expression is a good indicator of lymph node metastasis, depth of tumor invasion, and differentiation in case of ESCC.
 
Article
The mushroom Hericium erinaceus has been used as a food and herbal medicine since ancient times in East Asia. It has been reported that H. erinaceus promotes nerve growth factor secretion in vitro and in vivo. Nerve growth factor is involved in maintaining and organizing cholinergic neurons in the central nervous system. These findings suggest that H. erinaceus may be appropriate for the prevention or treatment of dementia. In the present study, we examined the effects of H. erinaceus on amyloid β(25-35) peptide-induced learning and memory deficits in mice. Mice were administered 10 µg of amyloid β(25-35) peptide intracerebroventricularly on days 7 and 14, and fed a diet containing H. erinaceus over a 23-d experimental period. Memory and learning function was examined using behavioral pharmacological methods including the Y-maze test and the novel-object recognition test. The results revealed that H. erinaceus prevented impairments of spatial short-term and visual recognition memory induced by amyloid β(25-35) peptide. This finding indicates that H. erinaceus may be useful in the prevention of cognitive dysfunction.
 
Article
Connexin (Cx) 43-mediated gap-junctional intercellular communication (GJC) mainly regulates the osteoblastic differentiation, but much of the function of Cx43 on the differentiation of bone marrow cells is unclear. This study is aimed to clarify relationship between the differentiation of rat bone marrow cells and the function of Cx43. Bone marrow cells derived from four-week-old Wistar strain rats were grown in the presence and absence of 18-alpha-glycyrrhetinic acid (AGA, 100 muM) to inhibit Cx43-mediated GJC. Expression of Cx43 gene and protein, and the level of intracellular cyclic adenosine monophosphate (cAMP) were determined as the assessment of the function in Cx43-mediated GJC, and alkaline phosphatase (ALP) activity and mineralization were measured as the assessment of osteoblastic differentiation. The Cx43 gene expression was first observed at 2 days, but under the condition in which rat bone marrow cells were treated with AGA, there was no significant effect on the Cx43 gene expression. By administrating AGA to rat bone marrow cells, all parameters of maturation but the Cx43 gene expression significantly decreased. The results of this experiment suggest that Cx43-mediated GJC plays a critical role in rat bone marrow cells, progress toward maturation.
 
Article
Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of ECM and loss of pulmonary function. However, no cure exists for this disease, and current treatments often fail to slow its progression or relieve its symptoms. We have previously reported that the anti-fibrotic agent SMP-534 has beneficial effects on renal fibrosis in animal model of nephropathy. In this study, we examined whether SMP-534 has beneficial effects on pulmonary fibrosis in bleomycin-treated hamsters. Treatment with SMP-534 [low dose (70 mg/kg) or high dose (110 mg/kg)] counteracted inhibition of body weight increase induced by bleomycin. In addition, SMP-534 significantly inhibited bleomycin-induced increase in lung hydroxyproline level, an index of collagen formation. Moreover, SMP-534 significantly ameliorated histological pulmonary fibrotic changes induced by bleomycin. The results of this study indicate that the anti-fibrotic agent SMP-534 may offer a new therapeutic option for the treatment of pulmonary fibrosis.
 
Article
Our previous study showed that tumor budding is a significant indicator of a poor prognosis in lung squamous cell carcinoma patients. Tumor budding-positive (Bud(+)) cases of lung squamous cell carcinoma (SqCC) showed locally aggressive growth, and the positivity was a useful indicator of the lymph node status and prognosis. The present study focused on the clinicopathologic significance of laminin-5γ2 chain expression for local aggressiveness in lung SqCC. Laminin-5γ2 chain immunohistochemical stains in tissue samples were divided into three distinct types: basement membrane (B type; laminin-5γ2 present in basement membrane), cytoplasmic (C type; laminin- 5γ2 present in intracellular matrix), and invasive front (F type; laminin-5γ2 present in cytoplasm, and strongly in part of peripheral nest). The F type was more common in Bud(+) cases than tumor budding-negative (Bud(-)) cases; B and C types were less common in Bud(+) cases (P 〈 0.001). The F type was more closely associated with decreased overall survival than the B and C types (P 〈 0.001 for both). Univariate analysis showed that the F type could be used to predict tumor size, lymph node metastasis, lymphatic invasion, tumor infiltrative patterns, tumor budding, and laminin-5γ2 chain staining. Multivariate analysis showed that laminin-5γ2 chain staining and tumor budding could be used to predict patient mortality (P 〈 0.001 and P = 0.005, respectively). The overall survival rate after curative resection was lower in patients with the F/Bud(+) type than in those with B+C/Bud(-) and B+C/Bud(+) types (P < 0.001 for both, log-rank test), and also lower with the F/Bud(-) type than the B+C/Bud(-) type. On the other hand, there was no significant difference between the F/Bud(+) and F/Bud(-) types. In conclusion, both laminin- 5γ2 chain staining and tumor budding are associated with tumor cell invasiveness and are independent predictors of mortality in lung SqCC patients.
 
Article
Our previous study demonstrated that the pT2 and pT3-4 gallbladder carcinomas can be classified into two groups, i.e. infiltrative growth type (IG type) and destructive growth type (DG type) and that the DG type is associated with poor differentiation, aggressive infiltration, and decreased postoperative survival. The present study focused on the clinicopathologic significance of laminin-5gamma2 chain expression as an indicator of local aggressiveness and Ki-67 labeling index (Ki-67 LI) as an indicator of the cell proliferation activity of gallbladder carcinoma. Ki-67 LI was higher in the DG type (26.3%) than in the IG type (21.4%), and the rate of high-grade cell proliferation cases (Ki-67 LI > or = 30%) was high in the DG type (P = 0.012). Gallbladder carcinoma cases with high Ki-67 LI were significantly associated with poorly differentiation (P = 0.089) and distant lymph node metastasis (P = 0.079). Laminin-5gamma2 expression patterns of gallbladder carcinoma were divided into two distinct types, extracellular staining and cytoplasmic staining. The extracellular staining was subclassified into two groups, basement membrane staining and stromal staining. In the basement membrane staining, laminin-5gamma2 was present in the basement membranes surrounding neoplastic glandular structures. The basement membrane staining of laminin-5gamma2 was more frequent in the IG type (40%) than in the DG type (12.9%) (P = 0.025). The stromal staining was more frequent in the DG type. Furthermore, the stroma-positive group was more closely associated with decreased overall survival than the stroma-negative group (P = 0.028). The cytoplasmic staining was not significantly correlated with invasion pattern in gallbladder carcinoma (P = 0.545). Univariate analysis demonstrated that laminin-5gamma2 stromal staining is a predictor of lymphatic invasion, venous invasion, neural invasion, the mode of subserosal infiltration, and lymph nodal status. Multivariate analysis revealed the mode of subserosal infiltration is the strongest predictor of stromal invasion (P = 0.068). In conclusion, high-grade cell proliferation and stromal laminin-5gamma2 staining were significantly correlated with a wall-invasion pattern of aggressive gallbladder carcinoma indicating destructive growth (DG type).
 
Article
Employing the DNA microarray technique, we previously reported the alteration in gene expression of nucleocytoplasmic transport factors, importins and exportins, induced by 1,25-dihydroxyvitamin D3 (DVD) in human leukemia HL-60 cells. Here, we used the quantitative reverse transcription-polymerase chain reaction method to confirm such previous findings, and compared them with those from the cells treated with all-trans-retinoic acid (ATRA). The results indicated that the gene expression of the transport factors examined was mostly down-regulated following differentiation induced by DVD and ATRA, but importin alpha5 gene expression was up-regulated in either case. The differences were found in the gene expression of importin alpha3 and exportin 6 between the cells after treatments with DVD and ATRA. These variations may be related to the difference between HL-60 cell lineages differentiating into monocytes/macrophages and granulocytes. The present findings provide further evidence to support the important roles of importins and exportins in cell differentiation.
 
Article
Green tea and its constituent (-)-epigallocatechin-3-O-gallate (EGCG) are known to have apoptosis-inducing activity on tumor cells including human leukemia HL-60 cells, providing an explanation for their anti-cancer effects. In the present study, we compared the sensitivity of undifferentiated cells and differentiated HL-60 cells with normal-like phenotypic characters. HL-60 cells treated with three differentiating agents were found to be resistant to EGCG-mediated apoptosis as compared with undifferentiated cells. Gene and protein expression of 67 kDa laminin receptor was down-regulated in differentiated HL-60 cells, suggesting its contribution to the difference in sensitivity in view of the fact that the receptor is a target of EGCG's action to induce apoptosis. The finding supports the view that EGCG induces apoptosis preferentially in cancer cells as compared with normal counterparts.
 
Article
A multiplex PCR assay was developed that enabled the simultaneous detection of DNA from 6 types of human herpes virus, HSV-1/2, VZV, EBV, CMV, HHV-6A/B, and HHV-7, using appropriate primer sets and conventional PCR techniques and instruments, with PCR products for each type of virus designed to be easily distinguishable by size. Electropherograms obtained from conventional agarose gels showed that, for each type, the observed number of base pairs corresponded to the intended product and that bands were easily distinguishable from each other. A minimum of 20 copies of viral DNA in a reaction was sufficient to confirm the existence of each of the 6 types of human herpes virus. Comparison of the data obtained from this method and the data obtained from conventional TaqMan PCR using clinical specimens from various sources showed consistent results. The multiplex PCR method reported here for the detection and differentiation of human herpes viruses did not require special equipment or techniques such as hybridization analysis and sequencing analysis and, therefore, enabled us to easily and rapidly detect and identify the 6 types of human herpes virus using conventional methods.
 
Article
Blood glucose and plasma insulin levels between C57BL/6J and ICR strain mice with nicotinamide (NA) and streptozotocin (STZ)-induced diabetes were compared to establish a suitable strain of the experimental diabetic mouse model. The mice were intraperitoneally treated twice with STZ (100 mg/kg) 15 min after injection of NA (120 mg/kg) at a 1-day interval, and non-fasting blood glucose level was then weekly monitored for 5 weeks. The blood glucose level in ICR mice gradually increased and was about 2-times higher than that in C57BL/6J mice at the end of the observation. The plasma insulin level in ICR mice was comparatively low, compared with that in C57BL/6J mice. ICR mice were also markedly glucose-intolerant when oral glucose tolerance test was performed. These results indicate that ICR strain is more sensitive than C57BL/6J strain as a mouse model with NA/STZ-induced mild diabetes.
 
Article
Based on studies of hypophosphatasia, which is a systemic skeletal disorder resulting from tissuenonspecific alkaline phosphatase (TNSALP) deficiency, TNSALP was suggested to be indispensable for bone mineralization. Recently, we demonstrated that there was a significant difference in bone mineral density (BMD) among haplotypes, which was lowest among TNSALP (787T [Tyr-246Tyr]) homozygotes, highest among TNSALP (787T > C [Tyr246His]) homozygotes, and intermediate among heterozygotes. To analyze protein translated from the TNSALP gene 787T > C, we performed the biosynthesis of TNSALPs using TNSALP cDNA expression vectors. TNSALP (787T) and TNSALP (787T > C) were synthesized similarly as a high-mannose-type 66-kDa form, becoming an 80-kDa form. Expression of the human 787T > C TNSALP gene using the cultured mouse marrow stromal cell line ST2 demonstrated that the protein translated from 787T > C exhibited an ALP-specific activity similarly to that of 787T. Interestingly, the Km value for TNSALP in ST2 cells transfected with the 787T > C TNSALP gene was decreased significantly compared to that of cells carrying the 787T gene (P < 0.01). These results suggest that the significant difference in Km values between the proteins translated from 787T > C and 787T may contribute to regulatory effects on bone metabolism.
 
Article
Circadian rhythm pervades in many aspects of the biological processes including basic cellular functions. Here we examined the circadian gene expression of two forms of 90 kDa heat shock proteins referred to HSP86 and HSP84 in the mouse suprachiasmatic nucleus, the circadian center. In both light-dark, and constant dark conditions, Hsp86 mRNA showed an overt circadian rhythm showing a peak at (subjective) night and a trough at (subjective) day. Hsp84 mRNA also showed the similar expression profile, but the amplitude was weaker. These results indicate that gene expression of molecular chaperone such as Hsp86 and Hsp84 are regulated by the circadian clock.
 
Article
Oncostatin M (OSM) is a multifunctional regulator of cell growth and differentiation. It inhibits the growth of many types of tumor cells, but its role in metastasis is unknown. We studied the human OSM expressed and purified from reconstructed E. Coli on its activity of inhibiting metastasis of tumor cells by a series of assays in vitro and in vivo. Clone formation assay in soft agar was used to measure the inhibition activity of OSM on the proliferation of high metastatic human lung cancer cells 95-D. Cell attachment assay, cell migration assay and cell invasion assay were used to evaluate inhibition by OSM on 95-D cells of the adhesion ability, the migration ability, and the ability of cells to cross tissue barriers, respectively. Inhibition of OSM on secretion of MMP-2 and -9 secretion in 95-D cells was determined by Western blot. The in vivo inhibitory effect of OSM on metastasis of murine melanoma cells B16BL6 was examined in the pulmonary metastasis model. In vitro studies showed that OSM inhibited the proliferation of 95-D cells at low concentration. OSM also reduced the adhesion and invasion ability of 95-D cells and inhibited the secretion of MMP-2 and MMP-9 in OSM treated cells. In vivo results showed that OSM (20 microg/kg/d for 7 days) inhibited pulmonary metastasis at a rate of 20.7%. There were no differences in animal weights among the groups. These results suggest that OSM has the potential of being a clinical inhibitor on metastasis of some cancer cells.
 
Article
Proteins of the Bcl-2 family are key regulators of apoptosis. Bax can be regarded as pro-apoptotic, whereas Bcl-2 is perceived as anti-apoptotic. It has been proposed that an increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2 can be associated with apoptosis. Since prostaglandin A2 (PGA2) and 2-methoxyestradiol (2-ME) play an active role in the induction of apoptosis, the influence of 20 microg/ml PGA2 and 1 microM 2-ME was investigated on Bax and Bcl-2 expression levels in cervical carcinoma cells. Both PGA2 and 2-ME exposure led to statistically significant increases in Bax expression levels. Cells were shown to be more susceptible to the effects of 2-ME than to the effects caused by PGA2. In contrast, no statistically significant effects were observed on Bcl-2 expression levels after exposure to PGA2 and 2-ME. The Bax/Bcl-2 ratios for PGA2- and 2-ME-exposed cells were 2.06 and 1.87 respectively, normalised against Bcl-2 levels. Further investigation of the function and regulation of the Bcl-2 family will allow researchers to consider potential pathways of apoptosis signaling mechanisms for diseases where apoptosis can potentially be controlled.
 
Article
The effects of 20 microg/mL exogenous prostaglandin A(2) (PGA(2)) were determined on Bax, Bcl-2 and proliferating cell nuclear antigen (PCNA) expression levels in MCF-7 cells. Flow cytometric analysis indicated a pronounced increase in the S phase and a decrease in the G(1) phase, whereas a significant increase in the DNA content preceding the G(0)/G(1) peak was also observed after 48 h of exposure to PGA(2). Confirmation of apoptosis was determined after 12 h, 36 h and 48 h of PGA(2) exposure employing the mitosensor reagent that detects potential changes in the mitochondrial membrane. Twenty-eight percent of PGA(2)-exposed cells were in apoptosis when compared to the 7.1% vehicle-treated cells after 48 h. PGA(2) exposure led to statistically significant increase (1.25-fold) over vehicle-treated controls in Bax expression levels. Decreases in Bcl-2 (0.79-fold), as well as PCNA (0.69-fold) expression levels over vehicle-treated controls were observed. The Bax/Bcl-2 ratio for PGA(2)-exposed cells was 2.7. The present study suggests that an accumulation in the S phase, a decrease in expression levels of PCNA, as well as an altered ratio in favor of Bax, could lead to the induction of apoptosis in these cells.
 
mmunohistochemistry for type X collagen and RT-PCR for PTHrP and FGFR3 expressions in epiphyseal cartilage. Control (A) and experiment (B) specimens show epiphyseal cartilages of the similar size and with a similar pattern of immunopositivity for type X collagen (brown). In panel C, the PCR products representing GAPDH, PTHrP and FGFR3 are similar among control (Co: freshly-prepared specimen), stationary cultured (S) and centrifuged (Ce) samples. Bars, 0.5 mm 
Semithin sections of the epiphyseal cartilages and their distinct zones in control (A, B and C) and experimental (D, E and F) specimens (resting zone; A, D, proliferative zone; B, E, hypertrophic zone; C, F). In the resting zone, control chondrocytes (A) are round, smooth-surfaced cells relatively distant one of the other. Radially spreading, toluidine blue-stained fibrillar strucutures can be seen among chondrocytes (arrows). In a matching area, the experiment sample (D) shows higher cell density, with rough-surfaced chondrocytes contacting neighboring cells. In the proliferative zone, control chondrocytes are flattened and distributed in columns (B), a pattern that cannot be seen in a matching region of the experiment specimen (E). Again, toluidine blue-stained fibrillar structures are present in the control samples, apparently connecting neighboring cells and running parallel to the epiphyseal longitudinal axis (arrows). In the hypertrophic zone, non-hypertrophic, irregularly shaped cells can be seen in the centrifuged specimen (F), contrasted by the control specimen (C), which shows the typical hypertrophic chondrocyte morphology. Bars, 10 µm 
TEM imaging of resting (A, C) and proliferative (B, D–G) zones in centrifuged specimens. The electron-dense fibrillar structures (See Figs. 3A and 3B) seen in control specimens are virtually absent (A, B). Sinuous extracellular fibrils distribute unevenly among chondrocytes (Ch), some loosely packed and with remarkably reduced density (C in resting zone, and E in the proliferative zone) and others displaying higher electron density (D). Notice electron-dense fibrillar structure in the tightly-packed matrix (an arrow in D), while faint trace of this structure in loose matrix (an arrow in C). Serpentine collagen fibrils populate the intercolumnar regions (F). When observed contacting cells, cytoplasmic electron-dense materials are identified beneath the cell membranes (small arrows in G). Bars, A, B; 10 µm, C-F; 1 µm, G; 0.5 µm 
TEM imaging of the hypertrophic zones in control (A, B) and experimental (C, D) specimens. Control hypertrophic chondrocytes (Ch) display translucent cytoplasm and nuclei (A), with the surrounding extracellular matrix featuring robust fibrils running parallel to the cell membranes (B) in a densely interconnected and compartmentalized manner (arrows in B). In contrast, centrifuged hypertrophic chondrocytes, despite being irregularly shaped, maintain the translucent appearance and organelle dispersion (C). Extracellular fibrils, however, are kinky and partially compressed in experimental samples (D). Bars, A, C; 10 µm, B, D; 0.2 µm, inset; 0.1 µm 
TEM imaging and fluorescence for actin (A–D) and tubulin (E, F) in control (A, B, E) and experimental (C, D, F) epiphyseal cartilages. Actin fluorescence is even along the cellular membranes in control proliferative chondrocytes (A). TEM figure of a control chondrocyte (Ch) shows small cytoplasmic processes that contain electron-dense materials (arrows in B), which are evenly distributed along the cell membranes, associating with extracellular fibrils (B). Centrifuged chondrocytes display a more intense, yet uneven actin labeling accompanying the outline of their membranes (C). These cells display, beneath their cell membranes, a thick layer of electron-dense materials (arrows in D) that associates with the extracellular matrix (arrowheads in D). Tubulin immunofluorescence unveils a mesh-like network of microtubules that is evident at the hypertrophic zones of both control (E) and experiment (F) samples. Bars, A, C, E, F; 10 µm, B, D; 0.5 µm 
Article
We have examined the morphological changes in chondrocytes after exposure to experimental hypergravity. Tibial epiphyseal cartilages of 17-days-old mouse fetuses were exposed to centrifugation at 3G for 16 h mimicking hypergravitational environment (experimental group), or subjected to stationary cultures (control group). Centrifugation did not affect the sizes of epiphyseal cartilage, chondrocyte proliferation, type X collagen-positive hypertrophic zone, and the mRNA expressions of parathyroid hormone-related peptide and fibroblast growth factor receptor III. However, centrifuged chondrocytes showed abnormal morphology and aberrant spatial arrangements, resulting in disrupted chondrocytic columns. Through histochemical assessments, actin filaments were shown to distribute evenly along cell membranes of control proliferative chondrocytes, while chondrocytes subjected to centrifugal force developed a thicker layer of actin filaments. Transmission electron microscopic observations revealed spotty electron-dense materials underlying control chondrocytes' cell membranes, while experimental chondrocytes showed their thick layer. In the intracolumnar regions of the control cartilage, longitudinal electron-dense fibrils were associated with short cytoplasmic processes of normal chondrocytes, indicating assumed cell-tomatrix interactions. These extracellular fibrils were disrupted in the centrifuged samples. Summarizing, altered actin filaments associated with cell membranes, irregular cell shape and disappearance of intracolumnar extracellular fibrils suggest that hypergravity disturbs cell-to-matrix interactions in our cartilage model.
 
Article
The aim of this study is to determine whether pramipexole hydrochloride hydrate (PHH) and atropine sulfate affect valproic acid (VPA) pharmacokinetics and to evaluate how plasma VPA concentrations are altered by different PHH administration routes. The following studies were conducted on rats: 1) changes in plasma VPA concentration after simultaneous oral administration (PO) of PHH and VPA-Na; 2) effects of intraperitoneal administration (IP) of PHH on plasma VPA concentration after VPA-Na PO; 3) effects of PHH PO on plasma VPA concentration after intravenous administration (IV) of VPA-Na; and 4) changes in plasma VPA concentration after simultaneous PO of atropine sulfate and VPA-Na. Atropine sulfate PO significantly decreased the area under the concentration-time curve up to 3 h (AUC0-3, the total amount of drug plasma concentration) of VPA, suggesting that atropine sulfate decreases VPA-Na absorption probably due to reduced gastrointestinal motility by its anticholinergic action. Similarly, by PHH PO or IP, VPA AUC0-3 was significantly decreased. However, in cases of VPA-Na IV, all VPA parameters were unchanged by PHH PO. These results indicate that the PHH inhibitory effect may be caused in the absorption phase of VPA by pharmacological action of PHH, and thus PHH decreases VPA-Na bioavailability.
 
Western blot of Tff3 in 14-month-old SAM-R1 and SAM-P6 colons. Tff3 appeared as a dimeric 14 kDa band in SAM-R1 colon samples, and a Tff3 trimer band was also detected under 25 kDa in these samples. Three colon samples were examined for both SAM-P6 and SAM-R1. M: Molecular marker (15, 25, 35, 50, 75 kDa).  
Age-dependent changes in the cellular levels of cell defense-related mRNAs (Itln1, Tff3, Dmbt1). The relative abundance of each gene transcripts in colon tissue level from young (3-month old: 3M) and aged (14-month old: 14M) SAM-P6 or SAM-R1 mice was compared with the tissue level of the Tff3 mRNA in 3-month-old SAM-P6 measured by quantitative real-time RT-PCR.  
Venn diagram of the number of genes altered by more than two-fold in each of two independent pair-wise experiments between senescence-accelerated-resistant mice (SAM-R1) and senescence-accelerated-prone mice (SAM-P6).  
Quantitative real-time RT-PCR changes in the cellular levels of cell defense-related mRNAs (Itln1, Tff3, Dmbt1) in SAM-P6 colon compared with SAM-R1 colon of the same age.  
Article
Global comparison of the colonic gene expression profiles between 14-month-old senescenceaccelerated mouse (SAM)-P6 mice and SAM-R1 mice, a wild-type control, was conducted with an oligonucleotide microarray containing more than 5,000 mouse genes. Eight genes were upregulated more than two-fold and 94 genes were downregulated more than two-fold in SAM-P6 mice. The three cell defense genes intelectin1 (Itln1), trefoil factor 3 (intestinal) (Tff3) and "deleted in malignant brain tumors 1" (Dmbt1) were among those extensively downregulated. Quantitative RT-PCR analysis confirmed that Itln1 mRNA was almost undetectable in SAM-P6 colon, whereas it was readily detected in SAM-R1 colon. Colonic expression of both Tff3 and Dmbt1 mRNA was also substantially decreased, to one third and two thirds of the levels in SAM-R1 mice, respectively. A 14 kDa Tff3 dimer was detected by Western blotting in the colon of all three SAM-R1 mice, but was not present in three SAM-P6 mice. No upregulation of 3 cell defense genes was detected in 3-month-old SAM-R1 as well as SAM-P6 mice. These results suggest that a diminution of the intestinal trefoil factor system may be involved in the acceleration of aging in SAM-P6 mice.
 
Article
Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of avian lumbosacral spinal cords. Histological evidence has shown that neurons are present in AL and behavioral evidence suggests that AL acts as a sensory organ of equilibrium during bipedal walking. However, there is little functional evidence to indicate that cells in AL have neuronal functions. To elucidate this point, we developed a method to dissociate cells from chick AL and made electrophysiological recordings with the whole-cell patch clamp technique. Cells dissociated by enzymatic digestion from chick AL contained two major types of cells. One was round with clear cytosol and the other had a round cell body, rich cytosolic structures and some processes. Rapidly activating inward currents and slowly activating outward currents were recorded in response to depolarizing pulses to -10 mV under the voltage clamp configuration only from the latter type of cells. TTX at 100 nM inhibited the inward current by 85%, indicating the functional expression of TTX-sensitive voltage-gated Na(+) channel (VGSC). Activation and inactivation kinetics of the inward currents in AL cells were similar to those of mammalian VGSC. The VGSC-expressing AL cells generated action potentials in response to depolarization under the current clamp configuration. These results clearly indicate that functional neurons expressing fast inactivating and TTXsensitive VGSC which generate action potentials exist in the AL of the chick. These lines of cellular evidence clearly indicate that functional neurons exist in ALs and further support the proposal that the chick ALs function as the sensory organ of equilibrium.
 
Article
Bone remodeling is regulated by local factors that regulate bone-forming osteoblasts and boneresorbing osteoclasts, in addition to hormonal activity. Recent studies have shown that reactive oxygen species (ROS) act as an intracellular signal mediator for osteoclast differentiation. However the role of ROS on osteoblast differentiation is poorly understood. Here, we investigated the impact of ROS on osteoblastic differentiation of MC3T3-E1 cells. Osteogenic induction resulted in notable enhancement of mineralization and expression of osteogenic marker gene alkaline phosphatase, which were accompanied by an increase in ROS production. Additionally, we found that mitochondrial morphology dynamically changed from tubular reticulum to fragmented structures during the differentiation, suggesting that mitochondrial morphological transition is a novel osteoblast differentiation index. The antioxidant N-acetyl cysteine prevented not only ROS production but also mineralization and mitochondrial fragmentation. It is therefore suggested that the ROSdependent signaling pathways play a role in osteoblast differentiation accompanied by mitochondrial morphological transition.
 
Article
Cell-surface F1F0-ATP synthase was involved in the cell signaling mediating various biological functions. Recently, we found that cell-surface F1F0-ATP synthase plays a role on intracellular triacylglycerol accumulation in adipocytes, and yet, the underlying mechanisms remained largely unknown. In this study, we investigated the role of extracellular ATP on the intracellular triacylglycerol accumulation. We demonstrated that significant amounts of ATP were produced extracellularly by cultured 3T3-L1 adipocytes and that the antibodies against α and β subunits of F1F0-ATP synthase inhibited the extracellular ATP production. Piceatannol, a F1F0-ATP synthase inhibitor, and apyrase, an enzyme which degrades extracellular ATP, suppressed triacylglycerol accumulation. The selective P2Y1 receptor antagonist MRS2500 significantly inhibited triacylglycerol accumulation, whereas the selective P2X receptor antagonist NF279 has less effect. The present results indicate that cell-surface F1F0-ATP synthase on adipocytes is functional in extracellular ATP production and that the extracellular ATP produced contributes, at least in part, to the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation in adipocytes through P2Y1 receptor.
 
Article
Although it is known that tea catechins exert potent effects in obese subjects, there is scant information concerning these effects on body weight gain and body fat accumulation in the non-obese. We studied normal rats fed a normal diet and water containing either 0.1% or 0.5% tea catechins to examine the effects on body fat content and serum cholesterol levels, as well as evaluating whether the effect is related to bile acids, which in recent years have emerged as an inducer of energy expenditure. The administration of 0.5% catechins decreased the accumulation of body fat and the serum levels of cholesterol and bile acids. These results indicate that tea catechins modulate lipid metabolism not only in obese subjects, but also in the non-obese.
 
Article
We examined age-related changes in the protein expression of carbonic anhydrase III (CAIII) in livers of Long-Evans with a cinnamon-like color (LEC) rats using an agouti color (LEA) rats as controls. The levels of the protein of CAIII in the liver of LEC male rats increased before 20 weeks of age, at the stage of acute hepatitis, and were decreased at 54 weeks of age, while those of CAIII in the liver of LEA male rats were highly expressed at all ages. In the normal LEA rats, CAIII showed sexual dimorphism. The level of CAIII in LEA male rat liver relative to female was four times higher. On the other hand, young LEC rat (at 4-12 weeks) showed a higher protein level of CAIII than LEA rats, and then decreased during development of hepatitis. CAIII mRNA also decreased in the LEC rat liver during hepatocarcinogenesis. The level of CAIII in the tumor region was lower than that in the tumor-free region. Immunohistochemical analysis showed that glutathione S-transferase P (GST-P) was positive and CAIII was negative in the precancerous region. The expression of CAIII was suppressed in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that suppression of CAIII accompanied hepatocarcinogenesis and it is a secondary consequence of the high copper levels in the liver.
 
Article
Fish protein is a source of animal protein that is consumed worldwide. Although it has been reported that the intake of Alaska pollack protein (APP) reduces serum triglyceride and body fat accumulation in rats, the mechanisms underlying these effects are poorly understood. In the present study, we fed 5-week-old male Sprague-Dawley rats a high-fat diet with APP or casein for 4 weeks. We reconfirmed that the intake of APP decreases serum triglycerides and inhibits visceral body fat accumulation in rats. We found that APP had a higher non-digestive protein content than casein, and the amount of protein in feces was higher in the APP group than in the casein group. However, the amount of total lipids in feces did not differ significantly between the groups. We also found that the gastrocnemius muscle, a fast-twitch muscle, tended to increase in weight, and that the epididymal fat weight correlated negatively with gastrocnemius muscle weight in the APP group. These results imply that the enhancement of basal energy expenditure by fast-twitch muscle hypertrophy, rather than the enhancement of lipid excretion via feces, partly causes APP-induced inhibition of lipid accumulation in rats.
 
Structural formulas of C 2 -ceramide and C 2 -dihydroceramide (analog)
Sequences of primers used for PCR amplification
Article
Ceramide is generated by the hydrolysis of membrane sphingomyelin by sphingomyelinase and is implicated in multiple signaling pathways, including those regulating differentiation, inflammation and immune responses. Excess formation of prostaglandin E(2) (PGE(2)) is thought to increase susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases. We investigated the inhibitory effect of C(2)-ceramide, a short-chain ceramide analog, on the PGE(2)-stimulated accumulation of cAMP in human gingival fibroblasts. In human gingival fibroblasts pre-treated with C(2)-ceramide for 18 h, the PGE(2)-stimulated accumulation of cAMP was reduced, but an inactive C(2)-ceramide analog had no such effect. The accumulation of cAMP induced by EP2 and EP4 receptor agonists (ONO-AE1-259 and ONO-AE1-329, respectively) was inhibited in cells treated with C(2)-ceramide. However, treatment with C(2)-ceramide had no effect on the expression of mRNAs encoding the EP2 and EP4 receptors. Accumulation of cAMP could be induced by cAMP-elevating agents (forskolin, isobutylmethylxanthine and mastparan) but was not reduced by treatment with C(2)-ceramide. These observations suggest that C(2)-ceramide attenuates PGE(2) receptor function and consequently inhibits the accumulation of cAMP in human gingival fibroblasts.
 
Article
The stem cell factor (SCF)-c-kit signal transduction pathway plays an important role in the proliferation and migration of neural progenitor cells, but little is known about its function during the development of the cerebral cortex. We investigated the effects of SCF by directly administering it into the telencephalic ventricular space of 13.5-day-old mouse embryos. SCF produced the heterotopic accumulation of cortical cells in several distinct area of the cerebral cortex at the postnatal stage, including the subcortical periventricular area, marginal zone, and lateral ventricular space. Additional analysis revealed that the heterotopia included both neurons and astrocytes and that SCF initially increased the number of neural stem cells without affecting that of intermediate progenitors and also disturbed their organization. These results suggest that SCF alters the timing of the genesis and migration of neural stem/progenitor cells, which may lead to formation of the observed heterotopia.
 
Article
Kale is a cruciferous vegetable (Brassicaceae) that contains a large amount of health-promoting phytochemicals. The chronic ingestion of cabbage of the same family is known to accelerate conjugating acetaminophen (AA) and decrease the plasma AA level. Therefore, we examined to clarify the effects of kale on the pharmacokinetics of AA, its glucuronide (AA-G) and sulfate (AA-S). AA was orally administered to rats pre-treated with kale or cabbage (2000 mg/kg/day) for one week. Blood samples were collected from the jugular vein, and the concentrations of AA, AA-G and AA-S were determined. In results, kale ingestion induced an increase in the area under the concentration-time curve (AUC) and a decrease in the clearance of AA, whereas cabbage had almost no influence. In addition, there were significant differences in the AUC of AA-G between the control and kale groups. mRNA expression levels of UDP-glucuronosyltransferases, the enzymes involved in glucuronidation, in the kale group were significantly higher than those in the control group. In conclusion, kale ingestion increased the plasma concentrations of both AA and AA-G. The results suggest that kale ingestion accelerates the glucuronidation of AA, but an increase of plasma AA levels has a different cause than the cause of glucuronidation.
 
Protective role of NAC and MESNA in acetaminophen-induced hepatic injury. (A-D) Serial sections of acetaminophen-treated mouse livers were stained with H&E (top) or TUNEL (bottom), and microscopically examined. Arrows: necrotic areas. Scale bar: 500 ?m. (E) Serum ALT levels of mice treated with vehicle only (lane 1, n = 4), acetaminophen (APAP) alone (lane 2, n = 5), APAP and NAC (lane 3, n = 5), and APAP and MESNA (lane 4, n = 5). Asterisk (*): P < 0.05. (F) qPCR analysis for Ho-1 gene expression in the liver of mice treated with vehicle only (lane 1, n = 2), APAP alone (lane 2, n = 3), APAP and NAC (lane 3, n = 3), and APAP and MESNA (lane 4, n = 3). Asterisk (*): P < 0.05. (Student's t-test).
Suppression of acetaminophen-induced acroleinadduct formation by NAC or MESNA treatments. Acroleinadducts (FDP-lysine) in liver extracts were detected with western blot analysis using anti-FDP-lysine antibody. Liver was excised from mice treated with vehicle only (lane 1), acetaminophen (APAP) alone (lanes 2 and 3), APAP and NAC (lanes 4 and 5), and APAP and MESNA (lanes 6 and 7). Gapdh protein level was shown as loading control. +/?: treatment with/without indicated reagents.
Acrolein-adduct formation in the damaged area of the liver after acetaminophen treatment. Serial sections of livers of acetaminophen-treated mice were stained with H&E (top) or anti-FDP-lysine antibody (bottom), and microscopically examined. The liver was excised from mice treated with vehicle only (A), acetaminophen alone (B), acetaminophen and NAC (C), and acetaminophen and MESNA (D). Necrotic areas surrounding central veins were shown by arrows (B). Scale bar: 500 ?m.
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Although acetaminophen-induced liver injury in mice has been extensively studied as a model of human acute drug-induced hepatitis, the mechanism of liver injury remains unclear. Liver injury is believed to be initiated by metabolic conversion of acetaminophen to the highly reactive intermediate N-acetyl p-benzoquinoneimine, and is aggravated by subsequent oxidative stress via reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and the hydroxyl radical (•OH). In this study, we found that a highly toxic unsaturated aldehyde acrolein, a byproduct of oxidative stress, has a major role in acetaminophen-induced liver injury. Acetaminophen administration in mice resulted in liver damage and increased acrolein-protein adduct formation. However, both of them were decreased by treatment with N-acetyl-L-cysteine (NAC) or sodium 2-mercaptoethanesulfonate (MESNA), two known acrolein scavengers. The specificity of NAC and MESNA was confirmed in cell culture, because acrolein toxicity, but not H2O2 or •OH toxicity, was inhibited by NAC and MESNA. These results suggest that acrolein may be more strongly correlated with acetaminophen-induced liver injury than ROS, and that acrolein produced by acetaminophen-induced oxidative stress can spread from dying cells at the primary injury site, causing damage to the adjacent cells and aggravating liver injury.
 
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(-)-Bornyl acetate is the main volatile constituent in numerous conifer oils and has a camphoraceous, pine-needle-like odor. It is frequently used as the conifer needle composition in soap, bath products, room sprays, and pharmaceutical products. However, the psychophysiological effects of (-)-bornyl acetate remained unclear. We investigated the effects of breathing air mixed with (-)-bornyl acetate at different doses (low-dose and high-dose conditions) on the individuals during and after VDT (visual display terminal) work using a visual discrimination task. The amounts of (-)-bornyl acetate through our odorant delivery system for 40 min were 279.4 µg in the low-dose and 716.3 µg in the high-dose (-)-bornyl acetate condition. (-)-Bornyl acetate induced changes of autonomic nervous system for relaxation and reduced arousal level after VDT work without any influences of task performance in low-dose condition, but not in high-dose condition.
 
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Prostaglandins (PGs) are well known as one of the chemical mediators of inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs), PG synthesis inhibitors, are used for anti-nociception and/or anti-inflammation. We examine the effect of loxoprofen, an NSAID, on micturiton in acetic acid-induced bladder inflammation of the rats. In cystometrogram study with saline infusion into the urinary bladder, loxoprofen did not alter the interval of bladder contraction (IC, 107% of the control). IC was shortened by acetic acid infusion (65% of the control) and loxoprofen prolonged the IC (162% of acetic acid infused period). This prolonged IC was approximately same as the control. Loxoprofen did not alter the threshold pressure and the maximal voiding pressure. These data suggest that PGE2 might not play a part of normal micturition and may play a part of the micturition reflex during acetic acid infusion. That is, loxoprofen might be useful for pathological hyperreflex of the micturition.
 
Top-cited authors
Hiroshi Kijima
  • Hirosaki University
Atsukazu Kuwahara
  • Ritsumeikann Univeristy
Shin-Ichiro Karaki
  • University of Shizuoka
Yasuyuki Fukami
  • Aichi Medical University
Huachun Weng
  • National Cerebral and Cardiovascular Center