High expression of ABCG2 induced by EZH2 disruption has pivotal roles in MDS pathogenesis.

Both proto-oncogenic and tumor-suppressive functions have been reported for enhancer of zeste homolog 2 (EZH2). To investigate the effects of its inactivation, a mutant EZH2 lacking its catalytic domain was prepared (EZH2-dSET). In a mouse bone marrow transplant model, EZH2-dSET expression in bone marrow cells induced a myelodysplastic syndrome (MDS)-like disease in transplanted mice. Analysis of these mice identified Abcg2 as a direct target of EZH2. Intriguingly, Abcg2 expression alone induced the same disease in the transplanted mice, where stemness genes were enriched. Interestingly, ABCG2 expression is specifically high in MDS patients. The present results indicate that ABCG2 de-repression induced by EZH2 mutations have crucial roles in MDS pathogenesis.

Expressions of local renin-angiotensin system components in chondrocytes.

In 2013, we reported that local renin-angiotensin system (local RAS) components express during the hypertrophic differentiation of chondrocytes and can modulate it, using ATDC5 cell line that involves differentiation from mesenchymal stem cells to calcified hypertrophic chondrocytes. However, the expressions of local RAS components in normal chondrocytes have not been revealed yet. The purpose of this study is to examine the expression of the local RAS components in chondrocytes in vivo and the conditions allowing the expression. We stained five major regions of 8-week-old C57BL/6 adult mice in which chondrocytes exist, including epiphyseal plates and hyaline cartilages, with antibodies to local RAS components. We also examined the expression of local RAS components in the cultured bovine's articular cartilage chondrocytes using quantitative reverse transcription polymerase chain reaction and western blot analysis. In result, hypertrophic chondrocytes of epiphyseal plates included in the tibia and the lamina terminals expressed local RAS components. However, hyaline chondrocytes, including the knee articular cartilages, the parenchyma of nasal septums and of the tracheal walls, did not express local RAS components. Cultured bovine's articular cartilage chondrocytes also did not express local RAS components. However, inducing hypertrophy by administering interleukin-1ß or tumor necrosis factor-α, the cultured articular chondrocytes also expressed angiotensin II type 1 receptor and angiotensin II type 2 receptor. In conclusion, local RAS components express particularly in chondrocytes which occur hypertrophy and do not in hyaline chondrocytes. The results are in accord with our previous in vitro study. We think this novel knowledge is important to investigate cartilage hypertrophy and diseases induced by hypertrophic changes like osteoarthritis.

Enhanced vagal modulation and exercise induced ischaemia of the inferoposterior myocardium.

OBJECTIVE: To determine whether the Bezold-Jarisch reflex or enhancement of vagal nerves, which are preferentially distributed in the inferoposterior myocardium, results from exercise induced ischaemia in this region. METHODS: On the basis of exercise myocardial scintigraphy and coronary angiography, 145 patients were classified as follows: group I, 34 patients with inferoposterior ischaemia; group A, 32 with anterior ischaemia; and control, 79 without ischaemia. The relation between ischaemic areas and ECG leads with ST segment changes and vagal modulation assessed by heart rate variability (HRV) (high frequency (HF) component (0.15-0.40 Hz) and coefficient of HF component variance (CCVHF), which is the square root of HF divided by mean RR interval) were assessed. RESULTS: The rate of ST segment depression in any lead did not differ between group I and group A. HF and CCV(HF) were similar before exercise but higher in group I than in group A and the control group after exercise (mean (SEM) HF: 94 (17) ms2, 41 (7) ms2, and 45 (6) ms2, respectively, p = 0.021; CCV(HF): 1.18 (0.09)%, 0.81 (0.07)%, and 0.89 (0.05)%, p = 0.0053). Furthermore, the percentage change in CCV(HF) before and after exercise was higher in group I than in group A or controls (mean (SEM) 22 (10)%, -24 (4)%, and -21 (3)%, p < 0.0001). The optimal cut off for diagnosis of inferoposterior ischaemia was -5% with a sensitivity of 74%, specificity 75%, and accuracy 75%. CONCLUSIONS: Vagal modulation as assessed by HRV analysis was enhanced in association with exercise induced inferoposterior ischaemia. Exercise ECG testing combined with HRV analysis would increase accuracy in the diagnosis of ischaemic areas in selected patients with angina pectoris.

Hypoxia-induced nitric oxide protects chondrocytes from damage by hydrogen peroxide.

OBJECTIVE: Because articular cartilage has no vascular supply, chondrocytes are hypoxic under normal physiological conditions. Nitric oxide (NO) plays an important role in chondrocyte damage, such as apoptosis. Although oxygen stress with hydrogen peroxide was found to cause chondrocyte damage, these data were obtained under normoxic (21% O2) conditions. We investigated the effects of hypoxia on hydrogen peroxide-induced chondrocyte damage METHODS: Bovine articular chondrocytes were used in this study. Proteoglycan (PG) synthesis and the induction of apoptosis were analyzed with [(35)S]-sulfate incorporation and annexin V staining, respectively. The induction of NO was examined using a fluorescent probe and RT-PCR. RESULTS: Cells maintained at 5% O2 had the maximum PG synthesis. Under normoxic conditions, hydrogen peroxide inhibited PG synthesis and induced annexin V positive cells in a dose-dependent fashion. However, in those cells cultured under hypoxic (5%) conditions, the hydrogen peroxide-induced annexin V expression was attenuated. Chondrocytes exposed to hypoxia showed induction of NO. When the hypoxia-induced NO was inhibited, the hypoxia-enhanced PG synthesis was abolished and hydrogen peroxide clearly induced cell damage. CONCLUSIONS: Endogenous NO induced by hypoxia protects chondrocytes from apoptosis induced by an oxidative stress.

Possible involvement of p38 mitogen-activated protein kinase in decidual function in parturition.

We designed the present study to elucidate the molecular mechanism for parturition, focusing on p38 mitogen-activated protein kinase (p38). The kinase activity of p38 in mouse uterus was gestation stage-dependent, and was markedly increased on day 19 of gestation and during labor. Immunohistochemical examination with anti-phospho p38 antibody revealed that activated p38 was predominantly localized in decidual stromal cells stained with anti-prolactin antibody. In human primary cultured decidual cells, a p38 inhibitor, SB202190, significantly inhibited both prostaglandin F(2alpha) production and COX-2 expression induced by stimulation with IL-1beta. These results suggest that the p38 signaling pathway is involved in decidual function at the late stage of gestation and may contribute to parturition.

Cyclic tensile stretch inhibition of nitric oxide release from osteoblast-like cells is both G protein and actin-dependent.

Recent reports indicate the alteration of nitric oxide (NO) synthesis with mechanical stress loaded on the osteoblast and NO is considered to have a significant role in mechanotransduction. We found the involvement of guanine-nucleotide-binding regulatory proteins (G proteins), especially Gi, in stress-inhibited NO release of osteoblast-like cells (JOR:17;593-597, 1999). To determine further the mechanism involved in this process, we measured c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activity under cyclic tensile stretch loaded on osteoblast-like cells. Cyclic stretch significantly enhanced JNK/SAPK activity and pertussis toxin clearly reversed stress-enhanced JNK/SAPK activity. Cytochalasin D, actin microfilament disrupting reagent, also abolished the stress activation of JNK/SAPK. We propose a model for signaling events induced by cyclic tensile stretch, namely a transmembrane mechanosensor which couples Gi-protein, actin cytoskeleton and finally activates JNK/SAPK activity of osteoblasts.

Hydrogen peroxide induces apoptosis of chondrocytes; involvement of calcium ion and extracellular signal-regulated protein kinase.

OBJECTIVE: Recent observations demonstrated that reactive oxygen species facilitate cartilage degradation. We demonstrated that hydrogen peroxide (H2O2) caused inhibition of proteoglycan synthesis, induction of apoptosis and stimulation of extracellular signal-regulated protein kinase (ERK) of the chondrocytes (Inflamm Res 48: 399-403, 1999). To determine whether activation of ERK is involved in the induction of chondrocyte apoptosis, we examined the signal transduction pathways in this hydrogen peroxide induced apoptosis. DESIGN: Bovine articular chondrocytes were cultured. To determine the induction of apoptosis, Annexin V staining and terminal deoxynucleotidyl transferase were used. The activity of caspase-3 was measured using an apopain assay kit. Intracellular Ca2+ imaging was observed after fura2-AM loading. RESULTS: Hydrogen peroxide enhanced annexin V positive apoptotic cells and caspase-3 activity, which is an executor of apoptosis. Hydrogen peroxide also enhanced intracellular Ca2+ and preincubation with the intracellular Ca2+ chelator protected chondrocytes against hydrogen peroxide-induced cell apoptosis, indicating that an increase in the cytosolic Ca2+ plays a decisive role in this action. When ERK activity was blocked with geldanamycin and PD098059, increased apoptosis was evident. CONCLUSION: Hydrogen peroxide induces chondrocyte apoptosis via Ca2+ signaling, and ERK is involved in these signal transduction pathways.

Age-related changes in manganese superoxide dismutase activity in the cerebral cortex of senescence-accelerated prone and resistant mouse.

In this paper, we showed that the oxidative stress in brain of senescence-accelerated prone mouse 8 (SAMP8) at earlier stages was increased compared with that of senescence-accelerated resistant mouse 1 (SAMR1) irrespective of the breeding conditions. Furthermore, we found that manganese superoxide dismutase (Mn-SOD) activity in the cerebral cortex of 10-week-old SAMP8 was decreased by about 50% compared with that in age-matched SAMR1. These results indicate that the decrease of Mn-SOD activity may be involved in the increased oxidative stress in the brain of SAMP8 at younger stages. However, there was no difference in the expression of this protein between the two strains at 10 weeks of age, suggesting that Mn-SOD protein in SAMP8 was post-translationally modified to reduce its enzymatic activity.

A single-chain Fv fragment 2A3 specific for native lysozyme: isolation from a human synthetic phage display antibody library and characterization.

We have isolated from a human synthetic phage display library a clone, 2A3, which discriminates native lysozyme from denatured forms. Binding of single-chain Fv fragments (scFvs) of the clone to native hen egg white lysozyme was competitively inhibited by native hen egg white (hew) and human (h) lysozymes. Dot blotting analysis indicated that scFv of the clone did not react with denatured lysozymes. The K(d) values for scFv of 2A3 binding to native hew- and h-lysozymes were 3.78 x 10(-9) and 9.31 x 10(-9) M, respectively, indicating that 2A3 binds more strongly to native hew-lysozyme than to native h-lysozyme. The deduced amino acid sequence of the V(H) chain-CDR3 region of 2A3 was RRYALDY, of which the Arg residues at positions 1 and 2 of the CDR3 region were observed to be extremely rare in other antibodies by homology analysis. Based on these observations, site-directed mutagenesis of the RRYALDY-coding region was carried out. The results, combined with biomolecular analyses, demonstrated that Arg residues at positions 1 and 2 of this region were important for native lysozyme-binding.

Minimally invasive removal of infected pacemaker lead.

A 37-year-old woman with sick sinus syndrome suffered complications with recurring local infection at the generator pocket. Repeated debridement and antibiotic therapy was ineffective. Several attempts to remove leads via the implantation vein by direct traction were unsuccessful. We operated using cardiopulmonary bypass and applied a minimally invasive lower ministernotomy to obtain pleasing cosmetic results. After a right atriotomy, leads were removed. The minimally invasive approach gave satisfactory results, especially cosmetically.

Divergent hTAFII31-binding motifs hidden in activation domains.

Activation domains are functional modules that enable DNA-binding proteins to stimulate transcription. Characterization of these essential modules in transcription factors has been hampered by their low sequence homology. Here we delineate the peptide sequences that are required for transactivation and interaction with hTAF(II)31, a classical target of the acidic class of activation domains. Our analyses indicate that hTAF(II)31 recognizes a diverse set of sequences for transactivation. This information enabled the identification of hTAF(II)31-binding sequences that are critical for the activity of the activation domains of five human transcription factors: NFAT1, ALL1, NF-IL6, ESX, and HSF-1. The interaction surfaces are localized in short peptide segments of activation domains. The brevity and heterogeneity of the motifs may explain the low sequence homology among acidic activation domains.

Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation.

Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly)(n), possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly)(n) has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-Pro-Gly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly)(n), does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.

Effect of HSP47 on prolyl 4-hydroxylation of collagen model peptides.

Prolyl 4-hydroxylation, the most important post-translational modification in collagen biosynthesis, is catalyzed by prolyl 4-hydroxylase, an endoplasmic reticulum-resident enzyme. HSP47 is a collagen-binding stress protein which also resides in the endoplasmic reticulum (Nagata, K. and Yamada, K.M. (1986) J. Biol. Chem., 261, 7531-7536). Both prolyl 4-hydroxylase and HSP47 interact with procollagen alpha-chains during their folding and/or modification in the endoplasmic reticulum. Recent study has revealed that a simple collagen model peptide, (Pro-Pro-Gly)n, is recognized by HSP47 as well as by prolyl 4-hydroxylase in vitro (Koide et al., manuscript submitted). In the present study, we investigated the effect of HSP47 on the prolyl 4-hydroxylation of such collagen model peptides. To monitor the enzymatic hydroxylation of the peptides, we developed a non-RI assay system based on reversed-phase HPLC. When HSP47 was added to the reaction mixture, substrate and less-hydroxylated materials accumulated. This effect depended on the peptide-binding activity of HSP47, because a mutant HSP47 without collagen-binding activity did not show any inhibitory effect on prolyl 4-hydroxylation. Kinetic analysis and other biochemical analyses suggest that HSP47 retards the enzymatic reaction competing for the substrate peptide.

Bis-N-nitroso-caged nitric oxides: photochemistry and biological performance test by rat aorta vasorelaxation.

Three new caged nitric oxides (NOs)-BNN3, BNN5Na, and BNN5M were tested for biological use. BNNs have a strong ultraviolet (UV) absorption band (lambda(max): 300 nm, epsilon: 13.5 mM(-1) cm (-1)) extended to 420 nm and produce NO upon irradiation with 300-360 nm light in quantum yields about 2. A photoexcited BNN molecule yields two NOs with time constants of less than 10 ns for phase 1 and less than 20 micros for phase 2 at 37 degrees C, suggesting usefulness of BNNs for measuring in vivo and in vitro fast NO reactions. Upon irradiating with UV light, caged nitric oxides-loaded rat aortic strips maintained in a state of active tonic contraction effectively relaxed ( < 3 microM BNN5M loading solution concentration). BNN3 is incorporated in the lipid membrane. BNN5Na, insoluble in organic solvents but water soluble, localizes in the water phase. BNN5M, is muscle-cell-permeable and hydrolysed to BNN5Na to remain in cytosol. BNNs were thermally stable and demonstrated no observable toxicity.

Effect of hydrogen peroxide on the metabolism of articular chondrocytes.

OBJECTIVE: To examine the effect of hydrogen peroxide on chondrocyte metabolism. MATERIALS AND METHODS: Bovine articular chondrocytes were used. Proteoglycan (PG) synthesis was measured with [35S] sulfate incorporation. For detection of apoptosis, the TdT-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V assay were used. Extracellular-regulated protein kinase (ERK) activity was measured using a mitogen-activated protein kinase assay system. RESULTS: Addition of hydrogen peroxide resulted in the inhibition of PG synthesis, apoptosis, and enhanced ERK activity. CONCLUSION: Hydrogen peroxide plays an important role in regulating the metabolism of chondrocytes.

Cytodifferentiation potentiates aFGF-induced p21(ras)/Erk signaling pathway in rat cultured astrocytes.

MBP kinase detection assay revealed that acidic FGF (aFGF) augmented MBP kinase activity in a dose-dependent manner in astrocytes (AC). The molar potency of this action of aFGF in dibutyryl cyclic AMP (DBcAMP)-treated AC was significantly higher than that in quiescent AC. Consistently, the molar potency of accumulation of p21(ras)-GTP by aFGF was significantly higher in DBcAMP-treated AC than in quiescent AC. However, binding study showed that B(max) and K(D) for [(125)I]aFGF in DBcAMP-treated AC were quite similar to those in quiescent AC. Furthermore, the expression levels of Grb2, SOS, and p21(ras) were not changed by treatment of AC with DBcAMP. These results suggest that cytodifferentiation potentiates the p21(ras)/Erk signaling pathway in AC in response to aFGF without changing the expression levels of signaling molecules mediating from the FGF receptor to p21(ras).

Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells.

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene was disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecting biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduced this recombinant receptor into human embryonic kidney 293-T cells. Radioligand binding of [(125)I] angiotensin II to AT1a was specific, saturable, and reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium, and angiotensin II-induced phosphorylation of extracellular signal-regulated kinases (Erk) was found in a dose-dependent manner. After solubilization, Western blot analysis showed specific interactions between an anti-HA antibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of HA-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of this cell line with angiotensin II resulted in decrease in signals of the surface receptors. Based on these results, the cell line established here provides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.

Concurrent generation of nitric oxide and superoxide inhibits proteoglycan synthesis in bovine articular chondrocytes: involvement of peroxynitrite.

OBJECTIVE: Nitric oxide (NO), widely assumed to be a mediator of interleukin 1 (IL-1), inhibits proteoglycan synthesis in articular chondrocytes. IL-1 also produces superoxide anion. We hypothesized that the IL-1 inhibited proteoglycan synthesis is the result of peroxynitrite formed by the reaction of NO with superoxide. METHODS: Bovine articular chondrocytes were cultured in the presence of SIN-1, which leads to simultaneous generation of both NO and superoxide. Proteoglycan synthesis was measured based on the incorporation of [35S] sulfate, and the presence of peroxynitrite was confirmed using immunohistochemistry. RESULTS: SIN-1 inhibited proteoglycan synthesis and superoxide dismutase reversed SIN-1 inhibited proteoglycan synthesis, indicating the simultaneous generation of superoxide is essential to inhibit proteoglycan synthesis. IL-1 induced peroxynitrite in articular chondrocytes and addition of peroxynitrite inhibited proteoglycan synthesis. CONCLUSION: The concurrent generation of superoxide anion and NO is required for the action of IL-1 to inhibit proteoglycan synthesis. Peroxynitrite is a candidate for this underlying mechanism.