Ca2+/Calmodulin-dependent kinase II delta B is essential for cardiomyocyte hypertrophy and complement gene expression after LPS and HSP60 stimulation in vitro.

Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.

Ca2+/Calmodulin-dependent kinase II delta B is essential for cardiomyocyte hypertrophy and complement gene expression after LPS and HSP60 stimulation in vitro

Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.

Transgelin mediates transforming growth factor-ß1-induced proliferation of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Human periodontal ligament cells (HPDLCs) express transforming growth factor-ß1 (TGF-ß1) that regulates differentiation and proliferation, and plays key roles in homeostasis of PDL tissue. Transgelin is a cytoskeleton-associated protein with an Smad-binding element in its gene promoter region. In this study, we examined the localization and potential function of transgelin in PDL tissue and cells. MATERIAL AND METHODS: Microarray analysis of HPDLC lines (2-14, 2-23 and 2-52) was performed. Expression of transgelin in HPDLCs was examined by quantitative reverse transcription-polymerase chain reaction, immunofluorescence staining and western blot analysis. Effects of TGF-ß1 and its signaling inhibitor, SB431542, on transgelin expression in HPDLCs were examined by western blot analysis. The effects of transgelin knockdown by small interfering RNA (siRNA) on HPDLC proliferation stimulated by TGF-ß1 were assessed by WST-1 assay. RESULTS: In microarray and quantitative reverse transcription-polymerase chain reaction analyses, the expression levels of transgelin (TAGLN) in 2-14 and 2-23 cells, which highly expressed PDL markers such as periostin (POSTN), tissue non-specific alkaline phosphatase (ALPL), α-smooth muscle actin (ACTA2) and type I collagen A1 (COL1A1), was significantly higher than those in 2-52 cells that expressed PDL markers weakly. Immunohistochemical and immunofluorescence staining revealed expression of transgelin in rat PDL tissue and HPDLCs. In HPDLCs, TGF-ß1 treatment upregulated transgelin expression, whereas inhibition of the type 1 TGF-ß1 receptor by SB431542 suppressed this upregulation. Furthermore, TAGLN siRNA transfection did not promote the proliferation of HPDLCs treated with TGF-ß1. The expression levels of CCNA2 and CCNE1, which regulate DNA synthesis and mitosis through the cell cycle, were also not upregulated in HPDLCs transfected with TAGLN siRNA. CONCLUSION: Transgelin is expressed in PDL tissue and might have a role in HPDLC proliferation induced by TGF-ß1 stimulation.

HLA and SNP haplotype mapping in the Japanese population.

The genes that encode the human leukocyte antigen (HLA) class I and II molecules are highly polymorphic and located in the major histocompatibility complex (MHC) region, where there is a high density of immune-related genes. Numerous studies have identified disease susceptibility in this region; however, interpretation of the results is complicated because of the strong linkage disequilibrium (LD) among HLA alleles and single-nucleotide polymorphisms (SNPs). In this study, we evaluated the correlation between the HLA alleles of 6 loci (HLA-A, C, B, DRB1, DQB1 and DPB1) and 6502 SNPs within 8 Mb of the extended MHC region using 92 Japanese subjects to identify SNP single loci or haplotypes that tag HLA alleles. We found a total of 39 HLA alleles that showed strong LD (r(2)≥0.8) with SNPs, including 11 non-synonymous SNPs in non-HLA genes. In addition, we identified several SNP haplotypes in strong LD (r(2)≥0.8) with eight HLA alleles, which do not possess tag SNPs. Our detailed list of tag SNPs and haplotypes could be utilized for a better understanding of the results obtained by association studies in the Japanese population and for the characterization of the differences in LD structures between races.

Anti-influenza viral effects of novel nuclear export inhibitors from Valerianae Radix and Alpinia galanga.

Many pathogenic viruses, such as the influenza virus and the Human Immunodeficiency Virus (HIV)-1, are a threat to humans, thus leading to thousands of deaths annually. The development of antiviral drugs is urgent, and it is an essential strategy for the suppression of these infectious diseases. However, regardless of the rapid emergence of many infectious diseases, the development of novel antiviral drugs has been slow, except for the case of the AIDS. In addition, several viruses can easily mutate and escape the inhibitory activity of anti-viral drugs. It was already well-established that HIV escapes from anti-viral drug effects because of the lack of proofreading activity in its reverse transcriptase. It is known that the influenza virus, which is resistant to Tamiflu, is already spread all over the world. Viruses utilize the host cell environment and cellular factors to propagate. Therefore, the development of novel drugs which inhibit viral protein-host protein interactions or cellular functions appear to be good candidates. The influenza virus is unique in replicating in host nuclei, and we therefore focused on the nuclear export processes for the development of anti-influenza viral drugs. We previously reported that leptomycin B (LMB), which inhibited the nuclear export processes via the nuclear export signal (NES) inhibited the nuclear export of influenza viral RNP (vRNP), and resulted in the inhibition of influenza viral propagation. We herein examined novel CRM1 inhibitors, valtrate from Valerianae Radix, and 1'-acetoxychavicol acetate (ACA) from Alpinia galanga as potent inhibitors for the influenza virus replication.

Onecut transcription factor OC2 is a direct target of T-bet in type-1 T-helper cells.

T-box transcription factor, T-bet, has a central role in the differentiation of T-helper (Th) progenitor cells to Th1 or Th2 effector cells, partly by regulating the expression of genes such as interferon-gamma (IFN-gamma). However, the direct target genes, especially those mediating the transcriptional network initiated by T-bet, are not yet fully understood. By combining chromatin immunoprecipitation from Th1 cells with human cytosine-phosphate-guanine-island array analysis, Onecut 2 (OC2), which encodes a member of the ONECUT class of transcriptional activators, was identified as a direct target gene of T-bet. OC2 is expressed in Th1 but not Th2 cells and reporter assays showed that T-bet transactivates OC2 transcription through putative T-bet half-sites locating -451 to -347 of OC2 promoter region. Moreover, we found that OC2 binds and transactivates human T-bet promoter. These results suggest that not only cell-extrinsic regulation via the IFN-gamma/STAT1 pathway, but also cell-intrinsic transcriptional positive feedback loop between T-bet and OC2 could be involved in Th1 development.

High oxygen atmosphere improves human follicle development in organ cultures of ovarian cortical tissues in vitro.

BACKGROUND: Obtaining mature human follicles from cultured ovarian tissue may be beneficial for clinical use for women who wish to preserve fertile competence. However, the methodology of culture such as culture condition and gas atmosphere has not been well established in humans. Therefore, we investigated the effect of oxygen concentration in organ culture in order to establish an ovarian tissue culture method. METHODS: Ovarian tissue was obtained from 26-35-year-old women undergoing removal of a benign tumor (n = 12) or caesarean section (n = 16). The ovarian cortical tissues were cultured on a cell culture insert for 15 days under high (100%) and low (air, 20%) oxygen concentrations and then inspected for follicle development with light and electron microscopy. Estradiol and progesterone concentrations in the medium during culture were measured. RESULTS: The ultrastructure and the function of hormone secretion in the cultured tissues were well preserved after organ culture. The follicles developing under high oxygen were larger and more matured than those developing under low oxygen (P < 0.05). CONCLUSIONS: Human ovarian tissues can be cultured for 15 days under high oxygen concentration with the organ culture system used here. This technique could make it possible to utilize ovarian tissue for preservation of reproductive competence in cancer patients.

Successful delivery after the transfer of twice-vitrified embryos derived from in vitro matured oocytes: a case report.

We report here the first case of successful pregnancy and delivery after the blastocyst transfer of twice-vitrified embryos produced following in vitro maturation (IVM) and ICSI. The patient received 5000 IU hCG on day 12 of the treatment cycle, and oocyte retrieval was carried out 36 h after hCG injection. A total of 22 immature oocytes were obtained. Following incubation for 26 h in IVM medium, 15 oocytes (68.2%) reached metaphase II stage. In total, 13 oocytes (86.7%) were fertilized after ICSI with the husband's sperm, and 11 embryos at the pronuclear stage and two cleaved embryos on day 2 were vitrified because of thin endometrial thickness. Eight cryopreserved embryos at the pronuclear stage were warmed and cultured until the day 3 stage. Three embryos were transferred, and three embryos were twice vitrified. Unfortunately, these transferred embryos did not implant. Three twice-vitrified embryos were rewarmed and cultured until the day 5 stage, and two embryos were transferred. The second transfer attempt of twice-vitrified embryos resulted in the full-term delivery of a healthy infant. This case report demonstrates that twice-vitrified embryos, developed using an IVM protocol, retain the developmental competence for full-term, healthy infants.

Blockade of Angiotensin II type-1 receptor reduces oxidative stress in adipose tissue and ameliorates adipocytokine dysregulation.

Dysregulated production of adipocytokines may be involved in the development of atherosclerotic cardiovascular disease in metabolic syndrome and chronic kidney disease (CKD) associated with metabolic syndrome. The aim of this study was to determine the effects of treatment with angiotensin II (Ang II) type-1 receptor blocker (ARB) on the regulation of adipocytokines. Olmesartan, an ARB, significantly blunted the age- and body weight-associated falls in plasma adiponectin both in genetically and diet-induced obese mice, without affecting body weight, but had no effect on plasma adiponectin levels in lean mice. Olmesartan also ameliorated dysregulation of adipocytokines in obesity, such as tumor necrosis factor-alpha, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, and serum amyloid A3. Olmesartan significantly reduced reactive oxygen species originating from accumulated fat and attenuated the expression of nicotinamide adenine dinucleotide phospho hydrogenase oxidase subunits in adipose tissue. In cultured adipocytes, olmesartan acted as an antioxidant and improved adipocytokine dysregulation. Our results indicate that blockade of Ang II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue. Ang II signaling and subsequent oxidative stress in adipose tissue may be potential targets for the prevention of atherosclerotic cardiovascular disease in metabolic syndrome and also in metabolic syndrome-based CKD.

[Collateral blood flow showing dissection-like filling defect on coronary arteriography: a case report].

A 51-year-old man presented under a diagnosis of angina pectoris manifesting as exertional chest pain. First coronary angiography showed severe stenosis with ulceration and spontaneous dissection at the proximal right coronary artery and linear dissection-like filling defects extending to the distal right coronary artery. After about 3 months, repeat coronary angiography showed the previously observed stenosis with unclear dissection, and better developed collaterals from the left coronary artery to the right coronary artery showing the linear dissection-like filling defects. The bilateral coronary angiography did not clearly show filling defects. This phenomenon suggested that the collateral flows were related to filling of the defects. Intravascular ultrasonic imaging demonstrated severe atherosclerotic lesions at the proximal right coronary artery, but no dissection in the distal right coronary artery. Percutaneous transluminal coronary angioplasty for the stenosis was performed successfully with a stent. Coronary angiography after the angioplasty showed no collaterals, and the right ventricular branch appeared, suggesting that the linear dissection-like filling defects extending to the distal right coronary artery were due to the collateral flows. Filling defects extending distal to a severe stenosis must be distinguished carefully from coronary dissection.

Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins.

Peroxynitrite has been implicated in the oxidative modification of low-density lipoprotein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipoproteins modified by peroxynitrite lead to the onset of atherosclerotic vascular disease. We therefore prepared in vitro lipoproteins oxidatively modified by peroxynitrite (NO(2)-lipoprotein) and investigated the effect of NO(2)-lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular complexes of apolipoproteins in HDL were detected on immunoblotting with monoclonal antibodies against apolipoprotein AI and AII, suggesting that nitration of HDL by peroxynitrite causes intermolecular cross-linking of the apolipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prepared NO(2)-HDL, as quantitated by HPLC, and the amount in NO(2)-lipoprotein depended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whether or not NO(2)-lipoproteins affect the function of endothelial cells, we first examined the viability of cultured human aortic endothelial cells (HAECs) exposed to NO(2)-lipoproteins. Incubation with either NO(2)-HDL or NO(2)-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO(2)-HDL markedly suppressed at 48 h not only the expressed levels of mRNA and protein but also the activity of catalase in HAECs. In contrast, NO(2)-LDL significantly reduced the expression and activity of Cu(2+),Zn(2+)-superoxide dismutase (CuZn-SOD) in the cells. Neither NO(2)-HDL nor NO(2)-LDL interfered with nitric oxide production or expression of cyclooxygenases and NADPH oxidase in HAECs. Increased radical production in NO(2)-lipoprotein-treated HAECs implied that reactive oxygen species such as superoxide anions and hydroxyl radicals may contribute to the mechanism of the toxic effect induced in endothelial cells by NO(2)-lipoprotein. Overall, NO(2)-lipoprotein may lead to deterioration of the vascular function through these endothelial cell responses.

[Aneurysm-like wall motion abnormality in hibernating myocardium detected by stress thallium scintigraphy].

A 70-year-old female patient presented and ECG with QS patterns and ST elevation in V1-3. Left ventriculography showed severely abnormal wall motion of the anteroseptal similar to a left ventricular aneurysm. Based on previous experience that 201Tl myocardial scintigraphy revealed possible myocardial viability in a patient with left ventricular aneurysm suspected of having apparently no myocardial viability, percutaneous transluminal coronary angioplasty (PTCA) was performed for severe stenosis of the left anterior descending artery. Follow-up images 3 months later showed a remarkable improvement in parietal motility of the left ventricle and recovery of almost normal cardiac function. This case demonstrates that exercise myocardial scintigraphy is useful for diagnosing hibernating myocardium associated with severely abnormal parietal motility, such as left ventricular aneurysm.

Localization of NAD(P)H dehydrogenase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Antibodies raised against NdhH and NdhB detected these proteins in the thylakoid membrane of Synechocystis sp. strain PCC 6803, but not in a purified cytoplasmic membrane. We conclude that NAD(P)H dehydrogenase is largely, if not exclusively, confined to the thylakoid membrane.

Anthracycline-induced cardiomyopathies evaluated by tissue Doppler tracking system and strain rate imaging.

OBJECTIVES: Anthracycline cardiotoxicity is known to occur from the subendocardial side of the left ventricular wall. Recent advances of tissue Doppler echocardiography may allow the evaluation of anthracycline cardiotoxicity by dividing the left ventricular wall into the subendocardial half and subepicardial half. The present study assessed the feasibility using the tissue Doppler echo tracking system (M-mode) and myocardial strain rate imaging (B-mode) to noninvasively detect anthracycline cardiotoxicity. METHODS: The tissue Doppler echo tracking system (M-mode) was used to measure systolic thickening of the subendocardial layer (delta Endo), subepicardial layer (delta Epi), and whole wall (delta Total) of the left ventricular posterior wall in 41 normal subjects and three groups of patients receiving anthracycline: 34 patients in the low dose group, 19 in the middle dose group, and 12 in the high dose group. Strain rate is the spatial gradient of local velocities, reflecting local compression and expansion rates not affected by overall heart motion. Myocardial strain rate imaging (B-mode) was used in 25 normal subjects, 9 patients in the low dose group, and 10 patients in the high dose group. The ratio of peak systolic strain rate of subendocardium to that of subepicardium (peak strain rate endo/epi), and the ratio of integrated strain rate during ejection time of subendocardium to that of subepicardium (integrated strain rate endo/epi) were measured. RESULTS: Tissue Doppler echo tracking system (M-mode) measurement of delta Endo/delta Epi showed the most distinct difference and the least overlap of the data between normal subjects and patients, whereas delta Total failed to show significant differences. Myocardial strain rate imaging (B-mode) measurement of integrated strain rate endo/epi showed the most distinct difference and the least overlap of the data between normal subjects and patients, but ejection fraction failed to show statistically significant differences. CONCLUSIONS: These methods are highly sensitive tools for monitoring anthracycline cardiotoxicity.

Synthesis of differentially substituted hexaethynylbenzenes based on tandem Sonogashira and Negishi cross-coupling reactions.

[reaction: see text] Synthesis of polyethynyl-substituted aromatic compounds was achieved efficiently by the use of the Negishi cross-coupling reaction, and this method, coupled with the Sonogashira reaction, was applied to the synthesis of differentially substituted hexaethynylbenzenes from chloroiodobenzenes.

Heart rate decline after exercise in patients with hypertrophic cardiomyopathy.

To evaluate the heart rate recovery, submaximal exercise, echocardiographic examination, and Holter monitoring were performed on 30 patients with hypertrophic cardiomyopathy and 11 controls. The time constant of heart rate decline after exercise was calculated. Spectral analysis was performed on Holter recordings. The time constant did not correlate with heart rate, left ventricular end-diastolic pressure, ejection fraction, or wall thickness. There was no correlation between the time constant and any mean spectral indices over 24 hours in patients. However, the time constant correlated with high frequency component in the night. Nocturnal high frequency component in patients with short time constant was significantly less than in those with long time constant, but did not significantly differ from that in controls. In conclusion, the heart rate decline after exercise does not primarily reflect the severity of hypertrophy or hemodynamic impairment but is associated with nocturnal parasympathetic modulation in patients with hypertrophic cardiomyopathy.

Transient but marked ST elevation in precordial leads caused by ischemia of the isolated right ventricular branch.

The present case is a 64 year-old man in whom transient but marked ST elevation was confirmed in the contralateral precordial leads (V1-3) during percutaneous transluminal coronary angioplasty (PTCA) of the proximal right coronary artery, suggesting that the patient had anteroseptal ischemia. The ST elevation persisted even after the balloon was deflated, and no changes in the left coronary artery were detected. In addition, blood flow in the affected area of the right coronary artery was favorable and there was a transient delay only in the right ventricular branch. Once blood flow in the right ventricular branch improved, ST returned to baseline, and when the right ventricular branch was again occluded by the balloon, ST elevation occurred in a reproducible manner. Hence, the electrocardiographic changes in the precordial leads were caused by occlusion of the right ventricular branch. It is rare to observe ST elevation caused by isolated right ventricular branch ischemia.

Synthesis and association behavior of butadiyne-bridged

Butadiyne-bridged [4(4)](2,6)pyridinophane and [4(6)](2, 6)pyridinophane derivatives were synthesized, and their heteroassociations with the corresponding metacyclophanes and complexations with organic cations were investigated.

The effects of hyaluronan on the meniscus in the anterior cruciate ligament-deficient knee.

Anterior cruciate ligament (ACL) deficiency often induces meniscal tears and, ultimately, degenerative joint disease. The hypothesis of this study was that hyaluronan (HA; MW = 8 x 105) may have a protective effect on the medial meniscus following a period of ACL deficiency. The animal model consisted of creating an ACL deficiency by ACL transection (ACLT) in 51 mature New Zealand white rabbits. Postoperative injections started 4 weeks after ACLT to allow the ACL deficiency to create a degenerative change in the meniscus. The first group (n = 26) was injected with HA and the second group (n = 25) was injected with vehicle (phosphate-buffered saline) in their ACL-deficient knees once a week for 5 weeks, in a protocol similar to that used clinically. At the end of the injections, the HA-treated menisci showed a reduced meniscus area histomorphometrically (P<0.01), as well as a decrease in water content (P<0.01) when compared with the vehicle-treated menisci. The matrix composition of the menisci was assessed by the total glycosaminoglycans (GAGs) content, which decreased in the vehicle-treated menisci (P<0.05) but did not decrease in the HA-treated menisci. In our model, a positive effect of HA was observed biochemically on the preservation of the meniscus matrix composition in the ACL-deficient knee.