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• Integrated effect of EGFR and PAR-1 signaling crosstalk on airway hyperresponsiveness

  Authors: Takahiro Yoshikawa

  International Journal of Molecular Medicine. 30(1):41-48.

  Among previous genetic studies for asthma, inconsistent findings were often reported. We used a new approach to examine an integrated effect of haplotype blocks, newly termed ‘haplotype cluster’ overAmong previous genetic studies for asthma, inconsistent findings were often reported. We used a new approach to examine an integrated effect of haplotype blocks, newly termed ‘haplotype cluster’ over two different types of receptors which share common intracellular pathways on airway hyperresponsiveness (AHR). We recruited 165 young atopic adults without respiratory symptoms and measured airway responsiveness to methacholine and classified them into two groups, those with AHR (PC20 <8.0 mg/ml) and without AHR. In addition, we identified haplotype blocks or cluster tagging single nucleotide polymorphisms (SNPs) through two genes, epidermal growth factor receptor (EGFR) and protease activated receptor (PAR)-1, in all subjects, and compared the frequencies of haplotype block or cluster between subject groups. Significant differences in the frequencies were observed in the haplotype blocks within the EGFR gene (rs4947972, rs12718945 and rs2072454; rs4947972, rs12718945 and rs2227983; and rs4947972, rs12718945 and rs2293347) and the PAR-1 gene (rs37243 and rs253072). An integrated effect was also observed in one haplotype cluster consisting of both regions of the EGFR gene (rs2293347) and the PAR-1 gene (rs253072) (P=0.0426). Our results suggest the possibility that the integrated effect of functionally-related EGFR and PAR-1 genes (haplotype cluster) is associated with susceptibility to AHR.

• Nitric oxide prevents phosphorylation of neuronal nitric oxide synthase at Serine1412 by inhibiting the Akt/PKB and CaM-K II signaling pathways.

  Authors: Tao Song, Naoya Hatano, Katsuyoshi Sugimoto, Mariko Horii, Fuminori Yamaguchi, Masaaki Tokuda, Yoshiaki Miyamoto, Toshie Kambe, Yasuo Watanabe

  International journal of molecular medicine. 30(1):15-20.

  Neuronal nitric oxide synthase (nNOS) is an important regulatory enzyme in the central nervous system catalyzing the production of NO, which regulates multiple biological processes in the centralNeuronal nitric oxide synthase (nNOS) is an important regulatory enzyme in the central nervous system catalyzing the production of NO, which regulates multiple biological processes in the central nervous system. However, the mechanisms by which nNOS activity is regulated are not completely understood. In the present study, the effects of protein kinases on the phosphorylation of nNOS in GH3 rat pituitary tumor cells were evaluated. We show that phosphorylation of nNOS at Ser1412 could be induced by the phosphatidylinositol 3-kinase/protein kinase B (Akt/PKB) agonist insulin, the calcium/calmodulin-dependent protein kinase II (CaM-K II) agonist A23187 or the cAMP-dependent protein kinase A (PKA) agonist IBMX, respectively. The phosphorylation levels of nNOS at Ser1412, induced by activation of Akt/PKB or CaM-K II, but not by PKA signaling, were reduced by pre-treatment with the NO donor diethylamine-NONOate. This inhibitory effect could be reversed by addition of a reducing reagent, dithiothreitol. Furthermore, the levels of phosphorylation of nNOS at Ser1412, induced by Akt/PKB or CaM-K II but not by PKA signaling, were enhanced by inhibition of nNOS activity with 7-nitroindazole. These findings suggest that the activation of nNOS can be catalyzed by at least three protein kinases, Akt/PKB, CaM-K II or PKA. NO generated from nNOS feedback prevents the activation of nNOS by inhibiting either Akt/PKB or CaM-K II but not PKA signaling.

• Ethanol increases osteoclastogenesis associated with the increased expression of RANK, PU.1 and MITF in vitro and in vivo.

  Authors: Natsumi Iitsuka, Mamiko Hie, Atsuko Nakanishi, Ikuyo Tsukamoto

  International journal of molecular medicine. 30(1):165-172.

  Ethanol has been known to induce osteopenia. However, the cellular and molecular mechanisms responsible for its effect have not been well characterized. This study investigated the effects of ethanolEthanol has been known to induce osteopenia. However, the cellular and molecular mechanisms responsible for its effect have not been well characterized. This study investigated the effects of ethanol on bone metabolism and osteoclastogenesis using rats fed an ethanol-containing liquid diet (35% of calories from ethanol) for 3 weeks. Ethanol increased the activities of bone tartrate-resistant acid phosphatase (TRAP) and cathepsin K, without affecting the levels of serum osteocalcin or bone alkaline phosphatase activity. Histological analysis showed an increased number of osteoclasts in the proximal tibia, but no significant change in the number of osteoblasts. The mRNA levels of receptor for activation of NF-κB (RANK), c-fos, c-jun, TRAP and cathepsin K were significantly increased, although those of macrophage colony-stimulating factor and c-fms were unaltered. The mRNA and protein levels of PU.1 and microphthalmia-associated trascription factor (MITF) also increased. Further, the osteoclastic differentiation of bone marrow-derived macrophage/monocyte precursor cells (BMMs) in vitro was stimulated by ethanol. The increased osteoclastogenesis of BMMs was associated with increased levels of RANK, PU.1 and MITF expression, activated extracellular signal-regulated kinase (ERK), and reactive oxygen species (ROS). Higher lipid peroxide levels and lower glutathione levels were also observed in the serum of the ethanol-fed rats. These results suggested that ethanol promoted osteoclastogenesis by increasing RANK expression through increases in the production of ROS, activation of ERK and expression of PU.1 and MITF.

• Epigenetic inactivation of PLCD1 in chronic myeloid leukemia.

  Authors: Jun-Jun Song, Qiong Liu, Ying Li, Ze-Song Yang, Li Yang, Ting-Xiu Xiang, Guo-Sheng Ren, Jian-Bin Chen

  International journal of molecular medicine. 30(1):179-184.

  Phospholipase C δ1 (PLCD1), is located at the important tumor suppressor locus 3p22. It encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis andPhospholipase C δ1 (PLCD1), is located at the important tumor suppressor locus 3p22. It encodes an enzyme that mediates regulatory signaling of energy metabolism, calcium homeostasis and intracellular movements. PLCD1 has been studied in some human solid tumors relating to the CpG island methylation of the gene promoter as a functional tumor suppressor. However, no such information is available in chronic myeloid leukemia (CML). In this study, we investigated PLCD1 expression in the CML K562 cell line (0/1) and 15% (2/13) of bone marrow mononuclear cells with CML by using semi-quantitative PCR. The CpG island (CGI) methylation status of the PLCD1 promoter was detected in K562 (0/1) and 56% (23/41) of CML patients by methylation-specific PCR (MSP), but not in the normal adult bone marrow mononuclear cells. Furthermore, the DNA demethylation agent 5'-aza-2'deoxycytidine restored the expression of PLCD1 in K562 cells. Functional studies showed that ectopic expression of PLCD1 in K562 cells was able to dramatically inhibit their colony formation and induce cell cycle G1 arrest, suggesting that PLCD1 acts as a functional tumor suppressor and may serve as a biomarker for possible early detection and prognosis of CML.

• The sphingosine-1-phosphate receptor agonist FTY720 and its phosphorylated form affect the function of CD4+CD25+ T cells in vitro.

  Authors: Yong Liu, Jingjing Jiang, He Xiao, Xiaokui Wang, Yan Li, Yubo Gong, Yifei Huang

  International journal of molecular medicine. 30(1):211-219.

  The sphingosine-1-phosphate receptor agonist FTY720 and FTY720-P have a wide variety of fundamental functions. Many studies have demonstrated that CD4+CD25+ regulatory T (Treg) cells engage in theThe sphingosine-1-phosphate receptor agonist FTY720 and FTY720-P have a wide variety of fundamental functions. Many studies have demonstrated that CD4+CD25+ regulatory T (Treg) cells engage in the maintenance of immunological self-tolerance by actively suppressing self-reactive lymphocytes. Although FTY720 has also recently shown to possess an additional effect that increases the functional activity of Treg cells, the mechanism leading to the enhanced Treg activity after FTY720 treatment is still not clear. We isolated Treg cells, which were co-cultured with FTY720 or FTY720-P. The proliferation of co-cultured Treg cells was detected by the cell counting kit-8. The changes of the phenotype CD25+ and forkhead box P3 (Foxp3)+ of co-cultured Treg cells were measured by flow cytometry. The levels of IL-10 and TGF-β1 in the supernatants were detected by Elisa. Cytokine mRNA expressions in co-cultured Treg cells were analyzed by real-time quantitative PCR. Mixed lymphocyte reaction assay examined the suppressive function. We found that neither FTY720 nor FTY720-P affected the proliferation of co-cultured Treg cells. The percentages of CD25+ and Foxp3+ were enhanced in the high-dose FTY720-P group. The levels of TGF-β1 in the supernatants were enhanced in the high-dose FTY720 group. Medium and high-dose FTY720-P also enhanced the levels of TGF-β1. TGF-β1 and Foxp3 mRNA expression were upregulated in the high-dose FTY720-P group. The proliferation of effector T (Teff) cells was suppressed significantly in the medium and high-dose FTY720-P group at a Treg/Teff cell ratio of 1:1. At a ratio of 1:1, the proliferation of Teff cells was also suppressed in the high-dose FTY720 group. It can be concluded that high-dose FTY720-P can enhance the immune function of co-cultured Treg cells, and that medium-dose FTY720-P and high-dose FTY720 could partly enhance the function. The reason may be attributed to enhanced levels of TGF-β1 and Foxp3.

• Gene expression of ATP-binding cassette transporters during liver regeneration after 90% hepatectomy in rats.

  Authors: Norihisa Kimura, Kenichi Hakamada, Shojiro-Kazunori Ikenaga, Yutaka Umehara, Yoshikazu Toyoki, Mutsuo Sasaki

  International journal of molecular medicine. 30(1):28-34.

  Liver damage with hyperbilirubinemia during regeneration of the small liver is the major hurdle to expand the indications of adult living donor liver transplantation. We performed a large-scale geneLiver damage with hyperbilirubinemia during regeneration of the small liver is the major hurdle to expand the indications of adult living donor liver transplantation. We performed a large-scale gene expression analysis of the regenerating liver after a 90% hepatectomy in rats, and analyzed the changes in the gene expression patterns related to the ATP-binding cassette (ABC) transporters. RNAs were prepared from 3 rat livers at 0, 24, 72 and 168 h after a 90% hepatectomy. The gene expression profile was analyzed by the Rat Genome 230 2.0 array with special references to the ABC transporters. Among 31,042 probes, 1,587 reported genes were identified as either upregulated or downregulated more than 2-fold. Among 20 ABC transporter genes, multidrug resistance protein (MRP) 2 and organic anion transporting polypeptide (OATP) 1 were significantly downregulated, while MRP1 and MRP3 tended to be expressed. These genetic changes were confirmed by real-time PCR. A microarray analysis demonstrated not only an extensive gene expression profile in the regenerating liver but more specific molecular events related to bilirubin transport at the same time. Changes in the expression pattern of the ABC transporters, therefore, seem to be the key event in liver failure during liver regeneration.

• Altered long non-coding RNA expression profile in patients with IgA-negative mesangial proliferative glomerulonephritis.

  Authors: Weiguo Sui, Huan Li, Minglin Ou, Dong'e Tang, Yong Dai

  International journal of molecular medicine. 30(1):173-178.

  Mesangial proliferative glomerulonephritis (MsPGN) is one of the most common immune-mediated renal diseases. The mesangium is expanded and hypercellular, immuno-globulin deposits can be found in theMesangial proliferative glomerulonephritis (MsPGN) is one of the most common immune-mediated renal diseases. The mesangium is expanded and hypercellular, immuno-globulin deposits can be found in the mesangium, but the mechanism underlying its cause remains largely unclear. There is a large amount of evidence suggesting that long ﹥200 nucleotide) non-coding RNAs (lncRNA) have important regulatory functions in the epigenetic control of gene expression. Multiple lines of evidence increasingly link mutations and dysregulations of lncRNAs to a diverse number of human diseases. Through microarray expression analysis, tests show that thousands of lncRNAs and protein-coding genes are significantly differentially expressed in IgA-negative MsPGN. Some lncRNAs and their neighboring protein-coding genes are closely related and are cooperatively expressed. This may be part of a potential regulatory mechanism. The malfunction of regulation in the network of lncRNAs may be a possible mechanism for the development of IgA-negative MsPGN. Our observations suggest that some lncRNAs are closely related to IgA-negative MsPGN and may be playing an important role in this disease.

• Synergistic effects of genetic variants of APOA5 and BTN2A1 on dyslipidemia or metabolic syndrome.

  Authors: Mizuho Hiramatsu, Mitsutoshi Oguri, Kimihiko Kato, Hideki Horibe, Tetsuo Fujimaki, Sachiro Watanabe, Kei Satoh, Yukitoshi Aoyagi, Masashi Tanaka, Dong-Jik Shin, Jong Ho Lee, Yangsoo Jang, Yoshiji Yamada

  International journal of molecular medicine. 30(1):185-192.

  We previously showed that the -1131T→C polymorphism (rs662799) of the apolipoprotein A-V gene (APOA5) and the C→T polymorphism (rs6929846) of the butyrophilin, subfamily 2, member A1 gene (BTN2A1)We previously showed that the -1131T→C polymorphism (rs662799) of the apolipoprotein A-V gene (APOA5) and the C→T polymorphism (rs6929846) of the butyrophilin, subfamily 2, member A1 gene (BTN2A1) were significantly associated with an increased serum concentration of triglycerides, a decreased serum concentration of high density lipoprotein (HDL)-cholesterol, and the prevalence of metabolic syndrome (MetS) in Japanese individuals. The purpose of the present study was to examine whether these polymorphisms synergistically affect the prevalence of dyslipidemia and MetS in East Asian populations. The study populations comprised 7471 Japanese and 3529 Korean individuals in the dyslipidemia study, and 3474 Japanese and 1671 Korean individuals in the MetS study. Multivariable logistic regression analysis of combined genotypes with adjustment for age, gender and diabetes mellitus revealed that rs662799 and rs6929846 significantly and synergistically affected dyslipidemia. Japanese or Korean individuals with the C allele of APOA5 and the T allele of BTN2A1 had a 2.05- or 1.92-fold increased risk for hypertriglyceridemia and a 1.82- or 1.56-fold increased risk for hypo-HDL-cholesterolemia, respectively, compared to those with the TT genotype of APOA5 and the CC genotype of BTN2A1. Similar analysis with adjustment for age and gender revealed that Japanese individuals, but not Korean individuals, with the C allele of APOA5 and the T allele of BTN2A1 had a 2.87-fold increased risk for MetS compared to those with the TT genotype of APOA5 and the CC genotype of BTN2A1. Genetic variants of APOA5 and BTN2A1 may synergistically affect the prevalence of dyslipidemia in East Asian populations and of MetS in Japanese individuals.

• Interleukin gene polymorphisms in pneumoconiosis.

  Authors: Simone Helmig, Martin Grossmann, Jelena Wübbeling, Joachim Schneider

  International journal of molecular medicine.

  Inhaled asbestos fibres are known to cause inflammation processes with the result of lung or pleural fibrosis and malignancies. Interleukins (IL), such as IL-1β, IL-6 and IL-10, have variousInhaled asbestos fibres are known to cause inflammation processes with the result of lung or pleural fibrosis and malignancies. Interleukins (IL), such as IL-1β, IL-6 and IL-10, have various functions in the regulation of the inflammatory response and in proliferative processes after inhalation of silica dust and can, therefore, influence the pathogenesis of asbestos-induced fibrosis and carcinogenesis. Polymorphisms within these genes may be associated with susceptibility to silica and asbestos-induced lung diseases. Thus, IL-1β, IL-6 and IL-10 polymorphisms were examined to determine an association with asbestos or silica-induced fibrosis or malignancies. Association studies were performed in 1180 individuals, using control subjects (n=177), fibrosis patients (n=605), lung cancer (LC) patients (n=364) and malignant mesothelioma (MM) patients (n=34). IL-1β (C-511T; C+3954T), IL-6 (G-174C) as well as IL-10 (G-1082A) polymorphisms were investigated. Compared to a healthy (control) group, a higher risk was seen for malignant mesothelioma patients in all investigated polymorphisms. The IL-6 -174C allele showed a tendency towards a higher risk for fibrosis or asbestos-induced lung cancer (ORasbestosis, 1.338; 95% CI, 0.71-2.53; ORsilicosis, 1.226; 95% CI, 0.54-2.81; ORfibrosis other aetiology, 1.313; 95% CI, 0.58-2.98 and ORLC asbestos, 2.112; 95% CI, 0.75-5.92). The IL-10 -1082A carrier seemed to be at higher risk for silicosis (ORsilicosis, 2.064; 95% CI, 0.78-5.49) but not for asbestosis. In summary, this study did not reveal sufficient evidence for a significant association of the investigated interleukin polymorphisms with asbestos or silica-induced diseases in the population studied.

• Genistein inhibits MDA-MB-231 triple-negative breast cancer cell growth by inhibiting NF-κB activity via the Notch-1 pathway.

  Authors: Hong Pan, Wenbin Zhou, Wei He, Xiaoan Liu, Qiang Ding, Lijun Ling, Xiaoming Zha, Shui Wang

  International journal of molecular medicine.

  Genistein (Gen) has been reported as a protective factor against breast cancer. However, the molecular mechanism by which Gen elicits its effects on triple-negative breast cancer cells has not beenGenistein (Gen) has been reported as a protective factor against breast cancer. However, the molecular mechanism by which Gen elicits its effects on triple-negative breast cancer cells has not been fully elucidated. In our study, the breast cancer cell line MDA-MB-231 was selected to determine the action of Gen on triple-negative breast cancer cells. MTT assay, flow cytometric analysis, siRNA transfection, western blotting and nuclear factor-κB (NF-κB) activation-nuclear translocation assay were used to address the role of NF-κB activity and the Notch-1 signaling pathway on the effects of Gen. Our study revealed that Gen elicited a dramatic effect on cell growth inhibition, in a dose-dependent and time-dependent manner. Treatment of MDA-MB-231 cells with 0, 5, 10 or 20 µM Gen induced apoptosis of 6.78, 18.98, 30.45 and 60.64%, respectively. Exposure of MDA-MB-231 cells to Gen also resulted in G2/M phase accumulation of cells corresponding to 4.93, 12.54, 18.93 and 30.95%, respectively. Furthermore, our data demonstrated for the first time that Gen inhibited the growth of MDA-MB-231 triple-negative breast cancer cells by inhibiting NF-κB activity via the Nocth-1 signaling pathway in a dose-dependent manner. We also found that Gen downregulated the expression of cyclin B1, Bcl-2 and Bcl-xL, possibly mediated by NF-κB activation via the Notch-1 signaling pathway. In conclusion, our results suggest that inhibition of NF-κB activity via the Notch-1 pathway may be a novel mechanism by which Gen suppresses the growth of triple-negative breast cancer cells. Further preclinical and clinical studies are warranted to further investigate the application of Gen for the treatment of triple-negative breast cancer.

• Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase.

  Authors: Yeon Woo Jeong, Geun-Shik Lee, Joung Joo Kim, Sun Woo Park, Kyeong Hee Ko, Mina Kang, Yu Kyung Kim, Eui-Man Jung, Sang Hwan Hyun, Taeyoung Shin, Eui-Bae Jeung, Woo Suk Hwang

  International journal of molecular medicine.

  Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transferDogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.

• Serine protease inhibitor A3 in atherosclerosis and aneurysm disease.

  Authors: Dick Wågsäter, Daniel Johansson, Vincent Fontaine, Emina Vorkapic, Alexandra Bäcklund, Anton Razuvaev, Mikko I Mäyränpää, Charlotta Hjerpe, Kenneth Caidahl, Anders Hamsten, Anders Franco-Cereceda, Johannes Wilbertz, Jesper Swedenborg, Xinghua Zhou, Per Eriksson

  International journal of molecular medicine.

  Remodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such asRemodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such as elastase, cathepsin G and chymase derived from mast cells and neutrophils. In this study, we investigated the putative role of serpinA3 in atherosclerosis and aneurysm formation. SerpinA3 was expressed in endothelial cells and medial smooth muscle cells in human atherosclerotic lesions and a 14-fold increased expression of serpinA3n mRNA was found in lesions from Apoe-/- mice compared to lesion-free vessels. In contrast, decreased mRNA expression (-80%) of serpinA3 was found in biopsies of human abdominal aortic aneurysm (AAA) compared to non-dilated aortas. Overexpression of serpinA3n in transgenic mice did not influence the development of atherosclerosis or CaCl2-induced aneurysm formation. In situ zymography analysis showed that the transgenic mice had lower cathepsin G and elastase activity, and more elastin in the aortas compared to wild-type mice, which could indicate a more stable aortic phenotype. Differential vascular expression of serpinA3 is clearly associated with human atherosclerosis and AAA but serpinA3 had no major effect on experimentally induced atherosclerosis or AAA development in mouse. However, serpinA3 may be involved in a phenotypic stabilization of the aorta.

• Evolving applications of microarray technology in postnatal diagnosis (Review).

  Authors: Ji Un Kang, Sun Hoe Koo

  International journal of molecular medicine.

  Microarray-based cytogenetics is revealing the tremendous fluidity and complexity of the human genome, and is starting to illustrate the implications of genomic variability with respect to humanMicroarray-based cytogenetics is revealing the tremendous fluidity and complexity of the human genome, and is starting to illustrate the implications of genomic variability with respect to human health and disease. In the last few years, the robustness of array-based technologies has provided accurate diagnosis and appropriate clinical management in a timely and efficient manner for identifying genomic defects of congenital and developmental abnormalities including developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASD) and/or multiple congenital anomalies (MCA). The implementation of this technology in these categories of disorders has been thoroughly evaluated and is now recommended as a first-line diagnostic approach for clinically suspected genetic disorders. However, clinical application of array-CGH in postnatal evaluation raises the debate of whether array-CGH will replace traditional cytogenetics in the near future and whether there is still a role for karyotyping and FISH. In this article, we therefore review the current status of array-based technology use for postnatal diagnosis and predict that it will replace standard cytogenetics as a first-line test for clinical evaluation in these population groups.

• Cyclosporine A inhibits breast cancer cell growth by downregulating the expression of pyruvate kinase subtype M2.

  Authors: Kai Jiang, Baoming He, Lihua Lai, Qingyun Chen, Yang Liu, Qingming Guo, Qingqing Wang

  International journal of molecular medicine.

  The high proliferative rate of tumor cells leads to metabolic needs distinct from those of their normal counterparts. An embryonic- and tumor-specific isoform of the enzyme pyruvate kinase M2 (PKM2)The high proliferative rate of tumor cells leads to metabolic needs distinct from those of their normal counterparts. An embryonic- and tumor-specific isoform of the enzyme pyruvate kinase M2 (PKM2) is overexpressed in cancer cells to increase the use of glycolytic intermediates for macromolecular biosynthesis and tumor growth. We report that Cyclosporin A (CsA) can regulate the expression and activity of PKM2 in breast cancer cell lines MCF-7, MDA-MB-435 and MDA-MB-231. PKM2 was found to be highly expressed in the three breast cancer cell lines compared to normal primary breast cells. Treatment with CsA inhibited the viability of breast cancer cells in a time- and dose-dependent manner. CsA significantly downregulated the expression of PKM2 in breast cancer cells and decreased adenosine triphosphate (ATP) synthesis, which induced cancer cells to undergo necrosis. Furthermore, the growth suppression effect of CsA was impaired in MCF-7 cells when they were transfected with the PKM2 overexpression plasmid, suggesting that CsA was an effective inhibitor of PKM2-dependent proliferation of breast cancer cells. These results may provide new insights into the mechanism of CsA in cancer therapy.

• Differentially expressed microRNAs in Huh-7 cells expressing HCV core genotypes 3a or 1b: Potential functions and downstream pathways.

  Authors: Yu Gu, Yuqiao Xu, Lina Jiang, Xiangmei Cao, Fang Liu, Hang Li, Liying Zhang, Zengshan Li, Jing Li, Jing Ye, Qing Li

  International journal of molecular medicine.

  microRNA (miRNA) dysfunction is believed to play important roles in human diseases, including viral infectious diseases. Hepatitis C virus (HCV) infection promotes the development of steatosis,microRNA (miRNA) dysfunction is believed to play important roles in human diseases, including viral infectious diseases. Hepatitis C virus (HCV) infection promotes the development of steatosis, cirrhosis and hepatocellular carcinoma, which is genotype-specific. In order to characterize the miRNA expression profile of Huh-7 cells expressing the HCV core 3a vs. 1b, microarrays and real-time PCR were performed. Consequently, 16 miRNAs (5 miRNAs upregulated and 11 miRNAs downregulated) were found to be dysregulated. In addition, we generated the predicted and validated targets of the differentially expressed miRNAs and explored potential downstream function categories and pathways of target genes using databases of Gene Ontology (GO) and PANTHER and the database for annotation, visualization and integrated discovery (David). The computational results indicated that the dysregulated miRNAs might perform the functions of cellular metabolism and cellular growth. Finally, these biological effects were preliminarily validated. This study identifies a specific miRNA expression profile in cells expressing HCV core proteins of different genotypes (genotype 3a and 1b), which may account for the variable pathophysiological manifestation associated with HCV infection.

• The effects of siRNA against RPL22 on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells.

  Authors: Kai Sun, Hong Xue, Hui Wang, Qiang Wang, Xiang-Rong Zuo, Wei-Ping Xie, Hong Wang

  International journal of molecular medicine.

  The aim of this study was to investigate the effects of small interference RNA (siRNA) against ribosomal protein L22 (RPL22) on ET-1-induced proliferation of human pulmonary arterial smooth muscleThe aim of this study was to investigate the effects of small interference RNA (siRNA) against ribosomal protein L22 (RPL22) on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells (HPASMCs). HPASMCs were transfected with siRNA against RPL22, incubated in smooth muscle cell culture medium (SMCM) and ET-1. RPL22 gene expression and protein levels of HPASMCs were measured by real-time PCR and western blotting, respectively. PCNA, CCK-8 immunohistochemistry and flow cytometry analysis were used to evaluate HPASMC proliferation. Cyclin D1 (CCND1) expression was also assayed. Following transfection with siRNA against RPL22, RPL22 expression was significantly inhibited in the control and ET-1 groups. HPASMC proliferation was significantly suppressed by transfection with siRNA against RPL22. Moreover, CCND1 was also downregulated by inhibiting RPL22 expression. In conclusion, these data suggest that inhibition of RPL22 expression can suppress HPASMC proliferation and CCND1 expression. The effects of siRNA against RPL22 on HPASMC proliferation are considered to be mediated through inhibition of CCND1 expression.

• Bone marrow-derived cells contribute to cell turnover in aging murine hearts.

  Authors: Sebastian Szardien, Holger M Nef, Christian Troidl, Matthias Willmer, Sandra Voss, Christoph Liebetrau, Jedrzej Hoffmann, Andreas Rolf, Johannes Rixe, Albrecht Elsässer, Christian W Hamm, Helge Möllmann

  International journal of molecular medicine.

  The paradigm that cardiac myocytes are non-proliferating, terminally differentiated cells was recently challenged by studies reporting the ability of bone marrow-derived cells (BMCs) to differentiateThe paradigm that cardiac myocytes are non-proliferating, terminally differentiated cells was recently challenged by studies reporting the ability of bone marrow-derived cells (BMCs) to differentiate into cardiomyocytes after myocardial damage. However, little knowledge exists about the role of BMCs in the heart during physiological aging. Twelve-week-old mice (n=36) were sublethally irradiated and bone marrow from littermates transgenic for enhanced green fluorescent protein (eGFP) was transplanted. After 4 weeks, 18 mice were sacrificed at the age of 4 months and served as controls (group A); the remaining mice were sacrificed at the age of 18 months (group B). Group A did not exhibit a significant number of eGFP+ cells, whereas 9.4±2.8 eGFP+ cells/mm2 was documented in group B. In total, only five eGFP+ cardiomyocytes were detected in 20 examined hearts, excluding a functional role of BM differentiation in cardiomyocytes. Similarly, a relevant differentiation of BMCs in endothelial or smooth muscle cells was excluded. In contrast, numerous BM-derived fibroblasts and myofibroblasts were observed in group B, but none were detected in group A. The present study demonstrates that BMCs transdifferentiate into fibroblasts and myofibroblasts in the aging murine myocardium, suggesting their contribution to the preservation of the structural integrity of the myocardium, while they do not account for regenerative processes of the heart.

• Picroside II protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by activating the PI3K/Akt and CREB pathways.

  Authors: Fan-Ji Meng, Su-Min Jiao, Bo Yu

  International journal of molecular medicine.

  Picroside II, an iridoid glucoside found in the root of Picrorhiza scrophulariiflora Pennell (Scrophulariaceae), has been demonstrated to reduce apoptosis in neuronal cells and other cell types.Picroside II, an iridoid glucoside found in the root of Picrorhiza scrophulariiflora Pennell (Scrophulariaceae), has been demonstrated to reduce apoptosis in neuronal cells and other cell types. However, whether picroside II has a protective effect against cardiomyocyte apoptosis is poorly understood. In the present study, we investigated the cardioprotective role of picroside II and the underlying mechanisms in hypoxia/reoxy-genation-induced cardiomyocyte apoptosis. The pretreatment with picroside II markedly attenuated hypoxia/reoxygenation-induced cell damage dose-dependently, which was evident by the increased cell viability and the corresponding decrease in lactate dehydrogenase release (LDH). The pretreatment with picroside II inhibited apoptosis confirmed by Annexin V-FITC staining, Hoechst 33258 nuclear staining and by assessment of caspase-3 activity. In addition, we found that picroside II not only increased the expression of Bcl-2, while decreasing Bax expression, but also augmented Akt and cAMP response element-binding protein (CREB) phosphorylation and ultimately inhibited hypoxia/reoxygenation-induced apoptosis. Furthermore, the protective effects of picroside II were abrogated by pretreatment of the cells with wortmannin or LY294002, a specific PI3K inhibitor. The present study suggests that picroside II inhibits hypoxia/reoxygenation-induced apoptosis in cardiomyocytes by activating the PI3K/Akt and CREB pathways and modulating expression of Bcl-2 and Bax.

• Effect of a hot water extract of Chlorella vulgaris on proliferation of IEC-6 cells.

  Authors: Seo-Hyeon Song, In-Hye Kim, Taek-Jeong Nam

  International journal of molecular medicine. 29(5):741-6.

  Chlorella vulgaris, a unicellular microalgae, exerts various biological effects; however their effect on proliferation signaling pathways in normal cells has not been studied. We investigated theChlorella vulgaris, a unicellular microalgae, exerts various biological effects; however their effect on proliferation signaling pathways in normal cells has not been studied. We investigated the effect of hot water extracts of Chlorella vulgaris (CVE) on cell proliferation and related signaling pathways in rat intestinal epithelial cells (IEC-6). CVE increased the expression of insulin-like growth factor-I receptor (IGF-IR) and the phosphorylation of focal adhesion kinase (FAK) and Src. In addition, CVE induced activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We verified the increased phosphorylation of extracellular-signal-related kinase (ERK) and Akt and the increased expression of the PI3K regulatory subunit p85. CVE also influenced the canonical Wnt pathway through increased expression of the nuclear β-catenin, cyclin D1. Tyr-397 of FAK mediates interactions with Src homology 2 (SH2) domains in a number of other signaling proteins, including PI3K, PLC-γ, Shc, Grb7, Src and Nck2. Because CVE induced FAK activation, FAK may affect the Wnt pathway. Addition of a FAK inhibitor decreased the expression of nuclear β-catenin, cyclin D1 and c-myc, and increased the expression of cytosolic β-catenin. We conclude that CVE stimulated proliferation of IEC-6 cells via the MAPK, PI3K/Akt and canonical Wnt pathways, and that this affected the canonical Wnt pathway.

• Molecular profile of organ culture-stored corneal epithelium: Lgr5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells.

  Authors: Joanna Brzeszczynska, Kanna Ramaesh, Bal Dhillon, James A Ross

  International journal of molecular medicine. 29(5):871-6.

  Long-term preservation of corneal limbal epithelium may decrease its quality and change the molecular signature of the limbal epithelial stem cells. In this study we have investi-gated the molecularLong-term preservation of corneal limbal epithelium may decrease its quality and change the molecular signature of the limbal epithelial stem cells. In this study we have investi-gated the molecular profile of isolated corneal epithelial cells that have been in storage for an extended time. Isolated cells were characterised by the expression profile of different cytokeratins and markers of squamous metaplasia (vimentin and α‑actin). Furthermore, we examined global markers of adult stem cells including p63α and ABCG2 but also Lgr5 as a novel stem cell marker. Immunocytochemical staining and PCR analysis of p63α, ABCG2 and Lgr5 revealed the existence of side-population cells with a stem-cell phenotype and maintenance of corneal limbal stem cell properties. Lgr5 expression can be related to cellular stemness and can be considered as a new phenotypic marker of residual human corneal limbal stem cells. However, the existence of ck10 together with co-expressed α-actin and vimentin suggests that the corneas investigated were under oxidative stress and showed evidence of squamous metaplasia.

• Polysaccharide isolated from Triticum aestivum stimulates insulin release from pancreatic cells via the ATP-sensitive K+ channel.

  Authors: Sun-Hee Lee, Sung-Won Lim, Young-Mi Lee, Hoi-Seon Lee, Dae-Ki Kim

  International journal of molecular medicine. 29(5):913-9.

  Traditional natural plants have been used throughout the world for their antidiabetic effects. The aim of the present study was to investigate the stimulating activity of a polysaccharide extractTraditional natural plants have been used throughout the world for their antidiabetic effects. The aim of the present study was to investigate the stimulating activity of a polysaccharide extract derived from T. aestivum sprout (TASP) on insulin secretion in vitro using the RIN-5F pancreatic β-cell line and rat pancreatic islets. In these experiments, TASP (0.1 to 2 mg/ml) augmented glucose-stimulated insulin secretion in a dose-dependent manner in the presence of a stimulatory glucose concentration (16.7 mM), but not of a basal concentration (1.1 mM). Although TASP failed to enhance the high K+-induced insulin secretion, the insulinotropic effect of TASP was significantly inhibited by diazoxide, an opener of ATP-sensitive K+ channel blocking insulin release. TASP potentiated the insulin secretion induced by other secretagogues, such as IBMX and tolbutamide. Moreover, glucose-derived blood insulin levels were significantly elevated by oral administration of TASP to mice, similarly to antidiabetic drugs. We also demonstrated that TASP significantly increased glucose-induced 45Ca2+ uptake and proinsulin mRNA expression in rat islets. Overall, our results suggest that TASP has a stimulating effect on insulin secretion and production in pancreatic β-cells via K+ channel closure and calcium influx. These results suggest that TASP may be useful as a candidate for the therapy of diabetes mellitus.

• Expression of a humanized single-chain variable fragment antibody targeting chronic myeloid leukemia cells in Escherichia coli and its characterization.

  Authors: Xiaoying Zhu, Dong Wang, Shenfeng Li, Qing Xiao, Kun Tao, Jing Hu, Weixi Cao, Wenli Feng

  International journal of molecular medicine. 29(5):939-45.

  Chronic myeloid leukemia (CML) is a malignant blood disease originating from hematopoietic stem cells. Drug resistance and tumor recurrence have become major problems for the treatment of thisChronic myeloid leukemia (CML) is a malignant blood disease originating from hematopoietic stem cells. Drug resistance and tumor recurrence have become major problems for the treatment of this disease. Therefore, new therapeutic methods need to be developed. Antigens expressed on the surface of cancer cells are potential targets for antibody-mediated drug delivery. In our study, an anti-CML cell single-chain variable fragment (scFv) antibody has been produced and characterized because it is the first step towards the construction of a novel cancer-targeted agent for cancer diagnosis and treatment. Here, a 46 kDa antibody derivative was produced by genetic fusion of a humanized scFv antibody against a CML cell surface antigen with the 6xHis-tag, which can specifically bind to CML cells. The recombinant scFv against CML cells was expressed as a fusion protein containing the 6xHis-tag at its N-termini, and purified by Ni2+-NTA column chromatography. The recombinant scFv, which was soluble, was expressed and produced in bacteria, and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. Finally, its cell-binding activity and immunoactivity were demonstrated by enzyme-linked immunosorbent assay (ELISA). Furthermore, flow cytometry analysis demonstrated that this scFv specifically targeted CML cells expressing the associated antigen (47.9 and 34.4%) other than non-expressing tumor cells (1.25%) in vitro. The results presented in this study illustrate that the humanized anti-CML cell scFv antibody may function as a novel therapeutic tool for CML.

• Radiation-induced double strand breaks and subsequent apoptotic DNA fragmentation in human peripheral blood mononuclear cells.

  Authors: Myriam Ghardi, Marjan Moreels, Bernard Chatelain, Christian Chatelain, Sarah Baatout

  International journal of molecular medicine. 29(5):769-80.

  In case of accidental radiation exposure or a nuclear incident, physical dosimetry is not always complete. Therefore, it is important to develop tools that allow dose estimates and determination thatIn case of accidental radiation exposure or a nuclear incident, physical dosimetry is not always complete. Therefore, it is important to develop tools that allow dose estimates and determination that are based on biological markers of radiation exposure. Exposure to ionizing radiation triggers a large-scale activation of specific DNA signaling and repair mechanisms. This includes the phosphorylation of γH2AX in the vicinity of a double-strand break (DSB). A DNA DSB is a cytotoxic form of DNA damage, and if not correctly repaired can initiate genomic instability, chromosome aberrations, mutations or apoptosis. Measurements of DNA DSBs and their subsequent repair after in vitro irradiation has been suggested to be of potential use to monitor cellular responses. The bone marrow and the blood are known to be the most radiosensitive tissues of the human body and can therefore be of particular importance to find radiation-induced biological markers. In the present study, changes in H2AX phosphorylation and apoptosis of irradiated human peripheral blood mononuclear cells (PBMCs) were analyzed. Freshly isolated PBMCs from healthy donors were irradiated with X-rays (0.1, 0.25, 0.5, 1, 2 and 4 Gy). The phosphorylation of γH2AX was measured at different time points (0, 0.25, 1, 2, 4, 6 and 24 h) after irradiation. We detected a linear dose-dependency of γH2AX phosphorylation measured by γH2AX foci scoring using immunofluorescence microscopy as well as by γH2AX fluorescence detection using flow cytometry. Apoptosis was detected by measuring DNA fragmentation at different time points (0, 24, 48, 72, 96 h) after X-irradiation using DNA ladder gel electrophoresis. The apoptotic DNA fragmentation increased in a dose-dependent manner. In conclusion, DNA DSBs and subsequent apoptotic DNA fragmentation monitoring have potential as biomarkers for assessing human exposure in radiation biodosimetry.

• Autologous transplantation of adipose-derived mesenchymal stem cells attenuates cerebral ischemia and reperfusion injury through suppressing apoptosis and inducible nitric oxide synthase.

  Authors: Dehua Li, Yan Fang, Pan Wang, Wei Shan, Zhongfu Zuo, Ling Xie

  International journal of molecular medicine. 29(5):848-54.

  The purpose of this study was to investigate whether autologous transplantation of adipose-derived mesenchymal stem cells (ADMSCs) has a neuroprotective effect against cerebral ischemia/reperfusionThe purpose of this study was to investigate whether autologous transplantation of adipose-derived mesenchymal stem cells (ADMSCs) has a neuroprotective effect against cerebral ischemia/reperfusion (I/R) injury in rats and to explore the possible underlying mechanisms. Adult male Sprague-Dawley rats were randomly assigned into the sham, I/R injury model (I/R), and model plus autologous transplantation of ADMSCs (ADMSC) groups. Cerebral I/R injury was induced by 2 h middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. Rats in the I/R and ADMSC groups were intravenously injected with culture medium and ADMSCs (2.0x106), respectively, at the onset of reperfusion and 12 h after reperfusion. Cerebral infarct volume was detected by triphenyltetrazolium chloride (TTC) staining. The histopathological changes and neuronal apoptosis in the ischemic penumbra were evaluated with H&E staining and the TUNEL assay, respectively. The nitric oxide (NO) content, caspase-3 activity and the Bax/Bcl-2 protein ratio were also measured. Moreover, the inducible nitric oxide synthase (iNOS) expression in the ischemic regions of rats was determined by immunohistochemical staining, quantitative real-time RT-PCR and western blot analysis. We found that autologous transplantation of ADMSCs significantly reduced the cerebral infarct volume, improved the I/R injury-induced brain damages and inhibited the neuronal apoptosis. ADMSC implantation also decreased caspase-3 activity and the Bax/Bcl-2 protein ratio, and markedly downregulated the expression of iNOS and thus prevented NO release in response to cerebral I/R injury. Taken together, our results demonstrated that autologous transplantation of ADMSCs can protect the brain against cerebral I/R injury via the inhibition of neuronal apoptosis and iNOS expression.

• Relationship between serum undercarboxylated osteocalcin and hyaluronan levels in patients with bilateral knee osteoarthritis.

  Authors: Kiyohito Naito, Taiji Watari, Osamu Obayashi, Sadanobu Katsube, Isao Nagaoka, Kazuo Kaneko

  International journal of molecular medicine. 29(5):756-60.

  Serum undercarboxylated osteocalcin (s-ucOC) is a marker for vitamin K metabolism (deficiency). The aim of this study was to investigate the serum levels of ucOC in patients with bilateral kneeSerum undercarboxylated osteocalcin (s-ucOC) is a marker for vitamin K metabolism (deficiency). The aim of this study was to investigate the serum levels of ucOC in patients with bilateral knee osteoarthritis (K-OA), and the correlation between ucOC and other biomarkers for K-OA. A total of 25 patients (22 women, 3 men, mean age 76.0±7.8, range 54-88 years, mean BMI 24.9±4.7) with a Kellgren-Lawrence grade of 3 or 4 for bilateral knee were enrolled in this study. The levels of s-ucOC and other biomarkers were measured. The levels of s-ucOC (5.66±4.70 ng/ml) as well as other cartilage metabolism markers, were elevated in the patients; however, bone metabolism markers were within the normal ranges. Of interest, there was a significant correlation between s-ucOC and serum hyaluronan (a marker for synovitis) (P<0.05). Our findings suggest that vitamin K metabolism may be associated with synovitis in patients with K-OA, and s-ucOC could be a biomarker for K-OA.

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