ALBERT

Description: Mitochondrial function is achieved through the cooperative interaction of two genomes: one nuclear (nuDNA) and the other mitochondrial (mtDNA). The unusual transmission of mtDNA, predominantly maternal without recombination is predicted to affect the fitness of male offspring. Recent research suggests the strong sexual dimorphism in aging is one such fitness consequence. The uniparental inheritance of mtDNA results in a selection asymmetry; mutations that affect only males will not respond to natural selection, imposing a male-specific mitochondrial mutation load. Prior work has implicated this male-specific mutation load in disease and infertility, but new data from fruit flies suggests a prominent role for mtDNA in aging; across many taxa males almost invariably live shorter lives than females. Here we discuss this new work and identify some areas of future research that might now be encouraged to explore what may be the underpinning cause of the strong sexual dimorphism in aging. Editor's suggested further reading in BioEssays: Mitonuclear match: Optimizing fitness and fertility over generations drives ageing within generations Abstract Mitochondrial manoeuvres: Latest insights and hypotheses on mitochondrial partitioning during mitosis in Saccharomyces cerevisiae Abstract Mitochondria and the culture of the Borg Abstract The maternal inheritance of mtDNA results in a selection asymmetry; natural selection cannot act on mutations that affect only males. This asymmetry imposes a male-specific mutation load previously implicated in male disease and infertility. New work suggests this asymmetry underpins the strong sexual dimorphism in aging observed across taxa.

Description: Osteocalcin (OC) is a non-collagenous, Vitamin K-dependent protein secreted in the late stage of osteoblasts differentiation. The presence of the three residues of γ-carbossiglutamatic acid, specific of the active form of OC protein, allows the protein to bind calcium and consequently hydroxyapatite. The osteoblastic OC protein is encoded by the bone γ-carbossiglutamate gene whose transcription is principally regulated by the Runx2/Cbfa1 regulatory element and stimulated by Vitamin D 3 through a steroid-responsive enhancer sequence. Even if data obtained in literature are controversial, the dual role of OC in bone can be presumed as follows: firstly, OC acts as a regulator of bone mineralization; secondly, OC regulates osteoblast and osteoclast activity. Recently the metabolic activity of OC, restricted to the un-carboxylated form has been demonstrated in osteoblast-specific knockout mice. This effect is mediated by the regulation of pancreatic β-cell proliferation and insulin secretion and adiponectin production by adipose tissue and leads to the regulation of glucose metabolism and fat mass. Nevertheless clinical human studies only demonstrated the correlation between OC levels and factors related to energy metabolism. Thus further investigations in humans are required to demonstrate the role of OC in the regulation of human energy metabolism. Moreover it is presumable that OC also acts on blood vessels by inducing angiogenesis and pathological mineralization. This review highlights the recent studies concerning skeletal and extra-skeletal effects of OC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Invasive ductal adenocarcinoma (IDA) of the pancreas manifests poor prognosis due to the early invasion and distant metastasis. In contrast, intraductal papillary mucinous adenoma or carcinoma (IPMA or IPMC) reveals better clinical outcomes. Various molecular mechanisms contribute to these differences but entire picture is still unclear. Recent researches emphasized the important role of miRNA in biological processes including cancer invasion and metastasis. We previously described that miR-126 is down-regulated in IDA compared with IPMA or IPMC, and miR-126 regulates the expression of invasion related molecule disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). Assessing the difference of miRNA expression profiles of IDA, IPMA and IPMC, we newly identified miR-197 as an up-regulated miRNA specifically in IDA. Expression of miR-197 in pancreatic cancer cells resulted in the induction of epithelial-mesenchymal transition (EMT) along with the down-regulation of p120 catenin which is a putative target of miR-197. Direct interaction between miR-197 and p120 catenin mRNA sequence was confirmed by 3'UTR assay, and knockdown of p120 catenin recapitulated EMT induction in pancreatic cancer cells. In situ hybridization of miR-197 and immunohistochemistry of p120 catenin showed mutually exclusive patterns suggesting pivotal role of miR-197 in the regulation of p120 catenin. This miR-197 / p120 catenin axis could be a novel therapeutic target. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: DNA replication and transcription have structural and temporal organization within the cell nucleus [Spector et al., 1993; Berezney 2002; Stein et al., 2003; Berezney et al., 2005; Cremer et al., 2006; Zaidi et al., 2007; Misteli, 2007; Lanctôt et al., 2007; Stein et al., 2008; Malyavantham et al., 2008a; Malyavantham et al. 2008b; Malyvantham et al., 2010]. Regions within the nucleus are zoned for either transcription or replication during the S phase of the cell cycle [Wei et al., 1998; Berezney, 2002; Malyavantham et al., 2008b]. Moreover these regions within the genome are temporally organized so that genes which are highly active in transcription predominantly replicate earlier than those which are not [Schübeler et al., 2002; White et al., 2004; Woodfine et al., 2004]. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: MicroRNA (miRNA) is a family of small, non-coding RNA first discovered as an important regulator of development in Caenorhabditis elegans ( C. elegans ). Numerous miRNAs have been found in C. elegans , and some of them are well conserved in many organisms. Though, the biologic function of miRNAs in C. elegans was largely unknown, more and more studies support the idea that miRNA is an important molecular for C. elegans . In this review, we revisit the research progress of miRNAs in C. elegans related with development, aging, cancer and neurodegenerative diseases and compared the function of miRNAs between C. elegans and human. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: The transcription factor Runx1 has been studied in leukemia and blood for decades, but recently it has been also implicated in epithelial biology and pathology. Particularly in mouse skin Runx1 modulates Wnt signaling levels thereby regulating timely induction of hair follicle specification, proper maturation of the emerging adult hair follicle stem cells in embryogenesis, and timely stem cell (SC) activation during adult homeostasis. Moreover, Runx1 acts as a tumor promoter in mouse skin squamous tumor formation and maintenance, likely by repressing p21 and promoting Stat3 activation. Similarly, Runx1 is essential for oral epithelium tumorigenesis mediated in mice by Ras, and for growth of three kinds of human epithelial cancer cells. In contrast, Runx1 has a tumor suppressor function in the mouse intestine and shows tumor subtype specific behavior in human breast cancer. Multiple studies revealed Runx1 SNPs to be associated with human cancers and autoimmune disease. With this information as background, the field is poised for functional and mechanistic studies to elucidate the role of Runx1 in formation and/or progression of epithelial-based human disease. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: The identification and purification of murine multipotent mesenchymal stem cells (MSCs) have been difficult due to their low frequency, the presence of contaminating cell types and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOP) population that was also enriched for other mesenchymal precursors, including MSCs [Itoh and Aubin, 2009]. We now report that HipOPs are also highly enriched in vascular endothelial cells (VECs), which we hypothesized were an accessory cell type regulating osteogenesis. However, when VECs were immunodepleted from HipOPs with anti-CD31 antibodies, the resulting CD31(-) HipOP population had equal osteogenic capacity to the HipOPs in vitro and in vivo . Analysis of gene expression of Ncad , Pth1r , Ang1 , Cxcl12 , Jag1 , Pdgfr-β , α-sma , Desmin and Ng2 suggested that both HipOPs and CD31(-) HipOPs are hemopoietic stem cell (HSC) niche populations. However, the data support the view that osteoblast differentiation and depletion of VECs modulate the HSC niche. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: The extensive applications of cerium (Ce) increased the chance of human exposure to Ce and its compounds. It was reported that Ce was mainly deposited in the bone after administration. However, the potential effect and mechanism of Ce on bone metabolism are not well-understood. In this study, we investigated the cellular effects of Ce on the differentiation of mesenchymal stem cells (MSCs) and the associated molecular mechanisms. The results indicated that Ce promoted the osteogenic differentiation and inhibited the adipogenic differentiation of MSCs at cell level. Genes involved in transforming growth factor- β /bone morphogenetic proteins (TGF- β /BMP) signaling pathway were significantly changed when the MSCs were exposed to 0.0001 µM Ce by RT 2 Profiler™ PCR Array analysis. The expression of genes and proteins related to pathways, osteogenic and adipogenic biomarkers of MSCs upon interaction with Ce was further confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (Q-PCR) and western blot analysis. The results suggest that Ce exerts the effects by interacting with bone morphogenetic protein receptor (BMPR) and activates TGF- β /BMP signaling pathway, leads to the up-regulation of the osteogenic master transcription factor, runt-related transcription factor 2 (Runx 2), and the down-regulation of the adipocytic master transcription factor, peroxisome proliferator-activated receptor gamma 2 (PPARγ2). Runx2, which subsequently up-regulates osteoblast (OB) marker genes collagen I (Col I) and BMP2 at early stages, alkaline phosphatase (ALP) and osteocalcin (OCN) at later stages of differentiation, thus driving MSCs to differentiate into OBs. The results provide novel evidence to elucidate the mechanisms of bone metabolism by Ce. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: This study investigated the molecular mechanisms of liver cells with HBx expression on epithelium–mesenchymal transition (EMT) change using Western blot analysis and Transwell assay to assess EMT-related protein expression and cell mobility. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to test the Twist promoter containing different STAT3 binding loci. Electrophoretic mobility band-shift assay (EMSA) was used to detect Twist activity. Results showed that HBx expression affected the EMT-related protein expression and the cell mobility of liver cancer cells (MHCC97) and liver cells (HL-7702) in vitro or in vivo . These proteins exhibited reversed expression to a certain extent after Twist inhibition. In addition, the wound-healing capability and the mobility of HL-7702/HBx cells were lower than those treated with control-siRNA. The expressions of p-STAT3 and Twist were positively correlated with HBx expression. The second STAT-3 binding sequence in the Twist promoter region of the HL-7702/HBx cells was the first locus. Twist activity in the HL-7702/HBx2 cells was higher than that in HL-7702 cells. Moreover, the activity decreased when the cells were treated with HBx-siRNA to inhibit HBx expression, or with STAT3 inhibitor to reduce STAT3 activation. Therefore, Twist is essential for the regulation of the mobility of liver cells with HBx expression. HBx activates the Twist promoter by activating STAT3 and promotes EMT occurrence in liver cells. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: CXCL14 is a chemokine family member that is involved in various cellular responses in addition to immune cell activation. Although constitutive CXCL14 expression in normal epithelial cells may help protect against infection by activating immune systems, its expression in cancer cells has raised controversy regarding its possible role in tumorigenesis. However, the underlying mechanisms for this disparity remain unknown. Investigation of cellular CXCL14 binding properties might increase our understanding of the peptide's roles in tumorigenesis. In the present study, we found that CXCL14 binds to various cell types. Interestingly, binding to NCI-H460 cells was prevented by heparan sulfate and N-acetyl neuraminic acid. Next, we examined effect of CXCL14 binding in NCI-H460 and NCI-H23. CXCL14 enhanced proliferation and migration in NCI-H460 but had no effect on NCI-H23. A reporter gene assay with various transcription factor response elements revealed that only nuclear factor-κB (NF-κB) signaling was activated by CXCL14 in NCI-H460 cells, which was blocked by BAPTA-AM, TCPA, and brefeldin A. Exogenous expression of some glycoproteins such as syndecan-4, podoplanin, and CD43 in these cells enhanced CXCL14 binding and NF-κB activity. Collectively, these results demonstrate that CXCL14 binding to glycoproteins harboring heparan sulfate proteoglycans and sialic acids leads proliferation and migration of some cancer cells. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: Bone loss is a well documented phenomenon occurring in humans both in short and in long term spaceflights. This phenomenon can be also reproduced on the ground in human and animals and also modeled in cell-based analogs. Since space flights are infrequent and expensive to study the biomedical effects of microgravity on the human body, much of the known pathology of bone loss comes from experimental studies. The most commonly used in vitro simulators of microgravity are clinostats while in vivo simulators include the bed rest studies in humans and hindlimb unloading experiments in animals. Despite the numerous reports that have documented bone loss in wide ranges in multiple crew members, the pathology remains a key concern and development of effective countermeasures is still a major task. Thus far, the offered modalities did not show much success in preventing or alleviating bone loss in astronauts and cosmonauts. The objective of this review is to capture the most recent research on bone loss from spaceflights, bed rest and hindlimb unloading, as well as from in vitro studies utilizing cellular models in clinostats. Additionally, this review offers projections on where the research has to focus to ensure the most rapid development of effective countermeasures. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. Fluorescence technologies provide key to study molecular structure and dynamics with super spatial and temporal resolution. We discuss how synergistic developments of novel labeling technologies combined with optical engineering can make the biggest contribution to advance state of the art tools for both, in vitro and non-invasive in vivo measurements.

Description: Pronuclear microinjection of bacterial artificial chromosomes (BACs)is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here we describe a simpler pronuclear injection method that relies on transpositionto introducefull-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted repeats(ITRs) andco-injectedwith PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations was increased.Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that the BACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems. © 2012 Wiley-Liss, Inc.

Description: TGFβ1 is very important in the synthesis and degradation of extra cellular matrix (ECM), and also in the mediation of human lung fibroblasts proliferation, and miR-29 plays an important role in this process. To explore the interactions of miR-29 family members and TGFβ1, the effects of transforming growth factor TGFβ1 on the expression of miR-29 and whether miR-29 is involved in pro-survival signaling pathways mediated by TGFβ1 were examined in human lung fibroblasts. Treatment of the human embryonic lung fibroblast cell line IMR90 with TGFβ1 caused a decrease expression of miR-29a/b/c by real-time PCR analysis. TGFβ1 stimulation increased cell proliferation, colony formation and upregulated expression of COL1A1; transfecting with miR-29a/b/c mimics reverse TGFβ1-induced phenotype changes in IMR90 cells. Western blot analyses showed that TGFβ1 treatment unchanged total protein expression levels of PI3K or AKT, but the expression levels of p-PI3K, p-AKT and COL1A1 were increased; and miR-19a/b/c mimics interfering blocked phosphorylation of PI3K or AKT and decreased expression of COL1A1 after TGFβ1 treatment. The results indicate that TGFβ1 beta uses the PI3k-Akt pathway in these embryonic fibroblasts and miR29 blocks this activation pathway. It indicates a novel biological function of the PI3K-Akt pathway in IMR90. Elevated expression of miR-29 may play an important role in the pathogenesis of diseases related to fibrogenic reactions in human lung fibroblasts. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: RGS14 is a 60 kDa protein that contains a regulator of G protein signalling (RGS) domain near its N-terminus, a central region containing a pair of tandem Ras binding domains (RBD), and a GPSM (G protein signalling modulator) domain (a.k.a. Gi/o-Loco binding (GoLoco) motif) near its C-terminus. The RGS domain of RGS14 exhibits GTPase accelerating protein (GAP) activity toward Gαi/o proteins, while its GPSM domain acts as a guanine nucleotide dissociation inhibitor (GDI) on Gαi1 and Gαi3. In the current study, we investigate the contribution of different domains of RGS14 to its biochemical functions. Here we show that the full-length protein has a greater GTPase activating activity but a weaker inhibition of nucleotide dissociation relative to its isolated RGS and GPSM regions, respectively. Our data suggest that these differences may be attributable to an inter-domain interaction within RGS14 that promotes the activity of the RGS domain, but simultaneously inhibits the activity of the GPSM domain. The RBD region seems to play an essential role in this regulatory activity. Moreover, this region of RGS14 is also able to bind to members of the B/R4 subfamily of RGS proteins and enhance their effects on GPCR-activated Gi/o proteins. Overall, our results suggest a mechanism wherein the RBD region associates with the RGS domain region, producing an intramolecular interaction within RGS14 that enhances the GTPase activating function of its RGS domain while disfavoring the negative effect of its GPSM domain on nucleotide dissociation. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: We enthusiastically read the recent manuscript by Intapad et al. from the Catravas group in the August 227 issue of Journal of Cellular Physiology entitled “ Regulation of Asthmatic Airway Relaxation by Estrogen and Heat Shock Protein 90 ” [1]. The authors show impaired relaxation of murine tracheal rings sensitized with serum from human asthmatics to nitric oxide (NO) donors, and potentiation of such relaxation upon treatment with estradiol (E 2 ), or the ERa- or ERb-selective ligands PPT and DPN, respectively [1]. They conclude that estrogens can potentiate NO-mediated bronchodilation in normal and asthmatic airways. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: New genes have frequently formed and spread to fixation in a wide variety of organisms, constituting abundant sets of lineage-specific genes. It was recently reported that an excess of primate-specific and human-specific genes were upregulated in the brains of fetuses and infants, and especially in the prefrontal cortex, which is involved in cognition. These findings reveal the prevalent addition of new genetic components to the transcriptome of the human brain. More generally, these findings suggest that genomes are continually evolving in both sequence and content, eroding the conservation endowed by common ancestry. Despite increasing recognition of the importance of new genes, we highlight here that these genes are still seriously under-characterized in functional studies and that new gene annotation is inconsistent in current practice. We propose an integrative approach to annotate new genes, taking advantage of functional and evolutionary genomic methods. We finally discuss how the refinement of new gene annotation will be important for the detection of evolutionary forces governing new gene origination. Regardless of recent findings that new genes are important for human brain functions, we highlight that new genes are still generally under-characterized in functional studies and that new gene annotation is inconsistent in current practice. We propose an integrative approach to annotate new genes based on functional and evolutionary genomics.

Description: This study investigates whether the anti-metastasis effect of microRNA-139 (miR-139) on hepatocellular carcinoma (HCC) is mediated through regulating c-fos expression. The expression levels of miR-139 and c-fos in human HCC cell sublines with high (MHCC97H) and low (MHCC97L) spontaneous metastatic potentials were quantified using QPCR or Western blot. miR-139 mimics was transfected into MHCC97H cells to overexpress miR-139, and miR-139 inhibitor was transfected into MHCC97L cells to down-express miR-139. The effect of overexpression or down-expression of miR-139 on c-fos expression of MHCC97H and MHCC97L cells was evaluated using QPCR and Western blot. The 3′ untranslated region segments of FOS containing the miR-139 binding sites were amplified by PCR, and the luciferase activity in the transfected cells was assayed. In comparison with the expression level of miR-139 in MHCC97L cells, the expression level in MHCC97H cells was significantly decreased, whereas c-Fos was significantly up-regulated in MHCC97H. The overexpression of miR-139 significantly inhibited the expression of c-fos in MHCC97H cells, and the down-expression of miR-139 significantly promoted the expression of c-fos in MHCC97L cells. miR-139 suppressed the luciferase activity of the pGL-FOS by approximately 40% compared with the negative control. In vitro cell migration analysis demonstrated that depletion of c-fos or overexpression of miR-139 in MHCC97H cells reduced cell migration, whereas overexpression of c-fos or depletion of miR-139 in MHCC97L cells increased cell migration. Thus, we got the conclusion that miR-139 expression is down-regulated in human HCC cell sublines with high spontaneous metastatic potentials (MHCC97H). Derepression of c-Fos caused by miR-139 down-regulation contributes to the metastasis of HCC. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Colorectal cancer is one of the leading causes of tumour-related deaths. In the present study, the chemopreventive effect of green tea on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis was studied in male Wistar rats. The DMH group received subcutaneous injections of DMH (30 mg kg −1 body weight) once a week for 30 weeks, the normal group received the vehicle of DMH, and the DMH + green tea group received DMH simultaneously with 1% green tea as their sole source of drinking fluid throughout the experimental period. In the DMH group treated with green tea, significant reductions in gene overexpressions of colonic nuclear factor κB (NF-κB), tumour necrosis factor α, inducible nitric oxide synthase and cyclooxygenase 2, and NF-κB immunostaining indicates the anti-inflammatory effect of green tea in attenuating colon cancer. Moreover, the anti-angiogenic and anti-invasiveness effects of green tea were revealed as reductions of both vascular endothelial growth factor and matrix metalloproteinase-7 mRNA expression levels. These effects were confirmed by the significant reduction of serum tumour necrosis factor α, C-reactive protein levels, inhibition of tumour incidence, and nearly normal survival rate and colonic architecture. It can be concluded that green tea exerts a potent chemopreventive effect on colon carcinogenesis possibly due to the inhibition of NF-κB. Copyright © 2012 John Wiley & Sons, Ltd.

Description: The aim of this study was to investigate the effect of isocaloric intake from a high-fat diet (HFD) on insulin resistance and inflammation in rats. Male Wistar rats were fed on an HFD ( n  = 12) or control diet ( n  = 12) for 12 weeks. Subsequently, all animals were euthanized, and blood glucose, insulin, free fatty acids, C-reactive protein, lipid profile, cytokines and hepatic-enzyme activity were determined. Carcass chemical composition was also analyzed. During the first and the twelfth weeks of the experimental protocol, the oral glucose tolerance test and insulin tolerance test were performed and demonstrated insulin resistance ( P  〈 0.05) in the HFD group. Although food intake (g) was lower ( P  〈 0.05) in the HFD group compared with the control group, the concentration of total cholesterol, low-density lipoprotein, C-reactive protein and liver weight were all significantly higher. The kinase inhibitor of κB, c-Jun N-terminal kinase and protein kinase B expressions were determined in the liver and skeletal muscle. After an insulin stimulus, the HFD group demonstrated decreased ( P  = 0.05) hepatic protein kinase B expression, whereas the kinase inhibitor of κB phospho/total ratio was elevated in the HFD muscle ( P  = 0.02). In conclusion, the isocaloric intake from the HFD induced insulin resistance, associated with impaired insulin signalling in the liver and an inflammatory response in the muscle. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ΔNp63α isoform. Here, we investigated the regulation of ΔNp63α expression upon EGFR activation and the role of the EGFR-ΔNp63α axis in proliferation of SCC tumor-initiating cells (TICs). SCC cell lines A-431, Cal-27 and SCC-25 treated with EGF showed a time-dependent increase in ΔNp63α expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ΔNp63α expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ΔNp63α impaired the TICs ability to grow under non-differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ΔNp63α accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non-differentiating conditions. Together, our findings provide evidence that ΔNp63α expression is regulated by EGFR activation through STAT3 and that the EGFR-ΔNp63α axis is crucial for proliferation of TICs present in SCCs. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The archetype driving the drug targeting approach to cancer therapy is the success of imatinib against chronic phase chronic myeloid leukemia (CML-CP). Molecular targeting success of this magnitude has yet to be repeated for most solid tumors. To answer why imatinib remains an exception of cancer research, we summarize key features and patterns of evolution that contrast CML-CP from prostate cancer, an example of a solid tumor that also shares a signature fusion gene. Distinctive properties of CML-CP include: a large cell population size that is not geographically constrained, a highly penetrant dominant oncogene that sweeps the entire cell population, subsequent progressive and ordered clonal genetic changes, and the effectiveness of molecular targeting within the chronic phase, which is comparable to the benign phase of solid tumors. CML-CP progression resembles a clonal, stepwise model of evolution, whereas the pattern of solid tumor evolution is highly dynamic and stochastic. The distinguishing features and evolutionary pattern of CML-CP support why the success of imatinib does not carry over to most solid tumors. Changing the focus of cancer research from a gene-based view to a genome-based concept will provide insight into solid tumor evolutionary dynamics. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Epithelial plasticity characterizes embryonic development and diseases such as cancer. Epithelial-mesenchymal transition (EMT) is a reversible and guided process of plasticity whereby embryonic or adult epithelia acquire mesenchymal properties. Multiple signaling pathways control EMT, and the transforming growth factor β (TGFβ) pathway plays a central role as its inducer. Here, we analyzed the role of the tumor suppressor protein p53 in TGFβ-induced EMT in a well-established mammary epithelial cell model. We found that diploid NMuMG mammary cells express bi-allelically a wild type and a missense mutant (R277C) form of p53. Global reduction of both forms of p53 led to an enhanced EMT response to TGFβ. Conversely, stabilization of wild type p53 using the compound nutlin had a negative impact on EMT. After silencing both p53 forms, rescue experiments using either wild type or R277C mutant p53 revealed that wild type p53 inhibited, whereas the R277C mutant did not significantly affect, the TGFβ-driven EMT response. Under serum-free culture conditions, silencing of total p53 levels led to higher numbers of mammospheres characterized by larger size. Rescue of the silenced endogenous p53 with R277C mutant p53, in contrast, suppressed both size and numbers of the mammospheres. This work proposes that wild type p53 controls the efficiency by which mammary epithelial cells undergo EMT in response to TGFβ. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The aim of this study was to assess the anion transport in equine erythrocytes through the measurement of the sulfate uptake operating from band 3 using different experimental temperatures and buffer solutions. Blood samples of six clinically healthy horses were collected via jugular vein puncture, and an emochrome-citometric examination was performed. The blood was divided into four aliquots and by centrifugation and aspiration the plasma and buffy coat were carefully discarded. The red blood cells were washed with an isosmotic medium and centrifuged. The obtained cell suspensions were incubated with two different experimental buffer solutions (buffer A: 115 mM Na2SO4, 10 mM NaCl, 20 mM ethylenediaminetetraacetic acid, 30 mM glucose; and buffer B: 115 mM Na2SO4, 10 mM NaCl, 20 mM ethylenediaminetetraacetic acid, 30 mM MgCl2) in a water bath for 1 h at 25 °C and 37 °C. Normal erythrocytes, suspended at 3% hematocrit, were used to measure the influx by absorption spectrophotometry at 425 nm wavelength. Unpaired Student's t -test showed a statistically significant decrease ( P  〈 0.01) of rate constants in equine erythrocytes at 25 °C versus 37 °C using both experimental buffer solutions. Comparing the buffer A with buffer B unpaired Student's t -test showed statistically lower values ( P  〈 0.0001) for A solution versus B solution both at 25 °C and at 37 °C. The greater inhibition of SO 4 = influx measured in equine erythrocytes indicates the increased formation of the sulfydryl bonds in band 3 and the modulation of the sulfydryl groups, culminating in the conformational changes in band 3. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Gap junction channels mediate direct cell-cell communication via the exchange of second messengers, ions, and metabolites from one cell to another. Mutations in several human connexin ( cx ) genes, the subunits of gap junction channels, disturb the development and function of multiple tissues/organs. In particular, appropriate function of Cx43 is required for skeletal development in all vertebrate model organisms. Importantly, it remains largely unclear how disruption of gap junctional intercellular communication causes developmental defects. Two groups have taken distinct approaches towards defining the tangible molecular changes occurring downstream of Cx43-based gap junctional communication. Here, these strategies for determining how Cx43 modulates downstream events relevant to skeletal morphogenesis are reviewed. © 2012 Wiley Periodicals, Inc.

Description: Renal cell carcinoma (RCC) is the most common renal tumour in adults. Altered levels of secondary messengers, that is, intracellular calcium and cyclic AMP (cAMP), have been implicated in the pathogenesis of various malignancies. In the present study, we measured levels of intracellular calcium and cAMP in RCC. The intracellular calcium level was significantly reduced, whereas the cAMP level was significantly augmented in RCC as compared with adjacent grossly normal renal parenchyma. Copyright © 2012 John Wiley & Sons, Ltd.

Description: We have previously determined that integrin α11β1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11 -/- mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11 -/- mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11-deficient iPDL cells, including matrix metalloproteinase-13 (MMP-13) and cathepsin K. α11 -/- iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP-13 protein expression levels. The MMP-13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin-mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11β1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11β1-dependent matrix remodeling, involving MMP-13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Fat accumulation in obese individuals worsens the clinical outcomes of cardiovascular disease (CVD). Paradoxically, increased circulating adipocytokines secreted from visceral fat may confer cardioprotective effects. Visfatin, a novel adipocytokine, has anti-diabetic, anti-tumor, and pro-inflammatory properties. However, its effects on cardiomyocytes and the underlying mechanisms remain unknown. This paper demonstrated that visfatin counteracted H 2 O 2 -induced apoptotic damage in H9c2 cardiomyocytes in a time-dependent manner. Qualitative immunofluorescence approaches demonstrated that visfatin pretreatment attenuated H 2 O 2 -induced DNA fragmentation (TdT-mediated dUTP-biotin nick end-labeling), phosphatidyl serine exposure (Annexin V/PI staining), and mitochondrial membrane potential (ΔΨm) depolarization (JC-1 staining). Biochemical studies on cardiomyoctes showed improved cell viability and reduced caspase-3 activation caused by visfatin pretreatment. Visfatin did not inhibit the death receptor-dependent apoptotic pathways, as characterized by its absence in both Fas and TNFR1 down-regulation. Instead, visfatin specifically suppressed the mitochondria-dependent apoptotic pathways, as characterized by changed levels of p53 and its downstream Bcl-2 family genes. Visfatin also up-regulated the protein levels of phosphorylated AMPK, and the anti-apoptotic action of visfatin was attenuated by the AMPK-specific inhibitor compound C. These results suggested that visfatin plays a critical role in cardioprotection by suppressing myocardial apoptosis via AMPK activation. These findings may be the missing link between obesity and CVD. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Mollugin is a naphthohydroquine found in the roots of Rubia cordifolia , and has been reported to have a variety of biological activities, including anti-inflammatory and apoptotic effects. In the present study, we investigated the molecular mechanisms by which mollugin exerts anti-tumor effect in HER2-overexpressing cancer cells. Our results showed that mollugin exhibited potent inhibitory effects on cancer cell proliferation, especially in HER2-overexpressing SK-BR-3 human breast cancer cells and SK-OV-3 human ovarian cancer cell lines in a dose- and time-dependent manner without affecting immortalized normal mammary epithelial cell line MCF-10A. Furthermore, we found that a blockade of Akt/SREBP-1c signaling through mollugin treatment significantly reduced FAS expression and subsequently suppressed cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Mollugin treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level, potentially in part through suppression of NF-κB activation. The combination of mollugin with a MEK1/2 inhibitor may be required in order to achieve optimal efficacy in HER2-overexpressing cancers. These data provide evidence that mollugin inhibits proliferation and induces apoptosis in HER2-overexpressing cancer cells by blocking expression of the FAS gene through modulation of a HER2/Akt/SREBP-1c signaling pathway. Our findings suggest that mollugin is a novel modulator of the HER2 pathway in HER2-overexpressing cancer cells with a potential role in the treatment and prevention of human breast and ovarian cancer with HER2 overexpression. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Azithromycin is an antibiotic with anti-inflammatory properties used as an adjunct to treat periodontitis, a common inflammatory mediated condition featuring pathologic alveolar bone resorption. This study aimed to determine the effect of azithromycin on human osteoclast formation and resorptive activity in vitro . Osteoclasts were generated from peripheral blood mononuclear cells stimulated with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B (RANK) ligand. The effects of azithromycin at concentrations ranging from 0.5µg/mL to 40µg/mL were tested. Osteoclast formation and activity, acidification, actin ring formation and expression of mRNA and protein encoding for key osteoclast genes were assessed. The results demonstrated that azithromycin reduced osteoclast resorptive activity at all concentrations tested with osteoclast formation being significantly reduced at the higher concentrations (20µg/mL and 40µg/mL). mRNA and protein expression of key osteoclast transcription factor Nuclear Factor of Activated T cells (NFATc1) was significantly reduced by azithromycin at later stages of osteoclast development (day 17). Azithromycin also reduced tumour necrosis factor receptor associated factor-6 (TRAF6) mRNA expression at day 14, and cathepsin K mRNA expression at day 14 and 17. Integrin β3 and MMP-9 mRNA expression was reduced by azithromycin at day 17 in osteoclasts cultured on dentine. The osteoclast proton pump did not appear to be affected by azithromycin, however formation of the actin ring cytoskeleton was inhibited. This study demonstrates that azithromycin inhibits human osteoclast function in vitro, which may account for at least some of the beneficial clinical effects observed with azithromycin treatment in periodontitis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Tumor-associated macrophages (TAMs) are a key component of the tumor microenvironment and orchestrate various aspects of cancer. Diversity and plasticity are hallmarks of cells of the monocyte-macrophage lineage. In response to distinct signals macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a spectrum of activation states. Metabolic adaptation is a key component of macrophage plasticity and polarization, instrumental to their function in homeostasis, immunity and inflammation. Generally, TAMs acquire a M2-like phenotype that plays important roles in many aspects of tumor growth and progression. There is now evidence that also neutrophils can be driven towards distinct phenotypes in response to microenvironmental signals. The identification of mechanisms and molecules associated with macrophage and neutrophil plasticity and polarized activation provides a basis for new diagnostic and therapeutic strategies. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Low-power laser irradiation (LPLI) has been shown to exert promotive effects on cell survival and proliferation through activation of various signaling pathways. Estrogen receptors (ERs, ERa and ERβ) are ligand-activated transcription factors, which regulate target gene expression, promote cell proliferation and resist apoptosis. However, it is unclear whether LPLI could induce ligand-independent activation of ERs. In the present study, we investigated the subcellular pools, nuclear redistribution and transcriptional activity of ERs under LPLI (1.2 J/cm 2 , 633 nm) treatment using single-molecule fluorescence imaging and dual-luciferase reporter assay. We found that ERs were not only localized to nucleus, but also existed in mitochondria. Moreover, we found that LPLI induced nuclear redistribution and transcriptional activity of ERs in a ligand-independent manner. Our further investigation showed that PI3-K/Akt signaling cascade was involved in LPLI-induced activation of ERs. Wortmannin, a PI3-K inhibitor, or triciribine (API-2), a specific Akt inhibitor, potently suppressed the nuclear redistribution and transcriptional activity of ERs induced by LPLI, revealing that PI3-K/Akt signaling cascade was required for the activation of ERs induced by LPLI. Collectively, we demonstrated the first time that LPLI induced the ligand-independent nuclear redistribution and transcriptional activity of ERs, which were dependent on the activity of PI3-K/Akt. Our findings provide direct evidence for the molecular mechanisms of LPLI-induced transcription factor activation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias . In this study we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in 2-fold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na + /K + ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycoside specifically suppress steroidogenesis through strong inhibition of 11β-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21β-hydroxylase, but not 3β-hydroxysteroid dehydrogenase/isomerase. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: We used vertical growth phase (VGP) human VMM5 melanoma cells to ask whether the tumor microenvironment could induce Matrix Metalloproteinase-1 (MMP-1) in vivo , and whether this induction correlated with metastasis. We isolated two clones from parental VMM5 cells: a low MMP-1 producing clone (C4) and high producing clone (C9). When these clones were injected orthotopically (intradermally) into nude mice, both were equally tumorigenic and produced equivalent and abundant amounts of MMP-1. However, the tumors from the C4 clones displayed different growth kinetics and distinct profiles of gene expression from the C9 population. The C4 tumors, which had low MMP-1 levels in vitro , appeared to rely on growth factors and cytokines in the microenvironment to increase MMP-1 expression in vivo , while MMP-1 levels remained constant in the C9 tumors. C9 cells, but not C4 cells, grew as spheres in culture and expressed higher levels of Jarid 1b, a marker associated with melanoma initiating cells. We conclude that VMM5 melanoma cells exhibit striking intra-tumor heterogeneity, and that the tumorigenicity of these clones is driven by different molecular pathways. Our data suggest that there are multiple mechanisms for melanoma progression within a tumor, which may require different therapeutic strategies. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Numerous studies have reported that Vpr alters NF-κB signaling in various cell types, however, the findings have been largely conflicting with reports of both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells using an adenovirus based expression and indicator system. Our results show that Vpr is inhibitory to NF-κB, however, this effect is dependant on the particular manner of NF-κB stimulation. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF-α pathway, but not affecting the LPS pathway, suggesting that differential targets of Vpr may exist for NF-κB regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of IκBα in response to TNF-α stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Chronically HIV-1 infected U1 cells with knockdown constructs for Vpr were unexpectedly less responsive to TNF-α mediated viral replication, perhaps suggesting that other HIV-1 components may antagonize these anti-NF-κB effects in infected cells. We hypothesize that Vpr may serve an important role in the context of viral infection and immune function in vivo, through its selective inhibition of NF-κB pathways. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Colorectal cancer is ranked among the top leading causes of cancer death in industrialized populations. Polycomb group proteins (PcGs), including Suz12 and Ezh2, are epigenetic regulatory proteins that act as transcriptional repressors of many differentiation-associated genes and are overexpressed in a large subset of colorectal cancers. Retinoic acid (RA) acts as a negative regulator of PcG actions in stem cells, but has shown limited therapeutic potential in some solid tumors, including colorectal cancer, in part because of RARβ silencing. Through treatment with RA, Suz12 shRNA knockdown, or Ezh2 pharmacological inhibition with 3-deazaneplanocin A (DZNep), we increased TRAIL-mediated apoptosis in human colorectal cancer cell lines. This increased apoptosis in human colon cancer cells after RA or DZNep treatment was associated with a ∼2.5-fold increase in TNFRSF10B (DR5) transcript levels and a 42% reduction in the H3K27me3 epigenetic mark at the TNFRSF10B promoter after DZNep addition. Taken together, our findings indicate that pharmacological inhibition of PRC2 histone methyltransferase activity may constitute a new epigenetic therapeutic strategy to overcome RA non-responsiveness in a subset of colorectal tumors by increasing TRAIL-mediated apoptosis sensitivity. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The capacity of cartilage self-regeneration is considered to be limited. Joint injuries often evolve in the development of chronic wounds on the cartilage surface. Such lesions are associated with articular cartilage degeneration and osteoarthritis. Re-establishing a correct micro/macro-environment into damaged joints could stop or prevent the degenerative processes. This study investigated the effect of polydeoxyribonucleotides (PDRNs) on cartilage degradation in vitro and on cartilage extracted cells. The activities of matrix metalloproteinases 2 and 9 were measured in PDRN-treated cells and in controls at days 0 and 30 of culture. Human nasal cartilage explants were cultured, and the degree of proteoglycan degradation was assessed by measuring the amount of glycosaminoglycans released into the culture medium. The PDRN properties compared with controls were tested on cartilage tissues to evaluate deposition of extracellular matrix. Chondrocytes treated with PDRNs showed a physiological deposition of extracellular matrix (aggrecan and type II collagen: Western blot, IFA, fluorescence activated cell sorting, Alcian blue and safranin O staining). PDRNs were able to inhibit proteoglycan degradation in cartilage explants. The activities of matrix metalloproteinases 2 and 9 were reduced in all PDRN-treated samples. Our results indicate that PDRNs are suitable for a long-term cultivation of in vitro cartilage and have therapeutic effects on chondrocytes by protecting cartilage. Copyright © 2012 John Wiley & Sons, Ltd.

Description: The Pur proteins are an ancient family of sequence-specific single-stranded nucleic acid-binding proteins. They bind a G-rich element in either single- or double-stranded nucleic acids and are capable of displacing the complementary C-rich strand. Recently several reports have described Pur family member knockouts, mutations and disease aberrations. Together with a recent crystal structure of Purα, these data reveal conserved structural features of these proteins that have been adapted to serve functions unique to higher eukaryotes. In humans Pur proteins are critical for myeloid cell development, muscle development, and brain development, including trafficking of mRNA to neuronal dendrites. Pur family members have been implicated in diseases as diverse as cancer, premature aging and fragile-X mental retardation syndrome. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently we described an interplay between mitochondrial-derived oxidants and expression of FOXM1, a redox-responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito-carboxy-proxyl (MCP) and Mito-TEMPOL (MT) caused dose-dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox-responsive green fluorescent protein targeted to mitochondria (mito-roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi-1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Recent studies of prostate cancer and other tumor types have revealed significant support, as well as unexpected complexities, for the application of concepts from normal stem cell biology to cancer. In particular, the cell of origin and cancer stem cell models have been proposed to explain the heterogeneity of tumors during the initiation, propagation, and evolution of cancer. Thus, a basis of intertumor heterogeneity has emerged from studies investigating whether stem cells and/or non-stem cells can serve as cells of origin for cancer and give rise to tumor subtypes that vary in disease outcome. Furthermore, analyses of putative cancer stem cells have revealed the genetically diverse nature of cancers and expanded our understanding of intratumor heterogeneity and clonal evolution. Overall, the principles that have emerged from these stem cell studies highlight the challenges to be surmounted to develop effective treatment strategies for cancer. Concepts from stem cell biology can explain the basis for intertumoral heterogeneity between different tumors of the same tissue type, as well as intratumoral heterogeneity within a single tumor. In this review, we discuss the cell of origin, cancer stem cells, and clonal evolution, with a focus on prostate cancer.

Description: No abstract is available for this article.

Description: Adipose-derived stem cells (ASCs) have been discovered for more than a decade. Due to the large numbers of cells that can be harvested with relatively little donor morbidity, they are considered to be an attractive alternative to bone marrow derived mesenchymal stem cells. Consequently, isolation and differentiation of ASCs draw great attention in the research of tissue engineering and regenerative medicine. Cartilage defects cause big therapeutic problems because of their low self-repair capacity. Application of ASCs in cartilage regeneration gives hope to treat cartilage defects with autologous stem cells. In recent years, a lot of studies have been performed to test the possibility of using ASCs to re-construct damaged cartilage tissue. In this paper, we have reviewed the most up-to-date articles utilizing ASCs for cartilage regeneration in basic and translational research. Our topic covers differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes, increased cartilage formation by co-culture of ASCs with chondrocytes and enhancing chondrogenic differentiation of ASCs by gene manipulation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: OncostatinM (OSM) belongs to IL-6 subfamily and is mostly produced by T lymphocytes. High levels of OSM are detected in the pannus of rheumatoid arthritis (RA) patients and it may arouse the inflammation responses in joints and eventually leads to bone erosion. Placenta growth factor (PLGF) is an angiogenic factor and highly homologous with vascular endothelial growth factor (VEGF). It has been recently reported that PLGF is highly expressed in synovial tissue and enhances the production of proinflammatory cytokines including TNF-α and IL-6. Here we demonstrated that OSM increased mRNA and protein levels of PLGF in a time- and concentration-dependent manner in RA synovial fibroblasts. Inhibitors of JAK3 and PI3K antagonized OSM-induced production of PLGF. OSM enhanced the phosphorylation of Tyr705-STAT3, Ser727-STAT3, Ser473-Akt, and increased the nuclear translocation of phosphorylated STAT3 time-dependently. Transfection of dominant negative Akt or application of PI3K inhibitor LY294002 significantly inhibited p-Tyr705-STAT3, p-Ser727-STAT3 and PLGF expression, indicating that Akt is involved in JAK3/STAT3/PLGF signaling cascade. To further examine whether STAT3 binds to the promoter region of PLGF, Chip assay was used and it was found that OSM could bind with PLGF promoter, which was inhibited by JAK3 and PI3K inhibitors. Accumulation of PLGF in the pannus may contribute to the inflammation, angiogenesis and joints destruction in RA patients. These findings demonstrated the important role of OSM in the pathology network of RA and provided novel therapeutic drug targets for RA treatment. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The contribution of caveolae in Bone Morphogenetic Protein 2 (BMP2) activated Smad signaling was quantified using a system biology approach. BMP2 plays crucial roles during processes such as hematopoiesis, embryogenesis, and skeletal development. BMP2 signaling is tightly regulated on the plasma membrane by its receptors. The localization of BMP receptors in caveolae and endocytosis through clathrin coated pits are thought to regulate the signaling; however the conclusions in the current literature are inconsistent. Therefore published literature was used to establish a mathematical model that was validated using confocal AFM (atomic force microscopy), confocal microscopy, sucrose density centrifugation followed by western blots, and reporter gene assays. The model and experiments confirmed that both caveolae and CCPs regulate the Smad-dependent signaling pathway, however caveolae are centers at the plasma membrane where receptor - ligand interaction is crucial, Smad phosphorylation occurs, and a high degree of Smad signaling is regulated. This demonstrates a role for caveolae that needs to be considered and further studied. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: MicroRNAs are short non-coding RNAs that modulate gene expression by translational repression. Because of their high stability in intracellular as well as extracellular environments, miRNAs have recently emerged as important biomarkers in several human diseases. However, they have not been tested in the cerebrospinal fluid (CSF) of HIV-1 positive individuals. Here, we present results of a study aimed at determining the feasibility of detecting miRNAs in the CSF of HIV-infected individuals with and without encephalitis (HIVE). We also evaluated similarities and differences between CSF and brain tissue miRNAs in the same clinical setting. We utilized a high throughput approach of miRNA detection arrays and identified differentially expressed miRNAs in the frontal cortex of three cases each of HIV+, HIVE, and HIV- controls, and CSF of ten HIV-positive and ten HIV-negative individuals. For the CSF samples, the group of HIV+ individuals contained nine cases of HIV-Associated Neurological Disorders (HAND) and, among those, four had HIVE. All the HIV-negative samples had non-viral acute disseminate encephalomyelitis. A total of 66 miRNAs were found differentially regulated in HIV+ compared to HIV- groups. The greatest difference in miRNA expression was observed when four cases of HIVE were compared to five non-HIVE cases, previously normalized with the HIV-negative group. After statistical analyses, eleven miRNAs were fund significantly up-regulated in HIVE. Although more clinical samples should be examined, this work represents the first report of CSF miRNAs in HIV-infection and offers the basis for future investigation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The majority of hematopoietic stem/progenitor cells (HSPCs) reside in bone marrow (BM) surrounded by a specialized environment, which governs HSPC function. Here we investigated the potential role of bone remodeling cells (osteoblasts and osteoclasts) in homeostasis and stress-induced HSPC mobilization. Peripheral blood (PB) and BM in steady/mobilized state were collected from healthy donors undergoing allogeneic transplantation and from mice treated with granulocyte colony stimulating factor (G-CSF), parathyroid hormone (PTH), or receptor activator of nuclear factor kappa-B ligand (RANKL). The number and the functional markers of osteoblasts and osteoclasts were checked by a series of experiments. Our data showed that the number of CD45 - Ter119 - osteopontin (OPN) + osteoblasts was significantly reduced from 4085 ± 135 cells/femur on day 0 to 1032 ± 55 cells/femur on day 5 in mice (P = 0.02) and from 21.38 ± 0.66 on day 0 to 14.78 ± 0.65 on day 5 in healthy donors (P 〈 0.01). Decrease of osteoblast number leads to reduced level of HSPC mobilization regulators stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF) and OPN. The osteoclast number at bone surface (OC.N/B.s) was significantly increased from 1.53 ± 0.12 on day 0 to 4.42 ± 0.46 on day 5 ( P  〈 0.01) in G-CSF-treated mice and from 0.88 ± 0.20 on day 0 to 3.24 ± 0.31 on day 5 ( P  〈 0.01) in human. Serum TRACP-5b level showed a biphasic trend during G-CSF treatment. The ratio of osteoblasts number per bone surface (OB.N/B.s) to OC.N/B.s was changed after adding PTH plus RANKL during G-CSF treatment. In conclusion, short term G-CSF treatment leads to reduction of osteoblasts and stimulation of osteoclasts, and interrupting bone remodeling balance may contribute to HSPC mobilization. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Thyroid cancer is not very common, accounting for 1-2% of all cancers, with a population incidence of about 0.004%. Currently, the ability to discriminate between follicular adenoma and carcinoma represents the major challenge in preclinical diagnosis of thyroid proliferative lesions. Better discrimination between the two would help avoid unnecessary thyroidectomy and save valuable resources. Over the years, Galectin-3 has been proposed as a diagnostic marker with varied success. In this paper we used Environmental Scanning Electron Microscopy Immunogold Labelling (ESEM-IGL) to investigate the expression of galectin-3 on thin-prep fine needle cytology (FNAC). We optimized the ESEM-IGL method on thyroid cell lines (RO-82 and FTC-133) comparing our membrane Galectin-3 (Gal-3) labelling data with Western Blot. We evaluated 183 thyroid FNAC from Italian patients with a uncertain pre-surgical diagnosis. ESEM-IGL method marker sensitivity is 71.2%, while specificity is 53.3% and diagnostic efficacy is 61.2%. Our results confirmed that Galectin-3 expression is associated with situations of hypertrophy and/or cellular hyperproliferation, pathophysiological situations common both to adenomas and to thyroid carcinomas. The innovation of thyroid FNAC Thin-Prep ESEM-IGL shows the levels of Gal-3 immunolabeling clearly, even through the individual cells of a thyroid nodule. However, galectin-3 alone, as a molecular marker of thyroid cancer, can still have a limited application in pre-surgery diagnosis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Obesity has been shown to be associated with the risk of colorectal cancer (CRC). Adipokines produced by the adipose tissue are linked to some malignancies, including CRC. Visfatin is an adipokine shown to be a biomarker of CRC malignant potential. In addition, the stromal cell-derived factor-1 (SDF-1) has been reported to play a role in CRC progression. Although the relationship between visfatin and CRC has been established, the underlying mechanism has not been clarified. We investigated the molecular mechanism governing the interaction between visfatin stimulation and SDF-1 expression in human CRC cell lines. We found that visfatin stimulation led to an increase in the expression and secretion of SDF-1 in CRC DLD-1 and SW48 cells. Experiments involving specific inhibitors and small interfering RNA demonstrated that the activation of ERK and p38 mitogen-activated protein kinase (MAPK) pathways are critical for visfatin-induced SDF-1 expression. Analysis of transcription factor binding using ELISA and luciferase reporter assays revealed that visfatin increased NF-κB- and AP-1-DNA-binding activities in DLD-1 cells. Inhibition of NF-κB and AP-1 activation blocked the visfatin-induced expression and activity of the SDF-1 promoter. The effect of visfatin on DLD-1 signaling and SDF-1 expression was mediated by β1 integrin. In summary, these findings provide novel insights pertaining to the pathophysiological role of visfatin in CRC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative-RT-PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord-derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Previous studies revealed that gap junction intercellular communication (GJIC) between uterine stromal cells plays critical roles in modulating decidualization, neovasularization and embryo implantation. Connexin (Cx) proteins are the major component of gap junctions and Cx43 is the most widely expressed connexin in endometrium. Phosphorylation of Cx43 was found to impair gap junction communication in this tissue. Using primary human endometrial stromal cells (ESC) and a stable high telomerase-expressing ESC transfectant (T-HESC), we found that retinoic acid (RA) altered the phosphorylation status of Cx43 protein such that there was a decrease in the phosphorylated (P1 and P2) species accompanied by an increase in the non-phosphorylated (P0) form. This process is dependent on protein phosphatase 2A (PP2A) activity since selective PP2A inhibitors prevented the ability of RA to dephosphorylate Cx43. Although RA had no effect on total PP2A expression or activity, it significantly increased the intracellular association of Cx43 and PP2A. Inhibition of transcription and protein synthesis by actinomycin D and cycloheximide, respectively, had no effect on the RA-induced changes in the Cx43 phosphorylation pattern. Furthermore, BMS493, a potent antagonist of the classical RA-mediated transcriptional pathway, did not inhibit RA-induced Cx43 dephosphorylation. Our data indicate that RA stimulates physical association of PP2A with Cx43, resulting in the dephosphorylation of Cx43 and, as a consequence, up-regulation of GJIC in endometrial stromal cells. This process is independent of new mRNA and protein synthesis and suggests a novel mechanism by which aberrant retinoid metabolism can explain certain reproductive disorders manifested by dysfunctional endometrial cell GJIC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Tamoxifen is a triphenylethylene non-steroidal antiestrogen anticancer agent. It also shows inhibitory effects on metastasis of estrogen receptor (EsR)-independent tumors, but the underlying mechanism is unclear. It was demonstrated in this study that, in EsR-negative and highly metastatic human hepatocellular carcinoma MHCC97H cells, tamoxifen inhibited cell migration, volume-activated Cl - currents ( I cl,vol ) and regulatory volume decrease (RVD) in a concentration–dependent manner with a similar IC 50 . Analysis of the relationships between migration, I cl,vol and RVD showed that cell migration was positively correlated with I cl,vol and RVD. Knockdown of the expression of ClC-3 Cl - channel proteins by ClC-3 shRNA or siRNA inhibited I cl,vol , and cell migration, and these inhibitory effects could not be increased further by addition of tamoxifen in the medium. The results suggest that knockdown of ClC-3 expression may deplete the effects of tamoxifen; tamoxifen may inhibit cell migration by modulating I cl,vol and cell volume. Moreover, tamoxifen decreased the activity of Protein Kinase C (PKC) and the effects were reversed by the PKC activator PMA. Activation of PKC by PMA could competitively downregulate the inhibitory effects of tamoxifen on I Cl,vol . PMA promoted cell migration, and knockdown of ClC-3 expression by ClC-3 siRNA abolished the PMA effect on cell migration. The results suggest that tamoxifen may inhibit I Cl,vol by suppressing PKC activation; I cl,vol may be an EsR-independent target for tamoxifen in the anti-metastatic action on cancers, especially on EsR-negative cancers. The finding may have an implication in the clinical use of tamoxifen in the treatments of both EsR-positive and EsR-negative cancers. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Angiopoietin 1 (Ang1) plays an important role in various endothelial functions, such as vascular integrity and angiogenesis; however, less is known about its function outside of the endothelium. In this study, we examined whether Ang1 has direct effects on skeletal muscle cells. We found that Ang1 exhibited myogenic potential, as it promoted the proliferation, migration, and differentiation of mouse primary skeletal myoblasts. The positive effect of Ang1 on myoblast proliferation could have been mediated by the α7 and β1 integrins. We also found that Ang1 potentiated cellular Ca 2+ movements in differentiated myotubes in response to stimuli, possibly through the increased expression of two Ca 2+ -related proteins, namely, Orai1 and calmodulin. Ang1 also increased Orai1 and calmodulin expression in mouse hearts in vivo . These results provide an insight into the molecular mechanisms by which Ang1 directly affects the myogenesis of striated muscle. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: High mobility group box 1 (HMGB1) protein is a crucial cytokine that mediates response to infection, injury, and inflammation. Rosmarinic acid (RA) is an important component of the leaves of Perilla frutescens and has neuroprotective, anti-microbial, anti-oxidant, and anti-cancer effects but little is known of its effects on HMGB1-mediated inflammatory response. Here, we investigated this issue by monitoring the effects of RA on the lipopolysaccharide (LPS) or cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated modulation of inflammatory responses. RA potently inhibited the release of HMGB1 and down-regulated HMGB1-dependent inflammatory responses in human endothelial cells. RA also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. Furthermore, RA reduced CLP-induced HMGB1 release and sepsis-related mortality. Given these results, RA should be viewed as a candidate therapeutic agent for the treatment of various inflammatory diseases via inhibition of the HMGB1 signaling pathway. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including Osterix, Bone sialoprotein (Bsp), and Osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: We have recently shown that many mediators of the JAK/STAT signaling pathway are present in ejaculated human spermatozoa. Among them, STAT3 is detected mainly in membranes and flagellar cytoskeletal fractions. In order to determine the importance of STAT3-mediated signaling, sperm were incubated with Stattic V, a specific inhibitor. Effects on motility were evaluated by CASA, sperm acrosomal integrity was evaluated by FITC conjugated lectin (PSA or PNA) staining, and protein phosphotyrosine content was assessed by western blot using a monoclonal anti-phosphotyrosine antibody. INDO1-AM and JC-1 were used to measure sperm intracellular calcium and mitochondrial membrane potential, respectively, by flow cytometry, and reactive oxygen species (ROS) production was investigated by luminol-based assay. Percentages of motility and motility parameters were significantly affected by Stattic V. This later also significantly increased intracellular Ca 2+ levels, progesterone- and calcium ionophore (A23187)-induced acrosome reaction. On the other hand, a significant decrease in ATP content was measured when sperm were treated with Stattic V, associated with depolarization of mitochondrial membrane and elevated ROS production. These results suggest that STAT3 is involved in sperm functions, at least through regulation of mitochondrial activity. This further emphasizes that STAT3 mediates cellular activities in a manner different than strictly the activation of gene transcription. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: How fast? How strong? How many? So what? Why do numbers matter in biology? Chromatin binding proteins are forever in motion, exchanging rapidly between bound and free pools. How do regulatory systems whose components are in constant flux ensure stability and flexibility? This review explores the application of quantitative and mathematical approaches to mechanisms of epigenetic regulation. We discuss methods for measuring kinetic parameters and protein quantities in living cells, and explore the insights that have been gained by quantifying and modelling dynamics of chromatin binding proteins. Current models for chromatin mediated gene regulation often describe molecules as binding, modifying or recruiting other molecules, but with little reference to the quantitative differences between them. In this review we explore how quantitative and mathematical approaches can give insights into mechanisms of epigenetic regulation.

Description: Evolutionary theory assumed that mutations occur constantly, gradually, and randomly over time. This formulation from the “modern synthesis” of the 1930s was embraced decades before molecular understanding of genes or mutations. Since then, our labs and others have elucidated mutation mechanisms activated by stress responses. Stress-induced mutation mechanisms produce mutations, potentially accelerating evolution, specifically when cells are maladapted to their environment, that is, when they are stressed. The mechanisms of stress-induced mutation that are being revealed experimentally in laboratory settings provide compelling models for mutagenesis that propels pathogen–host adaptation, antibiotic resistance, cancer progression and resistance, and perhaps much of evolution generally. We discuss double-strand-break-dependent stress-induced mutation in Escherichia coli. Recent results illustrate how a stress response activates mutagenesis and demonstrate this mechanism's generality and importance to spontaneous mutation. New data also suggest a possible harmony between previous, apparently opposed, models for the molecular mechanism. They additionally strengthen the case for anti-evolvability therapeutics for infectious disease and cancer. Stress-induced mutation molecular mechanism in Escherichia coli : repair of DNA double-strand breaks is switched to a mutagenic mode using DinB and other error-prone DNA polymerases during stress, by the RpoS stress response. This increases genetic diversity, and the ability to evolve, when cells are maladapted to their environment: when stressed.

Description: Scientists should learn to communicate effectively with their colleagues through long-term, sustained training instead of ad hoc, one-off “interventions” that may or may not occur during graduate school or postdoctoral work. Since such training may place unreasonable demands on research advisors, institutions should create career opportunities for “peer-peer communication teachers.”

Description: Hedgehog is an important morphogenic signal that directs pattern formation during embryogenesis, but its activity also remains present through adult life. It is now becoming increasingly clear that during the reproductive phase of life and beyond it continues to direct cell renewal (which is essential to combat the chronic environmental stress to which the body is constantly exposed) and counteracts vascular, osteolytic and sometimes oncological insults to the body. Conversely, down-regulation of hedgehog signalling is associated with ageing-related diseases such as type 2 diabetes, neurodegeneration, atherosclerosis and osteoporosis. Hence, in this essay we argue that hedgehog signalling is not only important at the start of life, but also constitutes an important anti-geriatric influence, and that enhanced understanding of its properties may contribute to developing rational strategies for healthy ageing and prevention of ageing-related diseases. Also watch the Video Abstract Hedgehog is a morphogenic signal during embryogenesis and adult life with many vital biological functions such as neuromodulators and anti-adipogenesis. Downregulation of Hedgehog signalling is associated with ageing-related diseases like Alzheimer/Parkinson and T2D diseases and upregulation of this signalling reduces risk of these diseases. Thus, Hedgehog signalling is antagonist of ageing.

Description: The present study was designed to determine the modulatory effect of aqueous Azadirachta indica leaf extract (AAILE) on cell cycle–associated proteins during two-stage skin carcinogenesis in mice. Considering the dual role of reactive oxygen species in cancer and its chemoprevention, the levels of lipid peroxidation (index of peroxidative damage) were also determined. Skin tumours were induced by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) as a carcinogen followed by the repetitive application of 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Skin tumours obtained in the DMBA/TPA group exhibited enhanced expression of proliferating cell nuclear antigen (PCNA, index of proliferation), p21 and cyclin D1, with no alterations in p53 expression in comparison to the control group. Tumours in AAILE + DMBA/TPA group exhibited low PCNA and cyclin D1 expression and enhanced expression of p53 and p21 in comparison to the DMBA/TPA group. The skin tumours obtained in the AAILE + DMBA/TPA group exhibited high lipid peroxidation levels in comparison to the tumours obtained in the DMBA/TPA group. The observations of the present study suggest that AAILE behaves as a pro-oxidant in the tumours, thereby rendering them susceptible to damage, which eventually culminates into its anti-neoplastic action. Also, cell cycle regulatory proteins may be modulated by AAILE and could affect the progression of cells through the cell cycle. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Peroxiredoxins are ubiquitous proteins that recently attracted major interests in view of the strict correlation observed in several cell lines and/or tissues between different levels of their expression and the increased capacity of cells to survive in different pathophysiological conditions. They are recently considered as the most important enzymes regulating the concentration of hydroperoxides inside the cells. Most of neurodisorders such as Parkinson, Huntington, Alzheimer's diseases and ischemic injury are characterized by conditions of oxidative stress inside cells. In these pathophysiological conditions, a strict correlation between cell survival and Prx expression has been found. In CNS all the Prx isoforms are present though with different expression pattern depending on cell phenotype. Interestingly, neurons treated with amyloid beta peptide (Aβ), showed an overexpression of PrxI. In this study, the neuroprotective effect of PrxI after Aβ exposure and the underlying mechanisms by which PrxI expression counteracts cell death was investigated in a well established human AD in vitro model. Taking advantage on cells transfected by a construct where human PrxI is fused with a Green fluorescent protein (GFP) at the C-terminus, we report some events at the basis of cell survival after Aβ injury, suggesting possible new signal cascades dealing with the antiapoptotic effect of PrxI. The results obtained indicated a protective role for PrxI in counteracting Aß injury by increasing cell viability, preserving neurites and decreasing cell death. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: Human mesenchymal stromal or stem cells (hMSCs) are being investigated for cell therapy in a wide range of diseases. MSCs are a potent source of trophic factors and actively remodel their immediate microenvironment through the secretion of bioactive factors in response to external stimuli such as oxygen tension. In this study, we examined the hypothesis that hypoxia influences hMSC properties in part through the regulation of extracellular milieu characterized by the extracellular matrix (ECM) matrices and the associated fibroblast growth factor-2 (FGF-2). The decellularized ECM matrices derived from hMSC culture under both hypoxic ( e.g. , 2% O 2 ) and the standard culture ( e.g. , 20% O 2 ) conditions have different binding capacity to the cell-secreted and exogenenous FGF-2. The reduced hMSC proliferation in the presence of FGF-2 inhibitor and the differential capacity of the decellularized ECM matrices in regulating hMSC osteogeneic and adipogenic differentiation suggest an important role of the endogenous FGF-2 in sustaining hMSC proliferation and regulating hMSC fate. Additionally, the combination of the ECM adhesion and hypoxic culture preserved hMSC viability under serum withdrawal. Together, the results suggest the synergistic effect of hypoxia and the ECM matrices in sustaining hMSC ex vivo expansion and preserving their multi-potentiality and viability under nutrient depletion. The results have important implication in optimizing hMSC expansion and delivery strategies to obtain hMSCs in sufficient quantity with required potency and to enhance survival and function upon transplantation. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: Cyclin dependent kinase 9 (Cdk9) is a serine-threonine kinase, involved in many cellular processes. The regulatory units of Cdk9 are the T family Cyclins (T1, T2) and Cyclin K. Cyclin T2 has two forms termed Cyclin T2a and Cyclin T2b that arise by an alternative splicing of the primary transcript. Upon induction of muscle differentiation, MyoD recruits Cdk9/Cyclin T2 on muscle-specific gene promoter sequences. This complex is able to phosphorylate the C-terminal domain of RNA polymerase II, enhancing MyoD function and promoting myogenic differentiation. This work focuses on the characterization of two murine Cyclin T2 isoforms and the evaluation of the role of Cdk9/Cyclin T2 complexes during the skeletal muscle differentiation. This study demonstrated a predominant expression of isoform b in all stages of differentiation. Moreover, both isoforms of Cyclin T2 are able to activate the myogenic program but Cyclin T2b has a predominant role, in particular during the latest stages. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain Wild Type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (Streptozotocin Induced Diabetes, 200mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro . Non-transfected and hCOX-2-transfected cells were cultured at 5 mM and 25 mM of glucose by 72 hours. At 25 mM there was an increase in apoptosis in non-transfected cells vs those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE 2 in non-tranfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: G-protein coupled designer receptors that are specifically activated by designer drugs have been developed. Here, we have analyzed the regulation of gene transcription following activation of Gα q -coupled designer receptor (Rα q ). Stimulation of human embryonic kidney (HEK) 293 cells expressing Rα q with clozapine- N -oxide (CNO), a pharmacologically inert compound, induced the expression of biologically active Egr-1, a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor (TCF) Elk-1, a key transcriptional regulator of serum response element (SRE)-driven gene transcription, prevented Egr-1 expression. Stimulation of Rα q with CNO increased the transcriptional activation potential of Elk-1 and enhanced transcription of a SRE regulated reporter gene. In addition, AP-1 transcriptional activity was significantly elevated. AP-1 activity was controlled by TCFs and c-Jun in cells expressing an activated Gα q -coupled designer receptor. CNO stimulation did not increase Egr-1 and AP-1 activity in neuroblastoma cells expressing endogenous M3 muscarinic acetylcholine receptors, indicating that CNO did not function as a ligand for these receptors. Rα q stimulation also increased the transcriptional activation potential of CREB and cAMP response controlled gene transcription. Pharmacological and genetic experiments revealed that the protein kinases Raf and ERK were essential to connect Rα q stimulation with enhanced Egr-1 and AP-1 controlled transcription. In contrast, MAP kinase phosphatase-1 functioned as a nuclear shut-off device of stimulus-transcription coupling. The fact that Rα q stimulation activates the transcription factors Egr-1, Elk-1, AP-1, and CREB indicates that regulation of gene transcription is an integral part of Gα q -coupled receptor signaling. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.

Description: Control of osteogenesis by the canonical Wnt and BMP pathways in vivo: The cover image shows a transverse section through a Xenopus tropicalis metamorphosing tadpole phalange. This histological section was stained with Kernechtrot to reveal cell bodies (pink) and Alcian blue to show the cartilage (blue). The circular cartilaginous shaft is surrounded by a layer of bone mineralized matrix (grey ring) secreted by adjacent osteoblasts. On pages 953–962 Marcellini et al. review recent experimental evidence showing that the canonical Wnt and BMP pathways functionally interact as cells differentiate from osteochondroprogenitors to osteoblasts and osteocytes, in the context of the developing vertebrate embryo. BMP signalling specifies multipotent mesenchymal cells into osteochondroprogenitors which are subsequently driven towards the osteoblastic fate by the Wnt pathway. In osteoblasts, both pathways promote differentiation, albeit with notable mechanistic differences. Finally, in osteocytes, the Wnt and BMP pathways exert opposite effects on the control of bone resorption by osteoclasts. Cover by S. Marcellini.

Description: The purpose of this study was to investigate the activities of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and adenosine deaminase (E-ADA; EC 3.5.4.4) in lymphocytes from patients with rheumatoid arthritis (RA). Thirty patients diagnosed with RA through American College of Rheumatology criteria as well as 30 healthy patients were selected. Peripheral blood lymphocytes were isolated, and E-NTPDase and E-ADA activities were assayed. The results demonstrated an increased E-NTPDase activity (both ATP and ADP as substrates) and a decreased E-ADA activity in RA patients. These data suggest an organic effort to preserve the adenosine level, which is known to have anti-inflammatory and analgesic properties, working as a potent suppressor of immune response. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Tumour necrosis factor-α (TNF- α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H 2 S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF-α-induced insulin resistance is involved in endogenous H 2 S generation. The aim of the present study is to investigate the role of endogenous H 2 S in TNF-α-induced insulin resistance by studying 3T3-L1 adipocytes. We found that treatment of 3T3-L1 adipocytes with TNF-α leads to deficiency in insulin-stimulated glucose consumption and uptake and increase in endogenous H 2 S generation. We show that cystathionine γ-lyase (CSE) is catalysed in 3T3-L1 adipocytes to generate H 2 S and that CSE expression and activity are upregulated by TNF-α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF-α-induced insulin resistance in 3T3-L1 adipocytes, whereas H 2 S treatment of 3T3-L1 adipocytes impairs insulin-stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H 2 S system contributes to TNF-α-caused insulin resistance in 3T3-L1 adipocytes. Our findings suggest that modulation of CSE/H 2 S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Failure of surgery for glaucoma is usually due to post-surgical scarring (fibrosis), a process in which fibroblasts play a prominent role. We investigated the molecular mechanisms of such scarring by examining the expression of matrix metalloproteinases and cytokines in Tenon fibroblasts isolated from rats after glaucoma surgery. Filtration surgery was performed in one eye and implant surgery in the other; and Tenon fibroblasts were isolated from the tissue surrounding the bleb after surgery. The cells were cultured and examined for the expression of matrix metalloproteinases (MMPs) by reverse transcription-polymerase chain reaction, immunoblot and gelatin zymographic analyses. Culture supernatants were also assayed for cytokines with a multiplex array. The amounts of MMP-1 and MMP-3 mRNAs and proteins were greater in cells isolated after implant surgery than in those isolated after filtration surgery, with the progression of scar formation being more complete after the former surgery. The secretion of interleukin-6 (IL-6) by cells isolated after filtration surgery was greater than that for cells isolated after implant surgery. Depletion of IL-6 by RNA interference in cells isolated after filtration surgery increased the expression of MMP-1 and MMP-3 in these cells. These results thus suggest that the expression of MMP-1 and MMP-3 in Tenon fibroblasts is regulated by IL-6 during, and may play an important role in, scar formation after glaucoma surgery. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Regardless of the original causes and etiology, the progression to renal function declines follows a final common pathway associated with tubulointerstitial injury, in which the proximal tubular epithelial cells (PTEC) are instrumental. Kidney injury molecule-1 (KIM-1) is an emerging biomarker, and its expression and release are induced in PTEC upon injury. KIM-1 plays the role as a double-edged sword and implicates in the process of kidney injury and healing. Expression of KIM-1 is also associated with tubulointerstitial inflammation and fibrosis. More importantly, KIM-1 expressing PTEC play the role as the residential phagocytes, contribute to the removal of apoptotic cells and facilitate the regeneration of injured tubules. The precise mechanism of KIM-1 and its shedded ectodomain on restoration of tubular integrity after injury is not fully understood. Other than PTEC, macrophages (Mø) also implicate in tubular repair. Understanding the crosstalk between Mø and the injured PTEC, is essential for designing appropriate methods for controlling the sophisticated machinery in tubular regeneration and healing. This paper will review the current findings of KIM-1, beginning with its basic structure, utility as a biomarker, and possible functions, with focus on the role of KIM-1 in regeneration and healing of injured PTEC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The mechanisms whereby free fatty acids induce endothelial cell apoptosis are not yet understood. The present study aimed to investigate the role of PKCδ in free fatty acid–induced endothelial cell apoptosis. In addition, we looked for evidence of apoptosis-related interactions between PKCδ and Fas signal pathway. Human umbilical vein endothelial cells were treated with various concentrations of free fatty acids and transiently transfected with PKCδ siRNA or Fas siRNA to inhibit PKCδ or Fas expression. Cell proliferation was determined through colorimetric assays, and apoptosis was quantified using flow cytometry. Protein expression was determined from cell lysates using Western blots with antibodies against p-PKCδTyr512, PKCδ, and Fas. Statistical analyses were performed. Free fatty acids had multiple effects on human umbilical vein endothelial cells, including concentration-dependent inhibition of cell proliferation, induction of apoptosis, increased Fas expression, and increased PKCδ expression and phosphorylation. Inhibition of PKCδ mRNA expression by PKCδ siRNA led to a reduction in both free fatty acid–induced apoptosis and Fas expression. However, Fas siRNA treatment inhibited Fas, but not PKCδ, expression in human umbilical vein endothelial cells. The free fatty acid–induced apoptosis in endothelial cells are possibly mediated by PKCδ and may involve upregulation of its downstream Fas. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum . Some authors have suggested the involvement of a Th2-like immune response in the infected host, which leads to extensive tissue damage. The switch from a Th2 to a Th1 response pattern is one hypothesis to explain the curative properties of immunotherapy. Taking into account the importance of immunotherapy for pythiosis treatment and the contribution of adenine nucleotides in the immunoregulation of the host, we evaluated the ecto-adenosine deaminase (E-ADA; EC 3·5.4·4) activity in lymphocytes from rabbits inoculated with P .  insidiosum . Rabbits were inoculated with 1 milliliter of zoospores subcutaneously injected into the lateral thorax; after developing lesions, the rabbits received eight doses of immunotherapy. E-ADA activity was measured in lymphocytes and the adenine nucleotides and adenosine levels were quantitatively determined in serum. Rabbits with characteristic lesions of pythiosis showed a decreased E-ADA activity (82·36%), a decreased adenosine triphosphate concentration (54·04%) and a higher adenosine concentration (2·51 fold), when compared with controls, after 28 days of inoculation. However, after the immunotherapy, the rabbits showed an increase in the E-ADA activity when compared with control (78·62%), contributing for the change in the immune response. Our results reinforce the hypothesis that the change from a Th2 to a Th1 immune response with the participation of the purinergic system could be responsible for the curative properties of immunotherapy. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Voltage-gated Ca 2+ channels regulate cardiac automaticity, rhythmicity and excitation–contraction coupling. Whereas L-type (Ca v 1·2, Ca v 1·3) and T-type (Ca v 3·1, Ca v 3·2) channels are widely accepted for their functional relevance in the heart, the role of Ca v 2·3 Ca 2+ channels expressing R-type currents remains to be elucidated. We have investigated heart rate dynamics in control and Ca v 2·3-deficient mice using implantable electrocardiogram radiotelemetry and pharmacological injection experiments. Autonomic block revealed that the intrinsic heart rate does not differ between both genotypes. Systemic administration of isoproterenol resulted in a significant reduction in interbeat interval in both genotypes. It remained unaffected after administering propranolol in Ca v 2·3(−|−) mice. Heart rate from isolated hearts as well as atrioventricular conduction for both genotypes differed significantly. Additionally, we identified and analysed the developmental expression of two splice variants, i.e. Ca v 2·3c and Ca v 2·3e. Using patch clamp technology, R-type currents could be detected in isolated prenatal cardiomyocytes and be related to R-type Ca 2+ channels. Our results indicate that on the systemic level, the pharmacologically inducible heart rate range and heart rate reserve are impaired in Ca v 2·3 (−|−) mice. In addition, experiments on Langendorff perfused hearts elucidate differences in basic properties between both genotypes. Thus, Ca v 2·3 does not only contribute to the cardiac autonomous nervous system but also to intrinsic rhythm propagation. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Historically, our understanding of molecular genetic aspects of germ cell development has been limited. Recently, results demonstrated that the derivation of pluripotent stem cells may provide the necessary genetic system to study germ cell development. Here, we characterized an induced pluripotent stem cell (iPSC) line, which can spontaneously differentiate into embryonic bodies (EBs) after 3 days of suspension culture, expressing specific markers of three germ layers. Then, we induced the iPSCs to differentiate into germ cells by culturing adherent EBs in retinoic acid (RA) and porcine follicular fluid (PFF) differentiation medium or seminiferous tubule transplantation. Our results indicated that RA and PFF were beneficial for the derivation of germ cells and oocyte-like cells from iPSCs, and iPSCs transplantation could make a contribution to repairing the testis of infertile mice. Our study offers an approach for further study on the development and the differentiation of germ cells derived from iPSCs. Copyright © 2012 John Wiley & Sons, Ltd.

Description: The aims of this study were to assess the effects and potential mechanisms of parthenolide on the expression of vascular endothelial growth factor (VEGF), interleukin 8 (IL-8) and matrix metalloproteinase 9 (MMP-9) in human breast cancer cell line MDA-MB-231. After incubation with different concentrations of parthenolide for 24 h, MDA-MB-231 cells were collected, and the expressions of VEGF, IL-8 and MMP-9 were measured by real-time PCR and Western blot. The secretions of VEGF, IL-8 and MMP-9 in culture supernatant of MDA-MB-231 cells were then measured with ELISA assays. The NF-κB DNA-binding activity of breast cancer cells treated with parthenolide was analyzed using electrophoretic mobility assays. The real-time PCR and Western blot data showed that the expressions of VEGF, IL-8 and MMP-9 were significantly inhibited by parthenolide at both transcription level and protein level in MDA-MB-231 cells. ELISA results also confirmed these effects at a secretion level. The electrophoretic mobility assay results demonstrated that parthenolide can inhibit NF-κB DNA-binding activity of the breast cancer cells. Hence, the expression of VEGF, IL-8 and MMP-9 may be suppressed by parthenolide through the inhibition of NF-κB DNA-binding activity in MDA-MB-231 cells. Copyright © 2012 John Wiley & Sons, Ltd.

Description: The aim of the present study was to assess the effect of storage time at +4 °C on red blood cell count (RBC), haematocrit (Hct), haemoglobin (Hb), white blood cell count (WBC), thrombocyte count (TC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) in mullet ( Mugil cephalus ) using an automatic method. After blood collection ( T 0 ), all samples were analyzed using both the manual and automatic method. To test the validation of the automatic method, a paired t -test was applied, and no statistical difference was observed. The samples were successively divided into four different aliquots and stored at +4 °C to assess the haematological parameters using the automatic method. The first aliquot was refrigerated for 6 h, the second one for 24 h, the third one for 48 h and the last one at for 72 h. One-way repeated-measures ANOVA showed a significant effect of storage time ( P  〈 0.05) on Hb, WBC, TC, MCH and MCHC. These results suggest that haematological parameters can be assessed within 6 h from blood collection when samples are stored at +4 °C because long-term storage modifies the results of the analyses. Further studies on these parameters could be still needed in various fish species to validate an appropriate method for haematological analysis useful not only for the evaluation of the health status of animal living in captivity and in aquaculture but also to have reliability environmental haematological biomarkers. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Embryonic stem cells (ESCs) have the capacity to differentiate into nearly all sorts of cell types, including germ cells, which were regarded as one type of highly specialized cells in mammals, taking the responsibility of transferring genetic materials to the next generation. Studies on induction differentiation of murine embryonic stem cells (mESCs) into male germ cells, but with a low efficiency, basic reason is that the regulation mechanism of germ cell development in mammals is still unclear. miRNA might play an important role in spermatogenesis in mammals. In this study, several miRNAs, which might be related to spermatogenesis, were initially selected and detected in the mouse tissues by semi-polymerase chain reaction (PCR) and quantitative real time (qRT)-PCR to find a testis-specific miRNA. To study its effect on mESCs differentiation into male germ cells, miR-34c mimics were synthesized and pri-miR-34c-GFP plasmid was constructed, transfected into mESCs and combined with retinoic acid induction. The effects of miR-34c were analysed by morphology, alkaline phosphatase staining, qRT-PCR_and immunofluorescent staining. The results showed that miR-34c promoted mESCs differentiation into male germ-like cells, to some extent. Then miR-34c targeted genes were predicted by bioinformatics; Retinoic acid receptor gamma (RARg) was selected, and two dual-luciferase reporter vectors contained the normal and mutated 3′untranslated region of RARg were constructed, respectively. By miRNA mimics and vector co-transfection experiment, the predicted target gene-RARg was confirmed. In conclusion, we found a mammalian male germ cell specific miRNA—miR-34c, and it might be pivotal in mESCs differentiation into male germ cells through its target—RARg. Copyright © 2012 John Wiley & Sons, Ltd.

Description: MHC II, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHC II coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).

Description: Total parenteral nutrition (TPN) is essential for patients with postoperative impairing gastrointestinal function who are unable to receive and absorb oral/enteral feeding for at least 7 days. Oxidative stress plays a major role in the ethiopathogenesis of cancers. In this study, total antioxidant status (TAS), glutathione peroxidase (GPx), superoxide dismutase, malondialdehyde and ascorbic acid were studied in patients operated because of small intestine, colorectal or pancreatic cancer and subsequently receiving TPN in comparison with patients receiving standard nutrition after the operation. TAS level and GPx activity were decreased in patients with small intestine cancer but did not differ in patients with colorectal and pancreatic cancer before and after surgery. In all patient groups receiving TPN, superoxide dismutase activity after the surgery was kept at the same level as before. On the fifth day after the surgery, malondialdehyde concentration in each group was restored to the value observed before surgery. On the fifth day of TPN treatment, ascorbic acid concentration was increased in every group of patients. TPN applied during the postoperative period alleviates oxidative stress resulting from surgery. In the case of small intestine cancer, the addition of vitamins and antioxidants to the nutrition mixture seems to result in depletion of antioxidant enzymes' activities. Copyright © 2011 John Wiley & Sons, Ltd.

Description: JMJD3, a Jumonji C family histone demethylase, is induced by transcription factor, nuclear factor-kappa B (NF- κ B), in response to various stimuli. JMJD3 is crucial for erasing histone-3 lysine-27 trimethylation (H3K27me3), a modification associated with transcriptional repression and is responsible for the activation of a diverse set of genes. Here, we identify the genes in human leukaemia monocyte (THP-1) human monocytic cells that are significantly affected by the stable knockdown (kd) of JMJD3. Global gene expression levels were detected in stable JMJD3 knockdown THP-1 cells and in tumor necrosis factor-alpha (TNF-α)-stimulated JMJD3-kd THP-1 cells by using a 12-plex NimbleGen human whole genome array. In addition, datasets were analysed by using Ingenuity Pathway Analysis. Stable knockdown of JMJD3 in THP-1 cells affected particularly in expression levels and in downstream effects on inflammatory signalling pathways. JMJD3 attenuation down-regulates various key genes in NF-κB, chemokine and CD40 signalling, and mostly affects inflammatory disease response molecules. In addition, chromatin immunoprecipitation revealed that JMJD3-kd could inhibit several NF- κ B-regulated inflammatory genes by recruiting repressive histone-3 lysine-27 trimethylation to their promoters. Moreover, this study significantly highlights the connexion of NF- κ B with JMJD3, which suggests an epigenetic regulation in different signalling pathways. Finally, this study establishes novel JMJD3 targets through Ingenuity Pathway Analysis. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Triptolide is a diterpenoid triepoxide derived from the traditional Chinese medical herb Tripterygium wilfordii . In the present study, we demonstrated that this phytochemical attenuated colon cancer growth in vitro and in vivo . Using a proteomic approach, we found that 14-3-3 epsilon, a cell cycle- and apoptosis-related protein, was altered in colon cancer cells treated with triptolide. In this regard, triptolide induced cleavage and perinuclear translocation of 14-3-3 epsilon. Taken together, our findings suggest that triptolide may merit investigation as a potential therapeutic agent for colon cancer, and its anticancer action may be associated with alteration of 14-3-3 epsilon. Copyright © 2012 John Wiley & Sons, Ltd.

Description: Studies revealed that Nijmegen Breakage Syndrome protein 1 (NBS1) plays an important role in maintaining genome stability, but the underlying mechanism is controversial and elusive. Our results using clinical samples showed that NBS1 was involved in ataxia-telangiectasia mutated (ATM)-dependent pathway. NBS1 deficiency severely affected the phosphorylation of ATM as well as its downstream targets. BrdU proliferation assay revealed a delay of NBS cells in inhibiting DNA synthesis after Doxorubicin (Dox) treatment. In addition, under higher concentrations of Dox, NBS cells exhibited a much lower level of apoptosis compared to their normal counterparts, indicating a resistance to Dox treatment. Accelerated telomere shortening was also observed in NBS fibroblasts, consistent with an early onset of cellular replicative senescence in vitro . This abnormality may be due to the shelterin protein telomeric binding factor 2 (TRF2) which was found to be upregulated in NBS fibroblasts. The dysregulation of telomere shortening rate and of TRF2 expression level leads to telomere fusions and cellular aneuploidy in NBS cells. Collectively, our results suggest a possible mechanism that NBS1 deficiency simultaneously affects ATM-dependent DNA damage signaling and TRF2-regulated telomere maintenance, which synergistically lead to genomic abnormalities. Copyright © 2011 John Wiley & Sons, Ltd.

Description: Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, which is often hidden behind various sources of noise, convoluted with other information and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review some of the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem-specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but also an integral part of the design and implementation of a microscopy experiment.

Description: The use of super-resolution imaging techniques in cell biology has yielded a wealth of information regarding cellular elements and processes that were invisible to conventional imaging. Focusing on images obtained by stimulated emission depletion (STED) microscopy, we discuss how the new high-resolution data influence the ways in which we use and interpret images in cell biology. Super-resolution images have lent support to some of our current hypotheses. But, more significantly, they have revealed unexpectedly complex processes that cannot be accounted for by the simpler models based on diffraction-limited imaging. The super-resolution imaging data challenge cell biologists to change their theoretical framework, by including, for instance, interpretations that describe multiple functions, functional errors or lack of function for cellular elements. In this context, we argue that descriptive research using super-resolution microscopy is now as necessary as hypothesis-driven research.

Description: The current revolution in biological microscopy stems from the realisation that advances in optics and computational tools and automation make the modern microscope an instrument that can access all scales relevant to modern biology – from individual molecules all the way to whole tissues and organisms and from single snapshots to time-lapse recordings sampling from milliseconds to days. As these and more new technologies appear, the challenges of delivering them to the community grows as well. I discuss some of these challenges, and the examples where openly shared technology have made an impact on the field.

Description: The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs.

Description: Light sheet microscopy is an easy to implement and extremely powerful alternative to established fluorescence imaging techniques such as laser scanning confocal, multi-photon and spinning disk microscopy. By illuminating the sample only with a thin slice of light, photo-bleaching is reduced to a minimum, making light sheet microscopy ideal for non-destructive imaging of fragile samples over extended periods of time. Millimeter-sized samples can be imaged rapidly with high resolution and high depth penetration. A large variety of instruments have been developed and optimized for a number of different samples: Bessel beams form thin light sheets for single cells, and selective plane illumination microscopy (SPIM) offers multi-view acquisition to image entire embryos with isotropic resolution. This review explains how light sheet microscopy involves a conceptually new microscope design and how it changes modern imaging in biology.

Description: The role of the major histocompatibility complex (MHC) in mate choice in humans is controversial. Nowadays, the availability of genetic variation data at genomic scales allows for a careful assessment of this question. In 2008, Chaix et al. reported evidence for MHC-dependent mate choice among European American spouses from the HapMap 2 dataset. Recently, Derti et al. suggested that this observation was not robust. Furthermore, when Derti et al. applied similar analyses to the HapMap 3 European American samples, they did not see a significant effect. Although some of the points raised by Derti et al. are relevant, we disagree with the reported absence of evidence for MHC-dependent mate choice within the HapMap samples. More precisely, we show here that the MHC dissimilarity among HapMap 3 European American spouses is still extreme in comparison to the rest of the genome, even after multiple testing correction. This finding supports the hypothesis of MHC-dependent mate choice in some human populations. The MHC may influence mate choice in some human populations also depending on other variables such as socio-cultural factors, the level of genetic diversity and the strength of pathogenic pressures.

Description: Is a two-fold approach — preliminary studies based on small samples followed by a large-sample study to check reproducibility — in the search for biomarkers really prudent?