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Cellular Functions (cellular + function)
Kinds of Cellular Functions Selected AbstractsMatrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseasesPATHOLOGY INTERNATIONAL, Issue 7 2010Takayuki Shiomi Cellular functions within tissues are strictly regulated by the tissue microenvironment which comprises extracellular matrix and extracellular matrix-deposited factors such as growth factors, cytokines and chemokines. These molecules are metabolized by matrix metalloproteinases (MMP), a disintegrin and metalloproteinases (ADAM) and ADAM with thrombospondin motifs (ADAMTS), which are members of the metzincin superfamily. They function in various pathological conditions of both neoplastic and non-neoplastic diseases by digesting different substrates under the control of tissue inhibitors of metalloproteinases (TIMP) and reversion-inducing, cysteine-rich protein with Kazal motifs (RECK). In neoplastic diseases MMP play a central role in cancer cell invasion and metastases, and ADAM are also important to cancer cell proliferation and progression through the metabolism of growth factors and their receptors. Numerous papers have described the involvement of these metalloproteinases in non-neoplastic diseases in nearly every organ. In contrast to the numerous review articles on their roles in cancer cell proliferation and progression, there are very few articles discussing non-neoplastic diseases. This review therefore will focus on the properties of MMP, ADAM and ADAMTS and their implications for non-neoplastic diseases of the cardiovascular system, respiratory system, central nervous system, digestive system, renal system, wound healing and infection, and joints and muscular system. [source] Pod1 is required in stromal cells for glomerulogenesisDEVELOPMENTAL DYNAMICS, Issue 3 2003Shiying Cui Abstract Pod1 (capsulin/epicardin/Tcf21) is a basic-helix-loop-helix transcription factor that is highly expressed in the mesenchyme of developing organs that include the kidney, lung, gut, and heart. Null Pod1 mice are born but die shortly after birth due to a lack of alveoli in the lungs and cardiac defects. In addition, the kidneys are hypoplastic and demonstrate disrupted branching morphogenesis of the ureteric bud epithelium, a marked reduction in the number of nephrons, a delay in glomerulogenesis, and blood vessel abnormalities. To further dissect the cellular function of Pod1 during kidney development, chimeric mice were generated through aggregations of null Pod1 embryonic stem cells and murine embryos ubiquitously expressing enhanced green fluorescent protein (GFP). Histologic, immunohistochemical, and in situ hybridization analysis of the resulting chimeric offspring demonstrated both cell autonomous and non,cell autonomous roles for Pod1 in the differentiation of specific renal cell lineages that include peritubular interstitial cells and pericytes. Most strikingly, the glomerulogenesis defect was rescued by the presence of wild-type stromal cells, suggesting a non,cell autonomous role for Pod1 in this cell population. © 2003 Wiley-Liss, Inc. [source] Liposome-mediated transfection of mature taste cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2005Ana Marie Landin Abstract The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction-related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used ,-gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome-mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common "promiscuous" promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells. © 2005 Wiley Periodicals, Inc. J. Neurobiol, 2005 [source] Function of a long-term, GLP-1-treated, insulin-secreting cell line is improved by preventing DPP IV-mediated degradation of GLP-1DIABETES OBESITY & METABOLISM, Issue 5 2005B. D. Green Glucagon-like peptide-1 (GLP-1) is an important insulinotropic hormone with potential in the treatment of type 2 diabetes. However, the short biological half-life of the peptide after cleavage by dipeptidylpeptidase IV (DPP IV) is a major limitation. Inhibition of DPP IV activity and the development of resistant GLP-1 analogues is the subject of ongoing research. In this study, we determined cell growth, insulin content, insulin accumulation and insulin secretory function of a insulin-secreting cell line cultured for 3 days with either GLP-1, GLP-1 plus the DPP IV inhibitor diprotin A (DPA) or stable N -acetyl-GLP-1. Native GLP-1 was rapidly degraded by DPP IV during culture with accumulation of the inactive metabolite GLP-1(9,36)amide. Inclusion of DPA or use of the DPP IV-resistant analogue, N -acetyl-GLP-1, improved cellular function compared to exposure to GLP-1 alone. Most notably, basal and accumulated insulin secretion was enhanced, and glucose responsiveness was improved. However, prolonged GLP-1 treatment resulted in GLP-1 receptor desensitization regardless of DPP IV status. The results indicate that prevention of DPP IV action is necessary for beneficial effects of GLP-1 on pancreatic , cells and that prolonged exposure to GLP-1(9,36)amide may be detrimental to insulin secretory function. These observations also support the ongoing development of DPP-IV-resistant forms of GLP-1, such as N -acetyl-GLP-1. [source] Limits of life in hostile environments: no barriers to biosphere function?ENVIRONMENTAL MICROBIOLOGY, Issue 12 2009Jim P. Williams Summary Environments that are hostile to life are characterized by reduced microbial activity which results in poor soil- and plant-health, low biomass and biodiversity, and feeble ecosystem development. Whereas the functional biosphere may primarily be constrained by water activity (aw) the mechanism(s) by which this occurs have not been fully elucidated. Remarkably we found that, for diverse species of xerophilic fungi at aw values of , 0.72, water activity per se did not limit cellular function. We provide evidence that chaotropic activity determined their biotic window, and obtained mycelial growth at water activities as low as 0.647 (below that recorded for any microbial species) by addition of compounds that reduced the net chaotropicity. Unexpectedly we found that some fungi grew optimally under chaotropic conditions, providing evidence for a previously uncharacterized class of extremophilic microbes. Further studies to elucidate the way in which solute activities interact to determine the limits of life may lead to enhanced biotechnological processes, and increased productivity of agricultural and natural ecosystems in arid and semiarid regions. [source] Cardiovascular risk factors and collateral artery formationEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 12 2009D. De Groot Abstract Arterial lumen narrowing and vascular occlusion is the actual cause of morbidity and mortality in atherosclerotic disease. Collateral artery formation (arteriogenesis) refers to an active remodelling of non-functional vascular anastomoses to functional collateral arteries, capable to bypass the site of obstruction and preserve the tissue that is jeopardized by ischaemia. Hemodynamic forces such as shear stress and wall stress play a pivotal role in collateral artery formation, accompanied by the expression of various cytokines and invasion of circulating leucocytes. Arteriogenesis hence represents an important compensatory mechanism for atherosclerotic vessel occlusion. As arteriogenesis mostly occurs when lumen narrowing by atherosclerotic plaques takes place, presence of cardiovascular risk factors (e.g. hypertension, hypercholesterolaemia and diabetes) is highly likely. Risk factors for atherosclerotic disease affect collateral artery growth directly and indirectly by altering hemodynamic forces or influencing cellular function and proliferation. Adequate collateralization varies significantly among atherosclerotic patients, some profit from the presence of extensive collateral networks, whereas others do not. Cardiovascular risk factors could increase the risk of adverse cardiovascular events in certain patients because of the reduced protection through an alternative vascular network. Likewise, drugs primarily thought to control cardiovascular risk factors might contribute or counteract collateral artery growth. This review summarizes current knowledge on the influence of cardiovascular risk factors and the effects of cardiovascular medication on the development of collateral vessels in experimental and clinical studies. [source] The myeloid leukemia factor interacts with COP9 signalosome subunit 3 in Drosophila melanogasterFEBS JOURNAL, Issue 3 2008Wakana Sugano The human myeloid leukemia factor 1 (hMLF1) gene was first identified as an NPM,hMLF1 fusion gene produced by chromosomal translocation. In Drosophila, dMLF has been identified as a protein homologous to hMLF1 and hMLF2, which interacts with various factors involved in transcriptional regulation. However, the precise cellular function of dMLF remains unclear. To generate further insights, we first examined the behavior of dMLF protein using an antibody specific to dMLF. Immunostaining analyses showed that dMLF localizes in the nucleus in early embryos and cultured cells. Ectopic expression of dMLF in the developing eye imaginal disc using eyeless-GAL4 driver resulted in a small-eye phenotype and co-expression of cyclin E rescued the small-eye phenotype, suggesting the involvement of dMLF in cell-cycle regulation. We therefore analyzed the molecular mechanism of interactions between dMLF and a dMLF-interacting protein, dCSN3, a subunit of the COP9 signalosome, which regulates multiple signaling and cell-cycle pathways. Biochemical and genetic analyses revealed that dMLF interacts with dCSN3 in vivo and glutathione S -transferase pull-down assays revealed that the PCI domain of the dCSN3 protein is sufficient for this to occur, possibly functioning as a structural scaffold for assembly of the COP9 signalosome complex. From these data we propose the possibility that dMLF plays a negative role in assembly of the COP9 signalosome complex. [source] Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosiscFEBS JOURNAL, Issue 16 2005Melissa C. Miles Multiple ankyrin repeat motif-containing proteins play an important role in protein,protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s). [source] Role of Ca2+/calmodulin regulated signaling pathways in chemoattractant induced neutrophil effector functionsFEBS JOURNAL, Issue 18 2002Comparison with the role of phosphotidylinositol-3 kinase In human neutrophils, both changes in intracellular Ca2+ concentrations, [Ca2+]i, and activation of phosphatidylinositol-3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca2+]i and its effector molecule calmodulin in human neutrophils. Increased [Ca2+]i alone was sufficient to induce phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase-3, (GSK-3,). Inhibition of calmodulin using a calmodulin antagonist N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), did not effect N -formyl-methionyl-leucyl-phenylalanine (fMLP) induced ERK, p38 MAPK or GSK-3, phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca2+/calmodulin-dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca2+]i. Taken together, our data indicate critical roles for changes in [Ca2+]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca2+/calmodulin sensitive pathway in human neutrophils. [source] Group IID heparin-binding secretory phospholipase A2 is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissuesFEBS JOURNAL, Issue 11 2002Makoto Murakami Group IID secretory phospholipase A2 (sPLA2 -IID), a heparin-binding sPLA2 that is closely related to sPLA2 -IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA2 -IIA. Here we identified the residues of sPLA2 -IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA2 -IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA2s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA2 -IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA2 -IID and sPLA2 -X constitutively, the former of which was negatively regulated by IL-1. sPLA2 -IID, but not other sPLA2 isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA2 -IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA2 -IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions. [source] The localization change of Ybr078w/Ecm33, a yeast GPI-associated protein, from the plasma membrane to the cell wall, affecting the cellular functionFEMS MICROBIOLOGY LETTERS, Issue 1 2003Hiromichi Terashima Abstract The YBR078W/ECM33 gene of Saccharomyces cerevisiae encodes a glycosylphosphatidylinositol (GPI)-attached protein and its disruptant strain exhibited a temperature-sensitive (ts) growth defect. A HA-tagged Ybr078w protein, which complemented the ts growth phenotype of the ybr078w, strain, was predominantly located on the plasma membrane by GPI anchoring. To examine the requirement of the GPI anchoring on the plasma membrane for the function, the ,-minus region of Ybr078w was replaced with those of Ydr534c/Fit1 and Ynl327w/Egt2, which are known as GPI-dependent cell wall proteins. The replacement induced the change in localization of the mutant proteins from the plasma membrane to the cell wall and the mutant proteins lost the function to complement the ts cell growth defect of the ybr078w, strain. In addition, a similar result was obtained in a mutant protein, where the authentic SKKSK sequence at the ,-5 to ,-1 site of Ybr078w was replaced with a synthetic ISSYS sequence. It is concluded that the GPI anchoring on the plasma membrane is required for the Ybr078w function. [source] Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 2 2006Akira Ando Abstract Yeasts used in bread making are exposed to high concentrations of sucrose during sweet dough fermentation. Despite its importance, tolerance to high-sucrose stress is poorly understood at the gene level. To clarify the genes required for tolerance to high-sucrose stress, genome-wide screening was undertaken using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 273 deletions that yielded high sucrose sensitivity, approximately 20 of which were previously uncharacterized. These 273 deleted genes were classified based on their cellular function and localization of their gene products. Cross-sensitivity of the high-sucrose-sensitive mutants to high concentrations of NaCl and sorbitol was studied. Among the 273 sucrose-sensitive deletion mutants, 269 showed cross-sensitivities to sorbitol or NaCl, and four (i.e. ade5,7, ade6, ade8, and pde2) were specifically sensitive to high sucrose. The general stress response pathways via high-osmolarity glycerol and stress response element pathways and the function of the invertase in the ade mutants were similar to those in the wild-type strain. In the presence of high-sucrose stress, intracellular contents of ATP in ade mutants were at least twofold lower than that of the wild-type cells, suggesting that depletion of ATP is a factor in sensitivity to high-sucrose stress. The genes identified in this study might be important for tolerance to high-sucrose stress, and therefore should be target genes in future research into molecular modification for breeding of yeast tolerant to high-sucrose stress. [source] Capsosomes with Multilayered Subcompartments: Assembly and Loading with Hydrophobic CargoADVANCED FUNCTIONAL MATERIALS, Issue 1 2010Leticia Hosta-Rigau Abstract Therapeutic artificial cells or organelles are nanoengineered vehicles that are expected to substitute for missing or lost cellular function. The creation of capsosomes, polymer carrier capsules containing liposomal subcompartments, is a promising approach towards constructing such therapeutic devices using the layer-by-layer assembly method. Herein, the assembly of intact, nonaggregated capsosomes containing multiple liposome layers is reported. It is also further demonstrated that thiocoraline, a hydrophobic model peptide with antitumor activity, can be efficiently loaded into the membrane of the liposomal subcompartments of the capsosomes. Cell viability assays verify the activity of the trapped antitumor cargo. It is also shown that pristine capsosomes do not display inherent cytotoxic effects. The ability to tune the number of liposome layers and hence the drug loading in capsosomes as well as their noncytotoxicity provide new opportunities for the creation of therapeutic artificial cells and organelles. [source] Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3bGENES TO CELLS, Issue 7 2006Akiko Tsumura DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1,/, or Dnmt3a,/,Dnmt3b,/, ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases. [source] Shorter Telomere Length in Peripheral Blood Cells Associated With Migraine in WomenHEADACHE, Issue 6 2010Hua Ren PhD (Headache 2010;50:965-972) Objective., To evaluate relative telomere length of female migraine patients. Background., Migraine is a debilitating disorder affecting 6-28% of the population. Studies on the mechanisms of migraine have demonstrated genetic causes but the pathophysiology and subcellular effects of the disease remain poorly understood. Shortened telomere length is associated with age-related or chronic diseases, and induced stresses. Migraine attacks may impart significant stress on cellular function, thus this study investigates a correlation between shortening of telomeres and migraine. Methods., Relative telomere length was measured using a previously described quantitative polymerase chain reaction method. A regression analysis was performed to assess differences in mean relative telomere length between migraine patients and healthy controls. Results., The leukocyte telomeres of a cohort of 142 Caucasian female migraine subjects aged 18-77 years and 143 matched 17-77-year-old healthy control Caucasian women were examined. A significantly shorter relative telomere length was observed in the migraine group compared with the control group after adjusting for age and body mass index (P = .001). In addition, age of onset was observed to associate with the loss of relative telomere length, especially at early age of onset (<17 years old). No association was observed between relative telomere length and the severity and frequency of migraine attacks and the duration of migraine. Conclusion., Telomeres are shorter in migraine patients and there is more variation in telomere length in migraine patients. [source] Less-oxidative hemodialysis solution rendered by cathode-side application of electrolyzed waterHEMODIALYSIS INTERNATIONAL, Issue 3 2007Masaaki NAKAYAMA Abstract Electrolyzed water (EW) generated on the cathode side reportedly displays anti-oxidative properties, and application of EW to hemodialysis (HD) systems supposedly suppresses oxidative markers in patients on HD. However, most of the chemical properties and biological effects of such solutions remain unclear. This study aimed to examine those issues to clarify the scientific background for the clinical use of EW solution. Reverse osmosis water comprising EW from the cathode side (e-RO) was prepared and used to process a test HD solution (e-HD). Chemical and biological properties of these solutions were compared with controls. Redox properties were examined by chemiluminescence (CL) of the luminol-H2O2 system. Biological effects of e-RO on human polymorphonuclear leukocytes (PMNs) were tested with respect to the cellular protection against methylglyoxal, and with respect to the preservation of cellular function as to radical generation. Control HD solution presented the highest CL, followed by e-HD, control RO, suggesting a lower oxidative capacity for EW-based solutions. Increased levels of dissolved hydrogen were characteristic of e-RO and e-HD. Application of e-RO tended to be associated with less injury of PMNs by methylglyoxal, and with significantly higher levels of radical generation compared with the control. Compared with control HD, e-RO-based HD solution displays less-oxidative capacity in chemical terms, and may at least partly facilitate preservation of PMN viability. These results appear to offer a scientific basis for supporting the clinical challenge of applying this technology to HD treatment. [source] ,-Glutamyltranspeptidase,deficient knockout mice as a model to study the relationship between glutathione status, mitochondrial function, and cellular functionHEPATOLOGY, Issue 4 2000Yvonne Will ,-Glutamyltranspeptidase (GGT)-deficient mice (GGT,/,) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (<20 weeks). Using livers from these mice, we investigated the relationship between GSH content, especially mitochondrial, and mitochondrial and cellular function. We found that the GSH content of isolated liver mitochondria was diminished by ,50% in GGT,/, mice when compared with wild-type mice. Respiratory control ratios (RCRs) of GGT,/, mice liver mitochondria were ,60% those of wild-type mice primarily as a result of impaired state 3 respiration. Mitochondrial adenine nucleotide content was decreased by ,40% in mitochondria obtained from GGT,/, mice. We observed a strong correlation between mitochondrial GSH content and RCRs. Even moderate decreases (<50%) correlated with adverse effects with respect to respiration. Electron microscopy revealed that livers from GGT,/, knockout mice were deprived of fat and glycogen, and swollen mitochondria were observed in animals that were severely deprived of GSH. Thus, GGT,/, mice exhibit a loss of GSH homeostasis and impaired oxidative phosphorylation, which may be related to the rate of adenosine triphosphate (ATP) formation and subsequently leads to progressive liver injury, which characterizes the diseased state. We also found that supplementation of GGT,/, mice with N -acetylcysteine (NAC) partially restored liver GSH, but fully restored mitochondrial GSH and respiratory function. Electron microscopy revealed that the livers of NAC-supplemented GGT,/, mice contained fat and glycogen; however, slightly enlarged mitochondria were found in some livers. NAC supplementation did not have any beneficial effect on the parameters examined in wild-type mice. [source] Phospho-proteomic immune analysis by flow cytometry: from mechanism to translational medicine at the single-cell levelIMMUNOLOGICAL REVIEWS, Issue 1 2006Omar D. Perez Summary:, Understanding a molecular basis for cellular function is a common goal of biomedicine. The complex and dynamic cellular processes underlying physiological processes become subtly or grossly perturbed in human disease. A primary objective is to demystify this complexity by creating and establishing relevant model systems to study important aspects of human disease. Although significant technological advancements over the last decade in both genomic and proteomic arenas have enabled progress, accessing the complexity of cellular interactions that occur in vivo has been a difficult arena in which to make progress. Moreover, there are extensive challenges in translating research tools to clinical applications. Flow cytometry, over the course of the last 40 years, has revolutionized the field of immunology, in both the basic science and clinical settings, as well as having been instrumental to new and exciting areas of discovery such as stem cell biology. Multiparameter machinery and systems exist now to access the heterogeneity of cellular subsets and enable phenotypic characterization and functional assays to be performed on material from both animal models and humans. This review focuses primarily on the development and application of using activation-state readouts of intracellular activity for phospho-epitopes. We present recent work on how a flow cytometric platform is used to obtain mechanistic insight into cellular processes as well as highlight the clinical applications that our laboratory has explored. Furthermore, this review discusses the challenges faced with processing high-content multidimensional and multivariate data sets. Flow cytometry, as a platform that is well situated in both the research and clinical settings, can contribute to drug discovery as well as having utility for both biomarker and patient-stratification. [source] Improving cellular function through modulation of energy metabolismINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 5 2004D. Maes The ambivalent consequences of mitochondrial stimulation on cellular activity have been well established. Mitochondria supply the cell with energy through a process of oxidative phosphorylation but thereby generate free radicals, resulting in the accumulation of hydrogen peroxide in the cytoplasm. We have investigated the impact of cellular senescence as well as UV irradiation, on the balance between these two activities. The adenosine triphosphate (ATP) level, DNA and protein synthesis in fibroblasts obtained from donors between 30 and 90 years of age appeared to be significantly influenced by the aging process. Both DNA and protein synthesis could be stimulated by increasing intracellular ATP levels. In-vitro senescent fibroblasts showed a reduction in the level of ATP as well as a shift in mitochondrial membrane potential. At the same time, there was an increase in intracellular hydrogen peroxide with increasing population doubling, indicating a clear dysfunction of the metabolic machinery in the mitochondria of senescent cells. To counteract this degradation of the energy pool, we treated cells with creatine, which is known to restore the pool of phosphocreatine in the mitochondria. Creatine treatment significantly increased cell survival after UV exposure, stimulated the repair of UVB-induced DNA damage in keratinocytes and caused a significant reduction in the number of sunburn cells in a UVB-exposed reconstituted skin model. These results clearly indicate that restoration of the energy pool in mitochondria increased cellular self-defense mechanism. These data show the important role played by the mitochondrial energy metabolism on the aging process, and indicate a possible therapy that can be used to counteract this negative effect. Treatment with creatine seems to provide the necessary boost to the cellular metabolism, which leads to an induction of a significant amount of protection and repair to human skin cells. [source] Decorin and its galactosaminoglycan chain: Extracellular regulator of cellular function?IUBMB LIFE, Issue 11 2008Daniela G. Seidler Abstract A molecular network of extracellular matrix molecules determines the tissue architecture and accounts for mechanical properties like compressibility or stretch resistance. It is widely accepted that the elements of the cellular microenvironment are important regulators of the cellular behavior in vitro and in vivo. One large group comprising these molecules is the family of proteoglycans. Both, the core proteins and, in particular, the attached galactosaminoglycans, contribute to the regulation network as they bind a variety of signaling molecules, e.g. cytokines, chemokines, growth, and differentiation factors. We would like to emphasize specific patterns of epimerization and sulfation within the galactosaminoglycans chains, because these result in "motifs" that are responsible for the modulation of signal factor binding, release and activity. This property is crucial in physiological and pathological conditions, for example development and wound healing. © 2008 IUBMB IUBMB Life, 60(11): 729,733, 2008 [source] Immunohistochemical assessment of parafibromin in mouse and human tissuesJOURNAL OF ANATOMY, Issue 6 2006Andrea Porzionato Abstract Parafibromin is a protein encoded by the HRPT2 oncosuppressor gene, whose mutation causes the hyperparathyroidism,jaw tumour syndrome, characterized by the occurrence of parathyroid adenoma or carcinoma, fibro-osseous jaw tumours, and renal neoplastic and non-neoplastic abnormalities. Non-morphological techniques, such as Northern and Western blotting and reverse transcriptase-PCR, indicate that parafibromin is ubiquitously expressed, but extensive immunohistochemical studies have not been performed. To increase our knowledge of the distribution and patterns of expression of parafibromin, we examined its expression and location in many different mouse and human organs by immunohistochemistry. There were no substantial differences in parafibromin expression between mouse and human. We found widespread expression of parafibromin, except in connective tissue, smooth muscle, endothelium and some other types of epithelia (colonic, urinary, tubaric, uterine, thyroid). Heterogeneity of positivity intensity and subcellular location (nuclear, nucleocytoplasmic, cytoplasmic) was found between tissues and cell types, suggesting differential functional involvement of parafibromin. Moreover, higher parafibromin expression was found in cell types, such as hepatocytes, cells of the base of gastric glands, renal cortex tubules and the pars intermedia of the hypophysis, which are characterized by different proliferative capacity, thus indicating that the cellular function of parafibromin may not be reduced only to its anti-proliferative effect. [source] Cholesterol-Sensing Receptors, Liver × Receptor , and ,, Have Novel and Distinct Roles in Osteoclast Differentiation and ActivationJOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2006Kirsten M Robertson Abstract The liver × receptor (,,,) is responsible for regulating cholesterol homeostasis in cells. However, our studies using the LXR,,/,, LXR,,/,, and LXR,,/,,,/, mice show that both LXR, and , are also important for bone turnover, mainly by regulating osteoclast differentiation/activity. Introduction: The liver × receptors (,,,) are primarily responsible for regulating cholesterol homeostasis within cells and the whole body. However, as recent studies show that the role for this receptor is expanding, we studied whether the LXRs could be implicated in bone homeostasis and development. Materials and Methods: pQCT was performed on both male and female LXR,,/,, LXR,,/,, LXR,,/,,,/,, and WT mice at 4 months and 1 year of age. Four-month-old female mice were additionally analyzed with reference to qPCR, immunohistochemistry, histomorphometry, transmission electron microscopy, and serum bone turnover markers. Results: At the mRNA level, LXR, was more highly expressed than LXR, in both whole long bones and differentiating osteoblast-like MC3T3-E1 and osteoclast-like RAW 264.7 cells. Four-month-old female LXR,,/, mice had a significant increase in BMD because of an increase in all cortical parameters. No difference was seen regarding trabecular BMD. Quantitative histomorphometry showed that these mice had significantly more endosteal osteoclasts in the cortical bone; however, these cells appeared less active than normal cells as suggested by a significant reduction in serum levels of cross-linked carboxyterminal telopeptides of type I collagen (CTX) and a reduction in bone TRACP activity. Conversely, the female LXR,,/, mice exhibited no change in BMD, presumably because a significant decline in the number of the trabecular osteoclasts was compensated for by an increase in the expression of the osteoclast markers cathepsin K and TRACP. These mice also had a significant decrease in serum CTX, suggesting decreased bone resorption; however, in addition presented with an increase in the expression of osteoblast associated genes, bone formation markers, and serum leptin levels. Conclusions: Our findings show that both LXRs influence cellular function within the bone, with LXR, having an impact on osteoclast activity, primarily in cortical bone, whereas LXR, modulates trabecular bone turnover. [source] HSPA1A is an important regulator of the stability and function of ZNF198 and its oncogenic derivative, ZNF198,FGFR1JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2007Chitta S. Kasyapa Abstract Mass spectroscopy analysis demonstrated that the HSPA1A protein is found in complex with the ZNF198 protein which is involved in a chromosome rearrangement with the FGFR1 gene in an atypical myeloproliferative disease. HSPA1A is a member of the HSP70 family of genes which has been shown to be inducible in a variety of circumstances. Exogenous expression of the ZNF198,FGFR1 fusion kinase gene as well as ZNF198 in a model cell system results in a large (>650-fold) increase in HSP70 mRNA levels. Using KNK437, a specific inhibitor of HSP70 transcription, we have demonstrated that an important function of HSPA1A is to stabilize the ZNF198 and ZNF198,FGFR1 proteins. In the absence of HSPA1A, specific functions of ZNF198,FGFR1 such as STAT3 phosphorylation is also lost. Treatment of cells with KNK437 in the presence of MG132, an inhibitor of proteasomal degradation of proteins, suggested that only the ZNF198,FGFR1 protein is subject to the proteasomal degradation pathway, while ZNF198 is not. These observations suggest an important role for HSPA1A in ZNF198 and ZNF198,FGFR1 mediated cellular function. J. Cell. Biochem. 102: 1308,1317, 2007. © 2007 Wiley-Liss, Inc. [source] Protein modification and replicative senescence of WI-38 human embryonic fibroblastsAGING CELL, Issue 2 2010Emad K. Ahmed Summary Oxidized proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins build up and potentially affect cellular function during replicative senescence of WI-38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresis-based proteomic approach coupled with immunodetection of HNE-, AGE-modified and carbonylated proteins. Thirty-seven proteins targeted for either one of these modifications were identified by mass spectrometry and are involved in different cellular functions such as protein quality control, energy metabolism and cytoskeleton. Almost half of the identified proteins were found to be mitochondrial, which reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE-modified proteins could be explained by the senescence-associated decreased activity of glyoxalase-I, the major enzyme involved in the detoxification of the glycating agents methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age-related build-up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP-stimulated Lon-like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified mitochondrial proteins. [source] Conserved cellular function and stress-mediated regulation among members of the proteolipid protein familyJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2010María E. Fernández Abstract Chronic stress causes morphological alterations in the hippocampus of rodents and tree shrews, including atrophy of CA3 dendrites and loss of synapses. The molecular mechanisms underlying these structural changes remain largely unknown. We have previously identified M6a as a stress responsive gene and shown that M6a is involved in filopodium/spine outgrowth and, likely, synapse formation. M6a belongs to the proteolipid protein (PLP) family, all of their members having four transmembrane domains that allow their localization at the plasma membrane. In the present work, we analyzed other members of this family, the closely related M6b as well as PLP and its splice variant DM20. We found that chronic restraint stress in mice reduces M6b and DM20, but not PLP, mRNA levels in the hippocampus. In addition, M6b and DM20, but again not PLP, induce filopodium formation in primary cultures of hippocampal neurons. Several M6b protein isoforms were studied, all of them having similar effects except for the one lacking the transmembrane domains. Our results reveal a conserved cellular function and a stress-mediated regulation among members of the proteolipid protein family, suggesting an involvement of proteolipid proteins in the stress response. © 2009 Wiley-Liss, Inc. [source] Electrostatic binding of nanoparticles to mesenchymal stem cells via high molecular weight polyelectrolyte chainsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2009Boon C. Heng Abstract Combining stem cell transplantation with nanoparticle-mediated delivery of drugs and pharmaceuticals is envisioned to be one of the next major developmental steps in regenerative medicine. However, a major challenge would be to keep nanoparticles co-localized with stem cells upon transplantation or transfusion in situ. Since nanoparticles are physically much smaller in size than cells and would not specifically bind to extracellular matrix, it is easier for them to disperse from the transplantation site via the blood circulation. Conjugating nanoparticles directly to the cell membrane can potentially interfere with cellular function by physically obstructing cell surface receptors from interacting with the extracellular matrix, various growth factors and cytokines and other cells. Moreover, drug-loaded nanoparticles may be internalized into the cytoplasm via endocytosis or phagocytosis, which may wreak damage on the cellular machinery, leading to impaired physiological function or cell death. A novel solution may be to utilize high molecular weight polyelectrolyte chains to electrostatically bind nanoparticles to cells. For this purpose, hyaluronan, poly- L -lysine and chitosan are of special interest, because these molecules are generally recognized to be biocompatible for application in various pharmaceutical and surgical products. This study investigated the use of these molecules to bind nanoparticles to mesenchymal stem cells (MSCs), and a novel technique of conjugating half the cell surface with nanoparticles through the use of polyelectrolyte chains was also developed. This would avoid blocking MSC interaction with cytokines, growth factors, extracellular matrix and other cells within the recipient tissue/organ upon delivery in situ. Copyright © 2009 John Wiley & Sons, Ltd. [source] Cellulosomes: microbial nanomachines that display plasticity in quaternary structureMOLECULAR MICROBIOLOGY, Issue 6 2007Harry J. Gilbert Summary The assembly of proteins that display complementary activities into supramolecular intra- and extracellular complexes is central to cellular function. One such nanomachine of considerable biological and industrial significance is the plant cell wall degrading apparatus of anaerobic bacteria termed the cellulosome. The Clostridium thermocellum cellulosome assembles through the interaction of a type I dockerin module in the catalytic entities with one of several type I cohesin modules in the non-catalytic scaffolding protein. Recent structural studies have provided the molecular details of how dockerin,cohesin interactions mediate both cellulosome assembly and the retention of the protein complex on the bacterial cell surface. The type I dockerin, which displays near-perfect sequence and structural symmetry, interacts with its cohesin partner through a dual binding mode in which either the N- or C-terminal helix dominate heterodimer formation. The biological significance of this dual binding mode is discussed with respect to the plasticity of the orientation of the catalytic subunits within this supramolecular assembly. The flexibility in the quaternary structure of the cellulosome may reflect the challenges presented by the degradation of a heterogenous recalcitrant insoluble substrate by an intricate macromolecular complex, in which the essential synergy between the catalytic subunits is a key feature of cellulosome function. [source] Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditionsMOLECULAR MICROBIOLOGY, Issue 3 2004Mirna Mujacic Summary Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48°C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50°C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30°C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, ,clpB or ,ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding. [source] The many faces of nitric oxide: cytotoxic, cytoprotective or bothNEUROGASTROENTEROLOGY & MOTILITY, Issue 7 2007J. W. Wiley Abstract, Nitric oxide (NO) has emerged as a major modulator of cellular function in health and disease. In addition to its well-known role as a mediator of smooth muscle relaxation, a rapidly developing body of research suggests, paradoxically, that NO can have both cytotoxic and cytoprotective effects. In this issue of Neurogastroenterology and Motility, Choi et al. provide evidence that supports NO has a prosurvival effect on interstitial cells of Cajal in the mouse stomach. The objective of this short review is to place this interesting report in the context of the current literature. [source] Impairment of Eye Lens Cell Physiology and Optics by Broadband Ultraviolet A,Ultraviolet B Radiation,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2002O. M. Oriowo ABSTRACT The phototoxicity of ultraviolet A (UVA) alone and UVA plus ultraviolet B (UVB) combined on cultured porcine lenses was investigated by analyzing cellular function as measured with a fluorescence bioassay approach and optical integrity, in terms of sharpness of the lens focus as measured with a scanning laser system. The bioassay consisted of carboxyfluorescein diacetate-acetoxymethyl ester and alamarBlue fluorescent dyes. Aseptically dissected porcine lenses were maintained in modified medium 199 without phenol red supplemented with 1% penicillin,streptomycin and 4% porcine serum. At 1 week of preincubation, baseline measurements were obtained. Then the lenses were treated with single exposures of different UVA and UVB energy levels. The lenses treated with 86 J/cm2 UVA alone showed a significant (P < 0.05) decrease in cellular and optical integrity at 48 h after exposure, whereas those treated with 43 J/cm2 UVA alone did not show significant phototoxic effect. Lenses treated with 15.63 J/cm2 UVA plus 0.019 J/cm2 UVB combined showed significant adverse effects beginning from 48 h after exposure. Also, there was no recovery. These findings show that a high UVA dose alone and relatively low UVA in combination with low UVB radiant exposure can impair lens cellular and optical functions, respectively. [source] | |||||||||||||||||||||||||||||||||||||||||||