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Cell Homeostasis (cell + homeostasi)
Selected AbstractsThiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detectionELECTROPHORESIS, Issue 10 2005Angelo Zinellu Abstract Thiols and in particular glutathione (GSH) play a central role in human metabolism, including the detoxification of xenobiotics, cell homeostasis, radioprotection, and antioxidant defence. Here, a new method is provided for the measurement of reduced and total forms of thiols in red blood cells. In order to minimize oxidation of reduced thiols, a water erythrocyte lysis (15 min at 4°C) was performed followed by a protein precipitation step with acetonitrile. The supernatant was rapidly derivatized with 5-iodoacetoamidefluorescein that trapped thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 57 cm × 75 ,m ID capillary by using 5 mmol/L sodium phosphate, 4 mmol/L boric acid as electrolyte solution with 75 mmol/L N -methyl- D -glucamine at pH 11.0. Under these conditions, cysteinylglycine (CysGly), cysteine (Cys), glutathione, and ,-glutamylcysteine (GluCys) were baseline-resolved in , 4 min. Precision tests showed a good repeatability of our method both for migration times (coefficient of variation CV < 0.8%) and areas (CV < 3.3%). Furthermore, a good reproducibility of intrassay and interassay tests was obtained (CV < 5% and CV < 8%, respectively). The method was employed to investigate the effect of acidic precipitation on intracellular thiol concentration. Our data suggest that sample acidification causes a modification of the measured redox thiol status due to the development of a pro-oxidant environment; moreover, the thiol redox status of red blood cells was evaluated in 22 healthy volunteers. [source] Staying alive , naļve CD4+ T cell homeostasisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2007Jared F. Purton Abstract The immune system must maintain a broad repertoire of naļve T cells in order to respond to the diverse range of pathogens that it will encounter over the course of a lifetime. Although it is known that contact with IL-7 is crucial for the survival of naļve T cells, the precise intracellular signals that mediate its effects remain obscure. An article in this issue of the European Journal of Immunology has found that IL-7 requires the coordinated action of multiple pathways to maintain naļve CD4+ T cells. See accompanying article: http://dx.doi.org/10.1002/eji.200737234 [source] Expression and function of the adaptor protein Gads in murine B,cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2005Thomas Abstract Nearly all hematopoietic receptors are dependent on adaptor proteins for the activation of downstream signaling pathways. The Gads adaptor protein is expressed in many hematopoietic tissues, including bone marrow, lymph node, and spleen. Using intracellular staining, we detected Gads protein in a number cells, including B,cells, T,cells, NK cells, monocytes, and plasmacytoid DC, but not in macrophages, neutrophils, or monocyte-derived DC. In the B,cell compartment, Gads was first expressed after immature B,cells leave the bone marrow and was down-regulated after B,cell antigen receptor (BCR) ligation. Female Gads,/, mice had increased numbers of splenic B,cells, as compared to female Gads+/+ mice, suggesting a role for Gads in B,cell homeostasis. Although B,cell production and turnover of splenic B,cell subsets appeared normal in Gads,/, mice, homeostatic proliferation was significantly impaired in Gads,/, B,cells. Whereas BCR ligation can induce apoptosis in wild-type transitional stage 1 (T1) B,cells, Gads,/, T1 B,cells were resistant to BCR-induced apoptosis. Gads,/, B,cells also showed increased BCR-mediated calcium mobilization. We conclude that Gads may have a negative regulatory role in signaling through survival pathways, and is necessary for normal homeostatic proliferation in B,cells. [source] Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosisFEBS JOURNAL, Issue 2 2009Jeanette Blomberg Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-, stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-, stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation. [source] A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprinFEBS JOURNAL, Issue 18 2008Daniel Ambort In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin,Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin,Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unkown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively. [source] Mitochondria and Ca2+ signalingJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2000Emil C. Toescu Abstract Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca2+ signaling. Mitochondria are capable to sense the levels of cytosolic Ca2+ and generate mitochondrial Ca2+ responses. Specific mechanisms for both Ca2+ uptake and Ca2+ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca2+ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca2+ transport systems and some of the functional consequences of mitochondrial Ca2+ uptake [source] Defining ETS transcription regulatory networks and their contribution to breast cancer progressionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2007David P. Turner Abstract ETS factors are members of one of the largest families of evolutionarily conserved transcription factors, regulating critical functions in normal cell homeostasis, that when perturbed contribute to tumor progression. The well documented alterations in ETS factor expression and function during breast cancer progression result in pleiotropic effects manifested by the downstream effect on their target genes. Multiple ETS factors bind to the same regulatory sites present on target genes, suggesting redundant or competitive functions. Furthermore, additional events contribute to, or may be necessary for, target gene regulation. In order to advance our understanding of the ETS-dependent regulation of breast cancer progression and metastasis, this prospect article puts forward a model for examining the effects of simultaneous expression of multiple transcription factors on the transcriptome of non-metastatic and metastatic breast cancer. Compared to existing RNA profiles defined following expression of individual transcription factors, the anti- and pro-metastatic signatures obtained by examining specific ETS regulatory networks will significantly improve our ability to accurately predict tumor progression and advance our understanding of gene regulation in cancer. Coordination of multiple ETS gene functions also mediates interactions between tumor and stromal cells and thus contributes to the cancer phenotype. As such, these new insights may provide a novel view of the ETS gene family as well as a focal point for studying the complex biological control involved in tumor progression. J. Cell. Biochem. 102: 549,559, 2007. © 2007 Wiley-Liss, Inc. [source] Disruption of neurogenesis by amyloid ,-peptide, and perturbed neural progenitor cell homeostasis, in models of Alzheimer's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Norman J. Haughey Abstract Neurogenesis occurs in the adult mammalian brain and may play roles in learning and memory processes and recovery from injury, suggesting that abnormalities in neural progenitor cells (NPC) might contribute to the pathogenesis of disorders of learning and memory in humans. The objectives of this study were to determine whether NPC proliferation, survival and neuronal differentiation are impaired in a transgenic mouse model of Alzheimer's disease (AD), and to determine the effects of the pathogenic form of amyloid ,-peptide (A,) on the survival and neuronal differentiation of cultured NPC. The proliferation and survival of NPC in the dentate gyrus of the hippocampus was reduced in mice transgenic for a mutated form of amyloid precursor protein that causes early onset familial AD. A, impaired the proliferation and neuronal differentiation of cultured human and rodent NPC, and promoted apoptosis of neuron-restricted NPC by a mechanism involving dysregulation of cellular calcium homeostasis and the activation of calpains and caspases. Adverse effects of A, on NPC may contribute to the depletion of neurons and cognitive impairment in AD. [source] Expression of SLURP-1, an endogenous ,7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2009Kazuhide Horiguchi Abstract Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for ,7 nicotinic acetylcholine (ACh) receptors (,7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-, (TNF-,) release from keratinocytes and macrophages via ,7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-, release from macrophages in bronchial tissue. © 2009 Wiley-Liss, Inc. [source] Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2005Elena Fonfrķa Abstract Cerebellar granule cells are targeted selectively by mercury compounds in vivo. Despite the affinity of mercury for thiol groups present in all cells, the molecular determinant(s) of selective cerebellar degeneration remain to be elucidated fully. We studied the effect of mercury compounds on neuronal glutamate transport in primary cultures of mouse cerebellar granule cells. Immunoblots probed with an antibody against the excitatory amino acid transporter (EAAT) neuronal glutamate transporter, EAAT3, revealed the presence of a specific band in control and mercury-treated cultures. Micromolar concentrations of both methylmercury and mercuric chloride increased the release of endogenous glutamate, inhibited glutamate uptake, reduced mitochondrial activity, and decreased ATP levels. All these effects were completely prevented by the nonpermeant reducing agent Tris-(2-carboxyethyl)phosphine (TCEP). Reduction of mitochondrial activity by mercuric chloride, but not by methylmercury, was inhibited significantly by 4,4,-diisothiocyanato-stilbene-2,2,-disulfonic acid (DIDS) and by reduced extracellular Cl, ion concentration. In addition, DIDS and low extracellular Cl, completely inhibited the release of glutamate induced by mercuric chloride, and produced a partial although significant reduction of that induced by methylmercury. We suggest that a direct inhibition of glutamate uptake triggers an imbalance in cell homeostasis, leading to neuronal failure and Cl, -regulated cellular glutamate efflux. Our results demonstrate that neuronal glutamate transport is a novel target to be taken into account when assessing mercury-induced neurotoxicity. © 2005 Wiley-Liss, Inc. [source] Retinoids and retinoic acid receptors regulate growth arrest and apoptosis in human mammary epithelial cells and modulate expression of CBP/p300MICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2002Eric C. Dietze Abstract Retinoids and retinoic acid receptors (RARs) are important mediators of normal epithelial cell homeostasis. To assess the role of retinoids and RARs in regulating growth arrest and apoptosis in benign and malignant mammary epithelial cells, two model systems were developed: 1) RAR function was suppressed in retinoid-sensitive normal human mammary epithelial cells (HMECs) by the dominant-negative retinoic acid receptor, RAR,403 (DNRAR), and 2) retinoid-resistant MCF-7 breast cancer cells were transduced with a functional RAR,2. Inhibition of RAR function by the DNRAR in HMECs resulted in retinoid-resistance, increased proliferation, and dysregulated growth when cells were cultured in reconstituted extracellular matrix (rECM). Expression of RAR,2 in MCF-7 cells resulted in sensitivity to retinoid-induced growth arrest and apoptosis. The CREB-binding protein (CBP) and the homologous protein p300 are tightly regulated, rate-limiting integrators of diverse signaling pathways and are recruited during retinoid-mediated transcriptional activation. The relationship between retinoid receptor expression, growth regulation, and transcriptional regulation of CBP/p300 is poorly understood. Inhibition of RAR function in HMECs by DNRAR suppressed expression of CBP/p300 and expression of RAR,2 in MCF-7 cells promoted induction of CBP/p300 when cells were treated with 1.0 ,M all- trans -retinoic acid (ATRA). These results suggest that ATRA and RARs regulate growth arrest of HMECs and modulate CBP/p300 protein expression. Since CBP and p300 are normally present in limiting amounts, their regulation by ATRA and RARs may be an important element in the control of transcriptional activation of genes regulating growth arrest and apoptosis. Microsc. Res. Tech. 59:23,40, 2002. © 2002 Wiley-Liss, Inc. [source] Premature aging of the immune system in children with juvenile idiopathic arthritisARTHRITIS & RHEUMATISM, Issue 7 2008Martina Prelog Objective Juvenile idiopathic arthritis (JIA) is an autoimmune disease of the young. The pathogenesis is not completely understood. Premature aging, associated thymic involution, and compensatory autoproliferation could play important roles in the pathogenesis of autoimmunity. We undertook this study to determine whether patients with JIA demonstrate premature immunosenescence. Methods To test this hypothesis, we measured 3 indicators of aging: the percentages and total counts of peripheral blood naive T cells, the frequency of T cell receptor excision circles (TRECs) in naive T cells, and telomeric erosion and Ki-67 expression as estimates of the replicative history of homeostatic proliferation. Results JIA patients showed an accelerated loss of CD4+,CD45RA+,CD62L+ naive T cells with advancing age and a compensatory increase in the number of CD4+,CD45RO+ memory T cells. JIA patients demonstrated a significantly decreased frequency of TRECs in CD4+,CD45RA+ naive T cells compared with age-matched healthy donors (P = 0.002). TREC numbers correlated with age only in healthy donors (P = 0.0001). Telomeric erosion in CD4+,CD45RA+ naive T cells was increased in JIA patients (P = 0.01). The percentages of Ki-67,positive CD4+,CD45RA+ naive T cells were increased in JIA patients (P = 0.001) and correlated with disease duration (P = 0.003), which was also an independent factor contributing to telomeric erosion (P = 0.04). Conclusion Our findings suggest that age-inappropriate T cell senescence and disturbed T cell homeostasis may contribute to the development of JIA. In patients with JIA, dysfunction in the ability to reconstitute the T cell compartment should be considered when exploring new therapeutic strategies. [source] Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitroBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2007Caroline J. Marshall Summary The transforming growth factor- , -related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34+ cells with different levels of c-Kit expression and multipotency were identified. CD34+/c-Kithigh cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34+/c-Kitlow cells are Flk1+/CD45neg and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34+/c-Kitlow cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34+/c-Kithigh cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34+/c-Kithigh haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34+/c-Kithigh progenitors cultured with BMP4 also generated adherent colonies typical of c-Kitlow cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34+ AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche. [source] Lipooligosaccharide-independent alteration of cellular homeostasis in Neisseria meningitidis -infected epithelial cellsCELLULAR MICROBIOLOGY, Issue 6 2005Robert A. Bonnah Summary Neisseria meningitidis (MC) is an important cause of meningitis and septic shock. Primary loose attachment of MC to host epithelial cells is mediated by type IV pili. Lipooligosaccharide (LOS), opacity (Opa) proteins and glycolipid adhesins facilitate subsequent tight attachment. MC infection causes numerous changes in host epithelial cell homeostasis. These include cortical plaque formation, increased expression of proinflammatory cytokines and alterations in host iron homeostasis. Using both biochemical and genetic approaches, we examined the role of LOS in mediating these events. We first examined specific cellular iron homeostasis changes that occur following addition of purified MC LOS to epithelial cells. Using an MC mutant that completely lacks LOS (MC lps tbp), we examined pili-mediated attachment and cortical plaque formation in human endocervical epithelial cells (A431). We also tested whether the lack of LOS alters cellular homeostasis, including changes in the levels of host stress response factors and proinflammatory cytokines. MC lps tbp elicited the formation of cortical plaques in A431 cells. However, the plaques were less pronounced than those formed by the MC parent. Surprisingly, the proinflammatory cytokine TNF, was upregulated during infection in MC lps tbp -infected cells. Furthermore, alterations in iron homeostasis, including lower transferrin receptor 1 (TfR-1) levels, altered TfR-1 trafficking, an ,iron-starvation' gene expression profile and low iron regulatory protein (IRP) binding activity are independent of LOS. Our results demonstrate that LOS is partially involved in both the attachment to host cells and formation of cortical plaques. However, TNF, induction and changes in iron homeostasis observed in MC-infected epithelial cells are independent of LOS. [source] Shift of C3 deposition from localization in the glomerulus into the tubulo-interstitial compartment in the absence of secreted IgM in immune complex glomerulonephritisCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2008C. Vaculik Summary The role of secretory IgM in protecting kidney tissue from immune complex glomerulonephritis induced by 4 mg horse spleen apoferritin and 0·05 mg lipopolysaccharide has been investigated in mutant mice in which B cells do not secrete IgM, but are capable of expressing surface IgM and IgD and secreting other Ig isotypes. Glomerular size, number of glomeruli per cross-section, glomerular cellularity and urine content of protein and creatinine was comparable in treated secreted IgM (sIgM)-deficient and wild-type mice. Assessment of urinary proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a 30 kDa low molecular weight protein in treated sIgM-deficient animals only, reflecting dysfunction of proximal tubules. A shift of bound C3 from glomeruli to the tubulo-interstitial compartment in sIgM-deficient mice also suggests tubulo-interstitial damage. In contrast, local C3 synthesis within the kidney tissue did not differ between the two treated groups. Apoptosis physiologically present to maintain kidney cell homeostasis was increased slightly in treated wild-type mice. These results indicate that secretory IgM can protect the tubulo-interstitial compartment from immune complex-induced damage without having an effect on the glomerulus. [source] | |||||||||||||