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Various Cell Types (various + cell_type)
Selected AbstractsExpression of a Rho Guanine Nucleotide Exchange Factor, Ect2, in the Developing Mouse PituitaryJOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2010M. S. Islam The pituitary gland is a highly mitotically active tissue after birth. Various cell types are known to undergo proliferation in the anterior pituitary. However, little is known about the mechanisms regulating mitotic activity in this tissue. When searching for genes specifically expressed in the pituitary gland among those that we previously screened in Drosophila, we found epithelial cell-transforming gene 2 (Ect2). Ect2 is a guanine nucleotide exchange factor for Rho GTPases, which is known to play an essential role in cytokinesis. Although there have been many cellular studies regarding the function of Ect2, the temporal and spatial expression patterns of Ect2 in vivo have not been determined. In the present study, we examined the postnatal developmental expression of Ect2 in the mouse pituitary. Enhanced Ect2 expression was detected in the mouse pituitary gland during the first 3 weeks after birth, which coincided well with the period of rapid pituitary expansion associated with increased growth rate. Immunostaining analysis showed that Ect2-expressing cells were distributed in the anterior and intermediate lobes, but not the posterior lobe, of the pituitary. These Ect2-expressing cells frequently incorporated the thymidine analogue, EdU (5-ethynyl-2,-deoxyuridine), indicating that these cells were mitotically active. Taken together, the results demonstrate the functional role of Ect2 in postnatal proliferating cells in the two lobes of the pituitary, thereby suggesting roles in developmental growth of the mammalian pituitary. [source] Skeletal tissue engineering using embryonic stem cellsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 3 2010Jojanneke M. Jukes Abstract Various cell types have been investigated as candidate cell sources for cartilage and bone tissue engineering. In this review, we focused on chondrogenic and osteogenic differentiation of mouse and human embryonic stem cells (ESCs) and their potential in cartilage and bone tissue engineering. A decade ago, mouse ESCs were first used as a model to study cartilage and bone development and essential genes, factors and conditions for chondrogenesis and osteogenesis were unravelled. This knowledge, combined with data from the differentiation of adult stem cells, led to successful chondrogenic and osteogenic differentiation of mouse ESCs and later also human ESCs. Next, researchers focused on the use of ESCs for skeletal tissue engineering. Cartilage and bone tissue was formed in vivo using ESCs. However, the amount, homogeneity and stability of the cartilage and bone formed were still insufficient for clinical application. The current protocols require improvement not only in differentiation efficiency but also in ESC-specific hurdles, such as tumourigenicity and immunorejection. In addition, some of the general tissue engineering challenges, such as cell seeding and nutrient limitation in larger constructs, will also apply for ESCs. In conclusion, there are still many challenges, but there is potential for ESCs in skeletal tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd. [source] MSK regulate TCR-induced CREB phosphorylation but not immediate early gene transcriptionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2007Madlen Kaiser Abstract Stimulation of the T cell receptor activates the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) cascades. We demonstrate that TCR stimulation also activates the mitogen- and stress-activated kinases (MSK) downstream of ERK1/2 and p38 in both a T cell line and primary peripheral T cells. MSK1/2-knockout mice were found to have normal numbers of T cells in the thymus, and development of these cells appeared unaffected. Using naive T cells and T lymphoblasts from MSK1/2-knockout mice, it was found that MSK was the kinase responsible for phosphorylation of the transcription factor CREB in response to TCR stimulation. Phosphorylation of CREB by MSK has been linked to the transcription of nur77, nor1 and c-fos downstream of MAPK signalling in various cell types. In T cells, the TCR-dependent transcription of these genes was found to require a MAPK-dependent but MSK-independent signalling pathway. Nevertheless, the number of T cells present in the spleens of MSK1/2-knockout mice and the IL-2-induced proliferation of these cells was reduced compared to wild-type mice. This correlated to a reduction in the TCR-induced up-regulation of the IL-2 receptor CD25 and a requirement for MSK in IL-2-induced CREB phosphorylation. [source] A Method for the Real-Time Observation of Endodermal Cell Behavior on Micropatterned Surfaces,ADVANCED ENGINEERING MATERIALS, Issue 8 2009David C. Trimbach Surface chemistry and geometry have a strong influence on adhesion and proliferation of various cell types, including human embryonic stem cells (ES). Visceral endoderm like cells (END-2) is an important cell line which induces ES cells to differentiate into cardiomyocytes. In this study, we have investigated the effect of surface chemistry and geometry on the END-2 cell adhesion and proliferation on gold surface. [source] The role of stem cells in suppurative environmentsEXPERIMENTAL DERMATOLOGY, Issue 6 2006Dolores Herreros Purpose:,The management of suppurative perianal lesions presents an extremely challenging problem. Stem cells (SC) extracted from certain tissues, such as adipose tissue, can differentiate into various cell types. Therefore, we have tried to use such cells to stimulate healing in a purulent environment. Methods:,In the beginning, we designed a phase I clinical trial, involving five patients with Crohn's disease. We inoculated nine fistulas in four patients with autologous adipose-derived stem cells (ADSC) and were followed at least 8 weeks. Seventy-five percent became healed, and 25% showed a decrease in output flow. No adverse effects were observed in any patient. This study evidenced that such cells are safe. Then, we started a research line using SC in different suppurative environments. During the course of these studies, we had the opportunity to treat a patient with perianal hidradenitis suppurativa using our current protocol of ADSC transplantation. Eight weeks after injection, patient had no perianal suppuration, and a year later remains well. Discussion:,The biological mechanism that underlies the therapeutic success of ADSC transplantation is unknown. Cell differentiation, secretion of growth factors or immunomodulatory effects have been suggested. No ethical conflicts were identified by our Ethics Committee, because the cells were autologous. Conclusions:,Our study shows that ADSC are safe for the treatment of suppurative processes. The actual number of patients included and the uncontrolled nature of these pilot studies do not allow demonstration of the effectiveness of the treatment. However, the results encourage the performance of further studies. [source] Gene expression silencing with ,specific' small interfering RNA goes beyond specificity , a study of key parameters to take into account in the onset of small interfering RNA off-target effectsFEBS JOURNAL, Issue 11 2008Sébastien Vankoningsloo RNA-mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss-of-function experiments. However, several recent studies underscore the off-target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low-density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off-target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post-transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments. [source] RCAN1-1L is overexpressed in neurons of Alzheimer's disease patientsFEBS JOURNAL, Issue 7 2007Cathryn D. Harris At least two different isoforms of RCAN1 mRNA are expressed in neuronal cells in normal human brain. Although RCAN1 mRNA is elevated in brain regions affected by Alzheimer's disease, it is not known whether the disease affects neuronal RCAN1, or if other cell types (e.g. astrocytes or microglia) are affected. It is also unknown how many protein isoforms are expressed in human brain and whether RCAN1 protein is overexpressed in Alzheimer's disease. We explored the expression of both RCAN1-1 and RCAN1-4 mRNA isoforms in various cell types in normal and Alzheimer's disease postmortem samples, using the combined technique of immunohistochemistry and in situ hybridization. We found that both exon 1 and exon 4 are predominantly expressed in neuronal cells, and no significant expression of either of the exons was observed in astocytes or microglial cells. This was true in both normal and Alzheimer's disease brain sections. We also demonstrate that RCAN1-1 mRNA levels are approximately two-fold higher in neurons from Alzheimer's disease patients versus non-Alzheimer's disease controls. Using western blotting, we now show that there are three RCAN1 protein isoforms expressed in human brain: RCAN1-1L, RCAN1-1S, and RCAN1-4. We have determined that RCAN1-1L is expressed at twice the level of RCAN1-4, and that there is very minor expression of RCAN1-1S. We also found that the RCAN1-1L protein is overexpressed in Alzheimer's disease patients, whereas RCAN1-4 is not. From these results, we conclude that RCAN1-1 may play a role in Alzheimer's disease, whereas RCAN1-4 may serve another purpose. [source] Unmasking a hyaluronan-binding site of the BX7B type in the H3 heavy chain of the inter-,-inhibitor familyFEBS JOURNAL, Issue 3 2001Laetitia Jean The inter-,-inhibitor (I,I) family gathers together several plasma protease inhibitors such as I,I and pre-,-inhibitor (P,I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I,I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I,I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P,I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P,I may vary with the folding and/or fragmentation of this protein. [source] Changes of telomere length with agingGERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 2010Kaiyo Takubo We reviewed our methodology and results of telomere measurements, with reference to telomere length and aging. Human tissues always showed telomere shortening with age, except for the brain and myocardium. Yearly rates of telomere length reduction in various tissues were mostly within the range 20,60 bp, and thus compatible with that expected from only one round of mitosis. It was suggested that when telomeres were found to be longer in any specific organ in a given individual, then the other organs in that individual would also have longer telomeres. Using the quantitative fluorescence in situ hybridization (Q-FISH) method for telomere measurement, we were able to measure the telomere lengths of various cell types within tissues. Here we summarize the results obtained for various cell types in the stomach, tongue and breast. Our Q-FISH method using our original software program "Tissue Telo" is excellent for measuring telomere lengths using tissue sections and PNA probes. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S197,S206. [source] Ca2+ - and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membraneGLIA, Issue 12 2009J. W. Ou Abstract Large-conductance, voltage- and Ca2+ -activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming ,-subunits (MaxiK,) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiK, is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiK, was visualized by three independent labeling approaches: (1) MaxiK,-specific antibodies, (2) expressed EGFP-labeled MaxiK,, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiK, association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiK, labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+ -release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiK, surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a ,3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK,. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK, stores to the plasma membrane of developing murine astrocytes. © 2009 Wiley-Liss, Inc. [source] Natural killer T-cell characterization through gene expression profiling: an account of versatility bridging T helper type 1 (Th1), Th2 and Th17 immune responsesIMMUNOLOGY, Issue 1 2008Marcus Niemeyer Summary Natural killer T (NKT) cells constitute a distinct lymphocyte lineage at the interface between innate and adaptive immunity, yet their role in the immune response remains elusive. Whilst NKT cells share features with other conventional T lymphocytes, they are unique in their rapid, concomitant production of T helper type 1 (Th1) and Th2 cytokines upon T-cell receptor (TCR) ligation. In order to characterize the gene expression of NKT cells, we performed comparative microarray analyses of murine resting NKT cells, natural killer (NK) cells and naďve conventional CD4+ T helper (Th) and regulatory T cells (Treg). We then compared the gene expression profiles of resting and alpha-galactosylceramide (,GalCer)-activated NKT cells to elucidate the gene expression signature upon activation. We describe here profound differences in gene expression among the various cell types and the identification of a unique NKT cell gene expression profile. In addition to known NKT cell-specific markers, many genes were expressed in NKT cells that had not been attributed to this population before. NKT cells share features not only with Th1 and Th2 cells but also with Th17 cells. Our data provide new insights into the functional competence of NKT cells which will facilitate a better understanding of their versatile role during immune responses. [source] Ionizing radiation as a response-enhancing agent for CD95-mediated apoptosisINTERNATIONAL JOURNAL OF CANCER, Issue 4 2001Michael A. Sheard Ph.D. Abstract CD95 (Fas/APO-1) is a death receptor on the surface of a wide variety of cell types. In most cells examined, ionizing radiation acts as a response-enhancing agent for CD95-mediated cell death. Although DNA-damaging radiation appears to modulate CD95-mediated signals through multiple mechanisms, the only well-characterized mechanism is activation of the tumor-suppressor protein p53, which transcriptionally regulates the expression of CD95 on various cell types. The ligand for CD95 is expressed by activated lymphocytes and natural-killer cells, which produce factors that sensitize cells resistant to CD95-mediated cell death. Ligation of CD95 on irradiated tumor cells might be achievable using emerging modalities that reactivate the stalled anti-tumor immune response. © 2001 Wiley-Liss, Inc. [source] Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytesINVERTEBRATE BIOLOGY, Issue 3 2002Sally P. Leys Abstract. Early development and metamorphosis of Reniera sp., a haplosclerid demosponge, have been examined to determine how gastrulation occurs in this species, and whether there is an inversion of the primary germ layers at metamorphosis. Embryogenesis occurs by unequal cleavage of blastomeres to form a solid blastula consisting micro- and macromeres; multipolar migration of the micromeres to the surface of the embryo results in a bi-layered embryo and is interpreted as gastrulation. Polarity of the embryo is determined by the movement of pigment-containing micromeres to one pole of the embryo; this pole later becomes the posterior pole of the swimming larva. The bi-layered larva has a fully differentiated monociliated outer cell layer, and a solid interior of various cell types surrounded by dense collagen. The pigmented cells at the posterior pole give rise to long cilia that are capable of responding to environmental stimuli. Larvae settle on their anterior pole. Fluorescent labeling of the monociliated outer cell layer with a cell-lineage marker (CMFDA) demonstrates that the monociliated cells resorb their cilia, migrate inwards, and transdifferentiate into the choanocytes of the juvenile sponge, and into other amoeboid cells. The development of the flagellated choanocytes and other cells in the juvenile from the monociliated outer layer of this sponge's larva is interpreted as the dedifferentiation of fully differentiated larval cells,a process seen during the metamorphosis of other ciliated invertebrate larvae,not as inversion of the primary germ layers. These results suggest that the sequences of development in this haplosclerid demosponge are not very different than those observed in many cnidarians. [source] Steroid and thyroid hormone receptors in mitochondriaIUBMB LIFE, Issue 4 2008Anna-Maria G. Psarra Abstract Receptors for glucocorticoids, estrogens, androgens, and thyroid hormones have been detected in mitochondria of various cell types by Western blotting, immunofluorescence labeling, confocal microscopy, and immunogold electron microscopy. A role of these receptors in mitochondrial transcription, OXPHOS biosynthesis, and apoptosis is now being revealed. Steroid and thyroid hormones regulate energy production, inducing nuclear and mitochondrial OXPHOS genes by way of cognate receptors. In addition to the action of the nuclearly localized receptors on nuclear OXPHOS gene transcription, a parallel direct action of the mitochondrially localized receptors on mitochondrial transcription has been demonstrated. The coordination of transcription activation in nuclei and mitochondria by the respective receptors is in part realized by their binding to common trans acting elements in the two genomes. Recent evidence points to a role of the mitochondrial receptors in cell survival and apoptosis, exerted by genomic and nongenomic mechanisms. The identification of additional receptors of the superfamily of nuclear receptors and of other nuclear transcription factors in mitochondria increases their arsenal of regulatory molecules and further underlines the central role of these organelles in the integration of growth, metabolic, and cell survival signals. © 2008 IUBMB IUBMB Life, 60(4): 210,223, 2008 [source] A discourse on cancer cell chemotaxis: Where to from here?IUBMB LIFE, Issue 2 2007Lilian L. Soon Abstract The study of cancer cell chemotaxis on two-dimensional surfaces in vitro has relevance to the diverse migratory behaviours exhibited in vivo that involve a directed path. These may include translocation along collagen fibres, invasion into the basement membrane and across stroma, intravasation and extravasation to arrive at a secondary destination designated for cancer cell colonization. Chemotaxis invariably denotes the ability of cells to sense gradients, polarize, adhere and deadhere to substrate, and translocate in the right direction. Amongst these, the sensing function is perhaps the unifying aspect of different migration styles, permitting the cells to resolve its orientation and path. This review examines the decision-making processes that take place during chemotaxis and illustrates that a universal mechanism is involved. In various cell types from Dictyostelium to neutrophils, there are some unifying principles that dictate sensing and how the putative leading edge and trailing end of cells are determined. Some of these principles have recently been applied in the study of cancer cell chemotaxis albeit different pathways are substituted. In amoeboid-like cancer cells, local excitation of the EGFR/PLC,/cofilin pathway and parallel, global inhibition of cofilin by LIMK occur to promote the asymmetric distribution and amplification of these internal signals in response to an external EGF gradient. IUBMB Life, 59: 60-67, 2007 [source] In Vivo Function of a Differentiation Inhibitor, Id2IUBMB LIFE, Issue 4 2001Yoshifumi Yokota Abstract Cell differentiation is an essential process for the development of various cell types that constitute multicellular organisms. During development, the large family of factors bearing a helix-loop-helix (HLH) motif participates profoundly in this process and these factors serve as good experimental tools for investigating mechanisms underlying tissue-specific differentiation. The HLH family includes both positive and negative regulators of cell differentiation: basic HLH (bHLH)-type transcription factors and Id proteins, respectively. Following an exciting decade focusing on bHLH factors, advances achieved in studies of the inhibitory factors in the last couple of years have placed them in the front line of the research on differentiation and proliferation control. Here, we present and discuss recent results obtained using Id2 -deficient mice, which manifest intriguing phenotypes in various systems. [source] Evidence That Peroxynitrite Affects Human Osteoblast Proliferation and Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2002Francisco Airton Castro Da Rocha Abstract Peroxynitrite (PN), a nitric oxide (NO·)-derived anion, has been associated with NO· damage in various cell types. We examined the effects of adding PN to cultured human osteoblast-like (hOB) cells obtained after hip arthroplasty. Exposure to PN (0.1-0.4 mM) decreased both hOB proliferation and differentiation, measured by [3H]thymidine uptake and alkaline phosphatase production, respectively. Incubation with 3-morpholinosydnonimine (SIN-1; 0.25-1 mM), an NO· and O2, donor that leads to PN release, also reduced both hOB proliferation and differentiation. Coincubation with both superoxide dismutase (SOD; 100 U/ml) and catalase (CAT; 50 U/ml), rendering SIN-1 a pure NO· donor, reversed its effects on hOB proliferation and differentiation. However, SIN-1-induced NO· production, measured by nitrite release to the hOB medium, was not altered by cotreatment with SOD and CAT. Expression of nitrotyrosine by hOB, a marker of PN action, was significantly increased after SIN-1 addition, as compared with untreated cells, as revealed by Western blot analysis. Interleukin-1, (IL-1,) and interferon , (IFN-,) but not tumor necrosis factor , (TNF-,) also significantly increased nitrotyrosine expression in these cells. These data show that PN is at least partially responsible for osteoblast derangement by NO· and that cytokines released during inflammatory arthropathies can induce PN production in hOB cells. [source] HSP70 interacts with TRAF2 and differentially regulates TNF, signalling in human colon cancer cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2010Shengming Dai Abstract Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNF,-induced signalling pathway have not been fully elucidated. We report here that HSP70 over-expression in human colon cancer cells can inhibit TNF,-induced NF,B activation but promote TNF,-induced activation of c-Jun N-terminal kinase (JNK) through interaction with TNF receptor (TNFR)-associated factor 2 (TRAF2). We provide evidence that HSP70 over-expression can sequester TRAF2 in detergent-soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor-interacting protein (RIP1) and I,B, kinase (IKK) signalosome to the TNFR1,TRADD complex and inhibited NF,B activation after TNF, stimuli. In addition, we found that HSP70,TRAF2 interaction can promote TNF,-induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNF,-induced activation of NF,B and JNK through interaction with TRAF2, contributing to the pro-apoptotic roles of HSP70 in TNF,-induced apoptosis of human colon cancer cells. [source] Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Vijayalakshmi Thamilselvan Abstract Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0,±,3.0% (n,=,3; P,<,0.001) and primary cancer cells by 41.0,±,3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5,±,0.13 fold) and Western analysis (1.34,±,0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 µM), PP2 (20 µM), and actin-cytoskeleton stabilizer phalloidin (10 µM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0,±,2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection. Published 2004 Wiley-Liss, Inc. [source] Immunomodulation by mesenchymal stem cells and clinical experienceJOURNAL OF INTERNAL MEDICINE, Issue 5 2007K. Le Blanc Abstract Mesenchymal stem cells (MSCs) from adult marrow can differentiate in vitro and in vivo into various cell types, such as bone, fat and cartilage. MSCs preferentially home to damaged tissue and may have therapeutic potential. In vitro data suggest that MSCs have low inherent immunogenicity as they induce little, if any, proliferation of allogeneic lymphocytes. Instead, MSCs appear to be immunosuppressive in vitro. They inhibit T-cell proliferation to alloantigens and mitogens and prevent the development of cytotoxic T-cells. In vivo, MSCs prolong skin allograft survival and have several immunomodulatory effects, which are presented and discussed in the present study. Possible clinical applications include therapy-resistant severe acute graft-versus-host disease, tissue repair, treatment of rejection of organ allografts and autoimmune disorders. [source] Low-level plasma HIVs in patients on prolonged suppressive highly active antiretroviral therapy are produced mostly by cells other than CD4 T-cellsJOURNAL OF MEDICAL VIROLOGY, Issue 1 2009Gautam K. Sahu Abstract The cellular source(s) and the clinical significance of persistent low-level viremia, below 50 HIV RNA copies per ml of plasma, achieved in many patients with high adherence to highly active antiretroviral therapy (HAART) remain unclear. Also, it is not clear if residual plasma HIVs during HAART can become predominant populations in the rebounding plasma viral loads after therapy interruption. Since, different HIV quasispecies tend to compartmentalize in various cell types and tissue locations in patients during chronic infection, the phylogenetic relationships between HIV sequences amplified from residual plasma viruses and CD4 T cells of five patients on long-term suppressive therapy were examined. Three of these patients stopped therapy voluntarily for 3 weeks, but only one of them demonstrated viral load rebound in plasma. In phylogenetic analyses, the residual plasma viruses were found to be distinct genetically from the majority of CD4 T cell-associated virus populations in four of five patients. The compartmental analyses revealed that in all patients, plasma- and CD4 T cell-derived viral sequences were compartmentalized separately. Interestingly, the plasma sequences obtained before and after HAART-off in two patients were produced apparently from the same compartment, which was different from the circulating CD4 T cell-compartment. These results suggest the possibility that residual plasma viruses in patients on long-term suppressive HAART may be produced persistently from a cellular source yet to be identified, and are capable of spreading quickly in vivo, accounting for the rapid rebound of viral loads in plasma after therapy interruption. J. Med. Virol. 81:9,15, 2009. © 2008 Wiley-Liss, Inc. [source] Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides,JOURNAL OF MOLECULAR RECOGNITION, Issue 1 2005Johannes Oehlke Abstract In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides. Studies on structure,activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane. In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg3,Orn7 -SP and its D -amino acid analog, all- D -Arg3,Orn7 -SP, as well as by electron microscopic autoradiography using 3H-labelled SP and 125I-labelled all- D -SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature. Copyright © 2004 John Wiley & Sons, Ltd. [source] Fibroblast growth factor 9 prevents MPP+ -induced death of dopaminergic neurons and is involved in melatonin neuroprotection in vivo and in vitroJOURNAL OF NEUROCHEMISTRY, Issue 5 2009Jui-Yen Huang Abstract Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP+ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP+ intoxication and melatonin protection. We found that MPP+ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP+ -induced cell death of DA neurons. Melatonin co-treatment attenuated MPP+ -induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro. Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP+, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP+ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection. [source] Endogenous and Exogenous Fibroblast Growth Factor 2 Support Survival of Chick Retinal Neurons by Control of Neuronal Neuronal bcl-xL and bcl-2 Expression Through a Fibroblast Berowth Factor Receptor 1- and Erk-Dependent PathwayJOURNAL OF NEUROCHEMISTRY, Issue 1 2000Laurent Désiré Abstract : Fibroblast growth factor (FGF) 2 is a survival factor for various cell types, including retinal neurons. However, little is understood about the molecular bases of the neuroprotective role of FGF2 in the retina. In this report, FGF2 survival activity was studied in chick retinal neurons subjected to apoptosis by serum deprivation. Exogenous FGF2 supported neuronal survival after serum deprivation and increased neuronal bcl-xL and bcl-2 expression, through binding to its receptor R1 (FGF-R1), and subsequent extracellular signal-regulated kinase (ERK) activation. Endogenous FGF2 was transiently overexpressed after serum deprivation. Its down-regulation by antisense oligonucleotides and blockade of its signaling pathway (binding to FGF-R1, tyrosine phosphorylation, and ERK inhibition) decreased bcl-xL and bcl-2 levels and and enhanced apoptosis, suggesting that endogenous FGF2 supported neuronal survival through a pathway similar to that of exogenous FGF2. This pathway may serve to up-regulate, or maintain, bcl-xL and bcl-2 levels that normally decrease during the onset of apoptosis. Indeed, long-term ERK activation and high bcl-xL levels are necessary for the survival activity of both exogenous and endogenous FGF2. Because FGF2 is upregulated following retinal injury in vivo, we suggest that an injury-stimulated autocrine/paracrine FGF2 loop may serve to maintain high levels of survival proteins, such as Bcl-xL, through ERK activation in retinal neurons. [source] AUF-1 mediates inhibition by nitric oxide of lipopolysaccharide-induced matrix metalloproteinase-9 expression in cultured astrocytesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2006Wenlan Liu Abstract Neuroinflammatory diseases are associated with increased production of matrix metalloproteinase-9 (MMP-9) and excessive generation of nitric oxide (NO). NO hasbeen reported to have variable effects on MMP-9 gene expression and activation in various cell types. Inthe present study, we investigated the effect of NOon MMP-9 expression in primary cortical astrocytes. Zymography and real-time PCR showed that lipopolysaccharide (LPS) dramatically increased latent MMP-9 gelatinolytic activity and MMP-9 mRNA expression. By using the NO donor DETA NONOate, we observed a dose-dependent inhibition of MMP-9 induction by LPS. Active forms of MMP-9 were not found by zymography after NO treatment. The MEK1/2 inhibitor U0126 completely inhibited LPS-induced MMP-9, which was partially inhibited by the p38 MAPK inhibitor SB203580. NO had no effect on LPS-stimulated ERK1/2 and p38 MAPK activation, suggesting that the inhibitory action of NO occurs downstream of MAPK cascades. Real-time PCR analysis showed that NO accelerated the degradation of MMP-9 mRNA after LPS induction. Western blotting and pull-down assay demonstrated that NO increased AUF-1 expression as well as its specific binding to the MMP-9 gene 3,-untranslated region. Knockdown of AUF-1 with siRNA partially reversed the inhibitory action of NO on LPS-stimulated MMP-9 induction. We conclude that NO does not activate MMP-9 in astrocyte cultures but reduces LPS-induced MMP-9 expression via accelerating MMP-9 mRNA degradation, which is partially mediated by AUF-1. Our results suggest that elevated NO concentrations may suppress MMP-9 and restrict the inflammatory response in neurodegenerative diseases. © 2006 Wiley-Liss, Inc. [source] Osteoprotegerin induces osteopontin via syndecan-1 and phosphoinositol 3-kinase/Akt in human periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2009T. Yongchaitrakul Background and Objective:, Our previous study found that thrombin induced osteoprotegerin synthesis in human periodontal ligament cells. As elevated levels of osteoprotegerin can exert biological effects on various cell types, in the present study we investigated the effect of osteoprotegerin on the expression of osteopontin in human periodontal ligament cells. Material and Methods:, Cultured human periodontal ligament cells were treated with recombinant human osteoprotegerin (0,100 ng/mL) for 24,48 h. The expression of osteopontin mRNA and protein was analyzed using reverse transcription,polymerase chain reaction and western blot analyses, respectively. Phosphoinositol 3-kinase inhibitor, Akt inhibitor, heparinase, neutralizing antibody against receptor activator of nuclear factor-,B ligand (RANKL) and syndecan-1, and small interfering RNA against syndecan-1, were used to determine the mechanism involved. Results:, Osteoprotegerin up-regulated the mRNA and protein expression of osteopontin in human periodontal ligament cells in a dose-dependent manner. Addition of neutralizing antibody against RANKL attenuated the inductive effect of osteoprotegerin on osteopontin expression. In addition, the inductive effect of osteoprotegerin was abolished by phosphoinositol 3-kinase and Akt inhibitors, as well as by syndecan-1 antibody or syndecan-1 small interfering RNA. None of the inhibitors had any effect on the background level of osteopontin expression. Conclusion:, An increased level of osteoprotegerin can generate signals via a RANKL/syndecan-1/phosphoinositol 3-kinase/Akt pathway. The results also suggest that osteopontin is one of the downstream targets of the pathway mediated by osteoprotegerin in human periodontal ligament cells. Thus, in addition to counteracting RANKL in the RANKL,osteoprotegerin system, osteoprotegerin may play a role in periodontal tissue remodeling through modulation of the extracellular matrix. [source] Expression Pattern, Ethanol-Metabolizing Activities, and Cellular Localization of Alcohol and Aldehyde Dehydrogenases in Human Pancreas: Implications for Pathogenesis of Alcohol-Induced Pancreatic InjuryALCOHOLISM, Issue 6 2009Chien-Ping Chiang Background:, Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are major enzymes responsible for metabolism of ethanol. Genetic polymorphisms of ADH1B, ADH1C, and ALDH2 occur among racial populations. The metabolic effect and metabolites contribute to pathogenesis of pancreatic injury. The goal of this study was to determine the functional expressions and cellular localization of ADH and ALDH families in human pancreas. Methods:, Fifty five surgical specimens of normal pancreas as well as 15 samples each for chronic pancreatitis and pancreatic cancer from archival formalin-fixed paraffin-embedded tissue specimens were investigated. Class-specific antibodies were prepared by affinity chromatographies from rabbit antisera raised against recombinant human ADH1C1, ADH4, ADH5, ADH7, ALDH1A1, ALDH2, and ALDH3A1. The isozyme expression patterns of ADH/ALDH were identified by isoelectric focusing, and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting, and the cellular localizations were detected by immunohistochemistry and histochemistry. Results:, At 33 mM ethanol, pH 7.5, the activities were significantly different between allelic phenotypes of ADH1B. The activity of ALDH2-inactive phenotypes was slightly lower than ALDH2-active phenotypes at 200 ,M acetaldehyde. The protein contents were in the following decreasing order: ALDH1A1, ALDH2, ADH1, and ADH5. ADH1B was detected in the acinar cells and ADH1C in the ductular, islet, and stellate cells. The expression of ADH1C appeared to be increased in the activated pancreatic stellate cells in chronic pancreatitis and pancreatic cancer. Conclusions:, Alcohol dehydrogenase and ALDH family members are differentially expressed in the various cell types of pancreas. ADH1C may play an important role in modulation of activation of pancreatic stellate cells. [source] Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generationJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 7 2008Winston Costa Pereira Abstract Mesenchymal stem cells (MSCs) are considered to be a source of stem cells in tissue regeneration and therapeutics, due to their ability to undergo proliferation and differentiation. Complications associated with bone marrow-derived MSCs has prompted researchers to explore alternative sources of MSCs. The human umbilical cord is one such source; it is easily available and its collection is non-invasive. The sources of MSCs are non-controversial and thus they are not subjected to ethical constraints, as in the case of embryonic stem cells. MSCs are multipotent stem cells and has the ability to differentiate into various cell types of the mesodermal lineage. The aim of this study was to establish a reproducible method for the isolation of MSCs from human umbilical cord, as the few methods published till date gave inconsistent results and had a mixed population of contaminating endothelial cells. In our isolation strategy, we isolated a pure population of MSCs from Wharton's jelly of the human umbilical cord, which is very rich in collagen, and we used a high concentration of collagenase enzyme in the isolation of MSCs. Extensive phenotypic characterization analysis of these cells, using flow cytometry and antibody staining methods, have shown that we were able to isolate a pure population of the mesenchymal lineage cells that is devoid of haematopoietic and endothelial cell contaminants. When these MSCs were subjected to cardiomyocyte differentiation, we observed a change in the morphological characteristics, which was accompanied by the formation of myotube structures and spontaneous beating after 21 days. Copyright © 2008 John Wiley & Sons, Ltd. [source] Medium-sized peptides as built in carriers for biologically active compoundsMEDICINAL RESEARCH REVIEWS, Issue 6 2005Ferenc Hudecz Abstract A growing number of oligopeptides of natural and/or synthetic origin have been described and considered as targeting structures for delivery bioactive compounds into various cell types. This review will outline the discovery of peptide sequences and the corresponding mid-sized oligopeptides with membrane translocating properties and also summarize de novo designed structures possessing similar features. Conjugates and chimera constructs derived from these sequences with covalently attached bioactive peptide, epitope, oligonucleotide, PNA, drug, reporter molecule will be reviewed. A brief note will refer to the present understanding on the uptake mechanism at the end of each section. © 2005 Wiley Periodicals, Inc. Med Res Rev [source] The pars intercerebralis of the locust brain: A developmental and comparative studyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2002Peter Ludwig Abstract The anterior midline of the brain, also known as the pars intercerebralis, contains the largest collection of neurosecretory cells in the central nervous system of the grasshopper. In this study, we use immunocytochemical, intracellular staining, and histological methods to establish the ontogenies of the various cell types in the brain midline, and show how these cells contribute to the pars intercerebralis of the adult brain. We show that the adult pars intercerebralis develops from three distinct embryonic cell groups: (1) the median neurosecretory cells, which derive from a subset of neuroblasts in the protocerebral hemispheres, and which project axons to the corpora cardiaca; (2) the paired primary commissure pioneers, which derive directly from the mesectoderm of the dorsal median domain and whose axons project to the ventral nerve cord via the midline tract; and (3) the six progeny of the median precursor in the dorsal median domain, which share a common axonal projection with the primary commissure pioneers. Since the adult pars intercerebralis is a fusion product of these independent cellular components, it can only be understood in terms of its origins in the embryonic brain. When the expression pattern of the TERM-1 antigen is compared in subsets of median neurosecretory cells in a wide range of insect orders, the results suggests a common organizational Bauplan for the pars intercerebralis. This hypothesis is supported by the identification of putative homologs of the grasshopper primary commissure pioneers in all these insects. Microsc. Res. Tech. 56:174,188, 2002. © 2002 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||