Crucial Role In Adhesion Research Articles

EPIDERMOLYSIS BULLOSA: CLINICAL OBSERVATION AND PRACTICAL ASPECTS OF TREATMENT

Epidermolysis bullosa (EB) is a heterogenous group of hereditary diseases characterized by blisters on the skin and mucous membranes resulting in scarring. General occurrence rate of the disease is 8-10 cases per 1 000 000 of childbirths, with the 1:1 ratio between boys and girl. In the majority of patients clinical signs of the disease appear at birth and under 1 year. There are four main EB types: simple, functional, dystrophic and Kindler syndrome. Nowadays more than 1000 mutations of 14 structural genes have been identified resulting in the development of different EB phenotypes. Pathogenesis of the disease is based on abnormal, lost or insufficient level of a specific protein, which plays a crucial role in adhesion of the epidermis and dermis. This case report presents a clinical case of EB in a newborn girl and practical aspects of its treatment at the early neonatal period. The girl was born from the II pregnancy, II delivery in normal physiological term of gestation assessed as having 8-9 scores by Apgar scale. Some areas of disturbed skin integrity on the lower limb with some aplasia, penicilli and exfoliation of the oral mucosa were found at birth. These areas underwent changes in the dynamics of the disease, but new ones continuously appeared. Individual topical treatment was prescribed considering the stage of wound healing; partial parenteral feeding was carried out with gradual transition to enteral feeding with breast milk through the orogastric tube. Consultations with specialists providing medical aid to children suffering from EB and close contacts with parent organizations promote professional care to these patients, especially at the early neonatal period.

Spectroscopic Identification of Peptide Chemistry in the Caulobacter crescentus Holdfast.

The bacterium Caulobacter crescentus is known to attach irreversibly to underwater surfaces by utilizing an adhesive structure called the holdfast, which exhibits the greatest known adhesive strength of any organism. The very small size of the holdfast (∼400 nm wide and ∼40 nm high) has made direct chemical analysis difficult, and its structure remains poorly understood. In this study, we employ spectroscopic techniques, including attenuated total reflection infrared spectroscopy (ATR-IR) and X-ray photoelectron spectroscopy, to probe holdfast chemistry. The data indicate the presence of a peptide signal within the holdfast polymer. By comparing the ATR-IR spectrum of the holdfast to peptidoglycan spectra from other bacterial species, we demonstrate the similarity of the holdfast chemistry to that of peptidoglycan, suggesting peptide cross-linking may play a role in holdfast architecture. To probe the molecular groups at the interface, surface-sensitive sum frequency generation spectroscopy was used to show that aromatic and hydroxyl groups related to this protein content at the adhesive interface could be playing a crucial role in adhesion. On the basis of these results, we propose a model of the holdfast architecture with similarities to the peptide cross-linking observed in the peptidoglycan polymer of the bacterial cell wall. These results not only provide information about the development of adhesives that could be based on holdfast chemical architecture but also reveal a potentially yet unexplored biosynthetic pathway in holdfast synthesis that has not yet been revealed by genetic approaches, thereby opening up a potentially new avenue of research in holdfast synthesis.

Glycosphingolipids Recognized by Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic bacterial pathogen associated with hospital-acquired infections, including pneumonia, meningitis, bacteremia, urinary tract infection, and wound infections. Recognition of host cell surface carbohydrates plays a crucial role in adhesion and enables microbes to colonize different host niches. Here the potential glycosphingolipid receptors of A. baumannii were examined by binding of 35S-labeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby a selective interaction with two non-acid glycosphingolipids of human and rabbit small intestine was found. The binding-active glycosphingolipids were isolated and, on the basis of mass spectrometry, identified as neolactotetraosylceramide (Galβ4GlcNAcβ3Galβ4Glcβ1Cer) and lactotetraosylceramide (Galβ3GlcNAcβ3Galβ4Glcβ1Cer). Further binding assays using reference glycosphingolipids showed that A. baumannii also bound to lactotriaosylceramide (GlcNAcβ3Galβ4Glcβ1Cer) demonstrating that GlcNAc was the basic element recognized. In addition, the bacteria occasionally bound to galactosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, isoglobotriaosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide, in analogy with binding patterns that previously have been described for other bacteria classified as “lactosylceramide-binding”. Finally, by isolation and characterization of glycosphingolipids from human skin, the presence of neolactotetraosylceramide was demonstrated in this A. baumannii target tissue.

Expanding the DOPA Universe with Genetically Encoded, Mussel-Inspired Bioadhesives for Material Sciences and Medicine.

Catechols are a biologically relevant group of aromatic diols that have attracted much attention as mediators of adhesion of "bio-glue" proteins in mussels of the genus Mytilus. These organisms use catechols in the form of the noncanonical amino acid l-3,4-dihydroxyphenylalanine (DOPA) as a building block for adhesion proteins. The DOPA is generated post-translationally from tyrosine. Herein, we review the properties, natural occurrence, and reactivity of catechols in the design of bioinspired materials. We also provide a basic description of the mussel's attachment apparatus, the interplay between its different molecules that play a crucial role in adhesion, and the role of post-translational modifications (PTMs) of these proteins. Our focus is on the microbial production of mussel foot proteins with the aid of orthogonal translation systems (OTSs) and the use of genetic code engineering to solve some fundamental problems in the bioproduction of these bioadhesives and to expand their chemical space. The major limitation of bacterial expression systems is their intrinsic inability to introduce PTMs. OTSs have the potential to overcome these challenges by replacing canonical amino acids with noncanonical ones. In this way, PTM steps are circumvented while the genetically programmed precision of protein sequences is preserved. In addition, OTSs should enable spatiotemporal control over the complex adhesion process, because the catechol function can be masked by suitable chemical protection. Such caged residues can then be noninvasively unmasked by, for example, UV irradiation or thermal treatment. All of these features make OTSs based on genetic code engineering in reprogrammed microbial strains new and promising tools in bioinspired materials science.

Fungal Mannosyltransferases as Fitness Attributes and their Contribution to Virulence

Fungal pathogens affect a wide variety of hosts, such as human beings, plants, animals, and insects. The course of infection relies on the virulence grade of the fungus and the strength of the defense mechanisms of the host. Virulence factors are closely related to the cell surface; cell wall proteins have a crucial role in adhesion, hyphal development, hydrophobicity, biofilm formation, immunomodulation and surface variation. The enzymes involved in cell wall biosynthesis are not proper virulence factors, but they are necessary for cell function. The deletion of the genes encoding those enzymes often results in an attenuation of virulence. Secreted proteins and cell wall proteins are modified with sugar residues through the N- and O- glycosylation pathways. A set of glycosidases and glycosyltransferases from the Endoplasmic Reticulum and Golgi bodies determine the outcome of the protein. Proper protein glycosylation is important for folding, localization and protein function. In fungi, the glycoproteins are particularly enriched with mannose moieties. In this review, the role of mannosyltransferases from the Pmt, Ktr/Mnt, Mnn and Och1families for the full development of fungal virulence is summarized and discussed.

PP64. CD15S/CD62E HETEROPHILIC INTERACTION MEDIATES ADHESION OF NON-SMALL CELL LUNG CANCER CELLS TO BRAIN ENDOTHELIUM IN LUNG-BRAIN METASTASIS

AbstractINTRODUCTION: More than 50% of patients with lung cancer, including non-small cell lung cancer (NSCLC), will develop brain metastasis which ultimately results in a dismal prognosis. While there are several key steps in the metastastic process, we have focused on the stage where circulating cancer cells adhere to brain endothelium and extravasate through the Blood Brain Barrier (BBB). Sialyl Lewis x, also known as CD15s, is a cell adhesion molecule which is involved in lymphocyte homing and is overexpressed in non-central nervous system cancers. The purpose of our study was to investigate and characterise the interaction of CD15s and TNF-α induced CD62E (E-selectin) in brain metastatic lung cancer cells and brain derived endothelial cells. METHODS: Expression of CD62E and CD15s were evaluated in brain microvascular endothelial cells (hCMEC/D3), primary (A549 and COR-L105) and metastatic NSCLC cells (NCI-H1299, SEBTA-001 and SEBTA-005) using flow cytometry (FC), immunocytochemistry (ICC), immunohistochemistry (IHC) and Western blotting (WB). Qualitative and quantitative adhesion assays adhesion assays under static and physiological flow at shear stress 2.5 dyn/cm2 and volumetric flow rate 10mL/hr were conducted. Immunoblocking of CD15s was performed to investigate its role in adhesion on hCMEC/D3. RESULTS: While CD15s expression on brain endothelial cells was low, CD15s protein expression was detected on primary NSCLC cells, the highest CD15s expression was observed on metastatic NSCLC cells (P<0.001). CD62E expression was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC followed by primary NSCLC cells. Both CD15s and CD62E expression were detected in tissue sections of lung metastatic brain biopsies. Metastatic NSCLC adhered most strongly to hCMEC/D3 compared to primary NSCLC cells. CD15s immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p<0.001). CONCLUSIONS: This study provides information on the role of CD15s in adhesion of NSCLC cells to brain derived endothelial cells. Our findings suggest that CD15s is involved in the increase in adhesion of circulating lung cancer cells to endothelial cells. Our findings suggests CD15s plays a crucial role in adhesion in concert with TNF-α activation and its binding partner CD62E.

TNF-α enhancement of CD62E mediates adhesion of non-small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis.

BackgroundCD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non–small cell lung cancer (NSCLC).MethodsCD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry.ResultsCD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies.ConclusionThis study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E.

Up-regulation of connexin-43 expression in bone marrow mesenchymal stem cells plays a crucial role in adhesion and migration of multiple myeloma cells

Multiple myeloma (MM) cells accumulate mainly in the bone marrow (BM) and are closely related to the BM microenvironment. Connexin-43 (Cx43) is the main connexin present in bone marrow mesenchymal stem cells (BMSCs). We found that normal BMSCs expressed a lower level of Cx43 than those derived from patients with MM. Cx43 expression in BMSCs was up-regulated at both the gene and protein level, and gap junction-mediated intercellular communication was improved in BMSCs after co-culture with RPMI 8226 cells. The gap junction blocker 18α-glycyrrhetinic acid (18α-GA) inhibited the adherence of MM cells to BMSCs. Treatment with 18α-GA decreased stromal cell-derived factor-1α (SDF-1α) secretion by BMSCs and inhibited the migration of MM cells induced by BMSCs. These results suggest that alteration of Cx43 expression in BMSCs is involved in the interactions of MM cells with the BM environment and plays a crucial role in the adhesion and migration of MM cells.

Characterization of BCAM0224, a Multifunctional Trimeric Autotransporter from the Human Pathogen Burkholderia cenocepacia

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.

Isopeptide Bonds Mechanically Stabilize Spy0128 in Bacterial Pili

Pili on the surface of Streptococcus pyogenes play a crucial role in adhesion to and colonization in human cells. The major pilin subunit, Spy0128, features intramolecular covalent isopeptide bonds that autocatalytically form between the side chains of lysine and asparagine residues and are regarded as important factors in conveying structural stability. In support of this notion, single-molecule force spectroscopy experiments with Spy0128 recently demonstrated the inextensibility of these bonds under mechanical load. However, the molecular determinants of their apparent absolute durability remain unknown. Here, we studied the impact of the isopeptide bond in the Spy0128 C-terminal domain on the mechanical properties of this subunit using force-probe molecular dynamics simulations and force distribution analysis. Even in the presence of the covalent cross-link, the pili β-sandwich domain undergoes partial unfolding, albeit at ∼50% higher rupture forces and with the ability to rapidly refold on the nanosecond timescale. We find that the isopeptide bond is located right at the point of stress concentration in the protein, leading to relative, yet not absolute, mechanical stabilization by the additional cross-link. Our findings indicate how the isopeptide bond enhances the mechanical stability and refolding capability at the molecular level, ensuring that the domain remains predominantly in a potentially adhesive conformation.

Underwater locomotion in a terrestrial beetle: combination of surface de-wetting and capillary forces

For the first time, we report the remarkable ability of the terrestrial leaf beetle Gastrophysa viridula to walk on solid substrates under water. These beetles have adhesive setae on their feet that produce a secretory fluid having a crucial role in adhesion on land. In air, adhesion is produced by capillary forces between the fluid-covered setae and the substrate. In general, capillary forces do not contribute to adhesion under water. However, our observations showed that these beetles may use air bubbles trapped between their adhesive setae to walk on flooded, inclined substrata or even under water. Beetle adhesion to hydrophilic surfaces under water was lower than that in air, whereas adhesion to hydrophobic surfaces under water was comparable to that in air. Oil-covered hairy pads had a pinning effect, retaining the air bubbles on their feet. Bubbles in contact with the hydrophobic substrate de-wetted the substrate and produced capillary adhesion. Additional capillary forces are generated by the pad's liquid bridges between the foot and the substrate. Inspired by this idea, we designed an artificial silicone polymer structure with underwater adhesive properties.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle

BackgroundExtracellular matrix metalloproteinase inducer (EMMPRIN) regulates several biological functions involving the modulation of cell behaviors via cell-cell and cell-matrix interactions. According to its diverse functions, we hypothesized that EMMPRIN may play an important role in endometrial remodeling and establishment of pregnancy in cow.MethodsIn this study, endometrial tissues from the cyclic cows during before ovulation, after ovulation and middle of estrous cycle; and pregnant endometrial tissues from Day 19 to 35 of gestation have been used. Expression of mRNA was analyzed by RT-PCR, qPCR and in situ hybridization whereas protein expression by immunohistochemistry and western blot analysis.ResultsEMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at Day 35 of gestation than the cyclic endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium, and approximately 51 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on Day 19 conceptus. At Day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on Day 30 of gestation, slightly increased expression was found at the site of placentation. Expression of matrix metalloproteinase-2 (MMP-2) and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at Day 19 of gestation however it was also expressed at the site of placentation at Day 30 of gestation as observed for EMMPRIN. Expression of MMP-1 or -9 mRNA was very low and was below the detection limit in the cyclic and pregnant endometrium.ConclusionEMMPRIN from the luminal epithelium may regulate the expression of stromal MMP-2 and -14 suggesting its crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions.

Stromal cell‐derived factor‐1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF‐κB‐dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF-1 and CXCR4 (SDF-1 receptor). Treatment of osteosarcoma cells with SDF-1alpha increased the migration and cell surface expression of alphavbeta3 integrin. CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1alpha-induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF-1alpha-mediated migration and integrin expression. Stimulation of cells with SDF-1alpha increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited SDF-1alpha-mediated cell migration and integrin up-regulation. Stimulation of cells with SDF-1alpha induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the SDF-1alpha-mediated increasing kappaB-luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKalpha and IKKbeta mutants. Taken together, these results suggest that the SDF-1alpha acts through CXCR4 to activate MEK and ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of alphavbeta3 integrins and contributing the migration of human osteosarcoma cells.

Transforming growth factor-β1 increases cell migration and β1 integrin up-regulation in human lung cancer cells

Transforming growth factor-β1 (TGF-β1) plays a crucial role in adhesion and migration of human cancer cells. Besides, integrins are the major adhesive molecules in mammalian cells. Here we found that TGF-β1 increased the migration and cell surface expression of β1 integrin in human lung cancer cells (A549 cells). TGF-β1 stimulation increased phosphorylation of p85α subunit of phosphatidylinositol 3-kinase (PI3K) and Ser 473 of Akt was determined. Besides, we performed that PI3K inhibitor (Ly294002) or Akt inhibitor suppressed the TGF-β1-induced migration activities of A549 cells. Treatment of A549 cells with NF-κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also repressed TGF-β1-induced cells migration and β1 integrins expression. In addition, treatment of A549 cells with TGF-β1 induced IκB kinase α/β (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser 536 phosphorylation, and κB-luciferase activity. Furthermore, the TGF-β1-mediated increases in IKKα/β, IκBα phosphorylation and p65 Ser 536 phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85α and Akt mutants also reduced the TGF-β1-induced κB-luciferase activity. Taken together, our results suggest that TGF-β1 acts through PI3K/Akt, which in turn activates IKKα/β and NF-κB, resulting in the activations of β1 integrins and contributing the migration of human lung cancer cells.

Stromal cell-derived factor-1 enhances motility and integrin up-regulation through CXCR4, ERK and NF-κB-dependent pathway in human lung cancer cells

The chemokine stromal-derived factor-1α (SDF-1α) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that SDF-1α increased the migration and cell surface expression of β1 or β3 integrin in human lung cancer cells (A549 cells). CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1α-induced increase in the migration of lung cancer cells. Stimulation of cells with SDF-1α caused an increase in extracellular signal regulated kinase (ERK) phosphorylation in a time-dependent manner. In addition, treatment of A549 cells with ERK inhibitor (PD98059), NF-κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited SDF-1α-induced cells migration and integrins expression. Treatment of A549 cells with SDF-1α induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, IκBα degradation, p65 Ser 536 phosphorylation, and κB-luciferase activity. The SDF-1α-mediated increases in IKK α/β phosphorylation, p65 Ser 536 phosphorylation, and κB-luciferase activity were inhibited by PD98059 and ERK2 mutant. Taken together, these results suggest that SDF-1α acts through CXCR4 to activate ERK, which in turn activates IKKα/β and NF-κB, resulting in the activations of β1 and β3 integrins and contributing the migration of lung cancer cell.

Up-regulation of CXCR4 Expression in PC-3 Cells by Stromal-Derived Factor-1α (CXCL12) Increases Endothelial Adhesion and Transendothelial Migration: Role of MEK/ERK Signaling Pathway–Dependent NF-κB Activation

The chemokine stromal-derived factor-1alpha (SDF-1alpha/CXCL-12) and its receptor, CXCR4, play a crucial role in adhesion and transendothelium migration (TEM) of prostate cancer cells. We tested the hypothesis that enhanced expression of CXCR4 in prostate cancer cells is dependent upon SDF-1alpha-mediated activation of nuclear factor-kappaB (NF-kappaB). SDF-1alpha increased the CXCR4 mRNA and protein expression in PC-3 cells but not in LNCaP cells. Similarly, SDF-1alpha enhanced the NF-kappaB-dependent transcriptional activity in PC-3 cells but not in LNCaP cells. SDF-1alpha increased PC-3 cell adhesion to the human umbilical vein endothelial cell monolayer and enhanced TEM, which was abrogated with anti-CXCR4 monoclonal antibody (mAb). Suppression of NF-kappaB activity in PC-3 cells by a mutant IkappaBalpha super-repressor adenoviral vector decreased the CXCR4 mRNA expression and inhibited adhesion and TEM. Transient overexpression of p65 subunit of NF-kappaB in PC-3 cells up-regulated CXCR4 receptor expression and increased the adhesion and TEM of these cells in response to SDF-1alpha gradient. Treatment of PC-3 cells with SDF-1alpha leads to nuclear translocation of NF-kappaB protein within 15 to 30 minutes, which correlated with IkappaBalpha phosphorylation. A p42/44 mitogen-activated protein kinase [MAPK, extracellular signal regulated kinase-1/2 (ERK-1/2)] biphasic activation pattern was observed in these cells at 15 minutes and 3 hours after SDF-1alpha treatment. Phosphorylation of IkappaB kinase alpha was observed within 30 minutes, which was blocked by PD98059 [MAPK kinase (MEK) inhibitor]. PD98059 cotreatment significantly inhibited SDF-1alpha-induced NF-kappaB reporter activity and CXCR4 receptor expression as shown by flow cytometry. These data suggest that SDF-1alpha-induced expression of CXCR4 in PC-3 cells is dependent on MEK/ERK signaling cascade and NF-kappaB activation.

CXC chemokine receptor 3 expression on CD34+hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor: chemotaxis and adhesion induced by its ligands, interferon γ–inducible protein 10 and monokine induced by interferon γ

AbstractCXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon γ (IFN-γ)–inducible protein 10 (γIP-10) and monokine induced by IFN-γ (Mig). We report the novel finding that CXCR3 is also expressed on CD34+ hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34+ progenitors. Freshly isolated CD34+progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase–polymerase chain reaction technique. γIP-10 and Mig induced chemotaxis of GM-CSF–stimulated CD34+ progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block γIP-10–induced and Mig-induced CD34+ progenitor chemotaxis. These chemotactic attracted CD34+ progenitors are colony-forming units—granulocyte-macrophage. γIP-10 and Mig also induced GM-CSF–stimulated CD34+ progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of γIP-10 and Mig but not of chemokine stromal cell–derived factor 1α. γIP-10–induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF–stimulated CD34+progenitors. Moreover, γIP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF–stimulated CD34+progenitors. These results indicate that CXCR3–γIP-10 and CXCR3–Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34+ hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34+ hematopoietic progenitors.

CXC chemokine receptor 3 expression on CD34+hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor: chemotaxis and adhesion induced by its ligands, interferon γ–inducible protein 10 and monokine induced by interferon γ

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon gamma (IFN-gamma)-inducible protein 10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report the novel finding that CXCR3 is also expressed on CD34(+) hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34(+) progenitors. Freshly isolated CD34(+) progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase-polymerase chain reaction technique. gamma IP-10 and Mig induced chemotaxis of GM-CSF-stimulated CD34(+) progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block gamma IP-10-induced and Mig-induced CD34(+) progenitor chemotaxis. These chemotactic attracted CD34(+) progenitors are colony-forming units-granulocyte-macrophage. gamma IP-10 and Mig also induced GM-CSF-stimulated CD34(+) progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of gammaIP-10 and Mig but not of chemokine stromal cell-derived factor 1 alpha. gamma IP-10-induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF-stimulated CD34(+) progenitors. Moreover, gamma IP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF-stimulated CD34(+) progenitors. These results indicate that CXCR3-gamma IP-10 and CXCR3-Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34(+) hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34(+) hematopoietic progenitors. (Blood. 2000;96:1230-1238)

A monoclonal antibody disrupting calcium-dependent cell-cell adhesion of brain tissues: possible role of its target antigen in animal pattern formation.

The Ca2+-dependent cell-cell adhesion system (CDS) is thought to be essential for the formation and maintenance of cell adhesion in a wide variety of tissues. Previous studies suggested that CDS has some cell-type specificity; for example, the monoclonal antibody ECCD-1 selectively recognizes CDS of certain epithelial tissues in mouse embryos but not nervous tissues. In the present study, we have obtained a monoclonal antibody, designated NCD-1, that disrupts connections between brain cells of mouse embryos. A series of experiments suggested that NCD-1 specifically recognizes CDS. We then determined the distribution of the NCD-1 antigen in various mouse tissues. NCD-1 reacted with cells of the following tissues and cell lines: nervous tissues from various sources, lens, striated muscle, cardiac muscle, glioma G26-20, adrenocortical tumor Y1, and melanoma B16. None of these cells reacted with ECCD-1, and the cells reactive with ECCD-1 did not react with NCD-1. There was also a class of cells that did not react with either ECCD-1 or NCD-1. These results suggest that cells in the body can be classified into at least three groups containing CDS of differing specificities. A map of the tissue localization of these different classes of CDS also suggests that the expression of cell-type-specific cell adhesion molecules in each tissue plays a crucial role in adhesion between the same cell types and segregation of different cell types in processes essential for animal morphogenesis.