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PubMedDate not supplied

Localization of a structural defect in type I procollagen in a patient affected with the severe non-lethal form of Osteogenesis imperfecta.

A case of severe non-lethal Osteogenesis imperfecta was studied. The patient's cultured skin fibroblasts synthesised a mixed population of type I collagen chains some of which showed abnormal behaviour on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further analysis revealed that two types of alpha 1(I) chains were synthesised, both an abnormal, slower migrating and a normal species. A small defect in one allele of one of the type I procollagen chains could lead to the larger size of the abnormal chains, probably caused by overmodifications of the triple helical region. CNBr peptide mapping allowed us to localise the defect midway along the triple helix: the defect site could be assigned to the region between the alpha 1(I)CB-3 and CB-7 peptides. The abnormal alpha 1(I) chains synthesised by the patient's cells had a melting temperature which was about 2 degrees C lower than normal chains. The results appear to be in agreement with the defect localisation and the phenotype.

Amino Acids↗
PubMed2001

Differential regulation of sphingosine-1-phosphate- and VEGF-induced endothelial cell chemotaxis. Involvement of G(ialpha2)-linked Rho kinase activity.

We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by sphingosine-1-phosphate (S1P), a potent bioactive lipid released from activated platelets, and by vascular endothelial growth factor (VEGF), a well-recognized angiogenic factor. S1P-induced endothelial cell migration was maximum at 1 microM (approximately 8-fold increase with PA endothelium) and surpassed the maximal response evoked by either VEGF (10 ng/ml) (approximately 2.5-fold increase) or hepatocyte growth factor (HGF) (approximately 2.5-fold increase). Migration induced by S1P, but not by VEGF, was significantly inhibited by treatment with antisense oligonucleotides directed to Edg-1 and Edg-3 (endothelial differentiation gene) S1P receptors and by G protein modification. These strategies included pretreatment with pertussis toxin, or transfection with mini-genes encoding a betagamma subunit inhibitory peptide of the beta-adrenergic receptor kinase, or an 11-amino-acid peptide that inhibits G(1alpha2) signaling. Various strategies to interrupt Rho family signaling, including C(3) exotoxin, dominant/negative Rho, or the addition of Y27632, a cell-permeable Rho kinase inhibitor, significantly attenuated S1P- but not VEGF-induced migration. Conversely, pharmacologic inhibition of either myosin light chain kinase, src family tyrosine kinases, or phosphatidylinositol-3' kinase reduced basal endothelial cell migration and abolished VEGF-induced endothelial cell migration but did not inhibit the increase in S1P-induced migration. Whereas VEGF and S1P increased both p42/p44 extracellular regulated kinase and p38 mitogen-activated protein (MAP) kinase activities, only p38 MAP kinase inhibition significantly reduced VEGF- and S1P-stimulated migration. These data confirm S1P as a potent endothelial cell chemoattractant through G(1alpha2)-coupled Edg receptors linked to Rho-associated kinase and p38 MAP kinase activation. The divergence in signaling pathways evoked by S1P and VEGF suggests complex and agonist-specific regulation of endothelial cell angiogenic responses.

Animals↗
PubMed2001

The short arm of the laminin gamma2 chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion.

Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the gamma2 chain that removes the NH(2)-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the gamma2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the gamma2 chain short arm, we expressed mutated gamma2 cDNAs into immortalized gamma2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the gamma2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the gamma2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.

Basement Membrane↗
PubMed1995

Epidermal growth factor-binding protein activates soluble and receptor-bound single chain urokinase-type plasminogen activator.

Epidermal growth factor-binding protein (EGF-BP) is a serine proteinase that reversibly associates with epidermal growth factor (EGF). We analyzed the reaction of EGF-BP with urokinase type plasminogen activator (u-PA), a serine proteinase that promotes pericellular proteolysis and cellular migration. EGF-BP cleaved single chain u-PA (scu-PA) between Lys158 and Ile159, converting the zymogen into enzymatically active two-chain u-PA (tcu-PA), as shown by SDS-PAGE, N-terminal sequence analysis, and enzymatic assay. The kcat and Km of the activation reaction were (5.6 +/- 0.6) x 10(-2)s-1 and 2.0 +/- 0.3 microM, yielding a catalytic efficiency of 2.8 x 10(4) M-1.s-1. EGF-BP also activated scu-PA bound to receptors on U937 monocytes as demonstrated by the generation of amidase activity against a tcu-PA-specific fluorogenic substrate. By activating scu-PA, EGF-BP may initiate u-PA-dependent cell surface proteolysis and therefore enhance EGF activities that require cellular migration and/or tissue remodeling.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity.

Integrin cell surface adhesion receptors play a central role in mediating cell migration. We have developed a model system consisting of CHO cells ectopically expressing the alpha IIb beta 3 integrin to study integrin affinity and cytoskeletal interactions during cell migration. The alpha IIb beta 3 integrins are suited for study of integrin receptors during cell migration because they are well characterized with respect to ligand binding, cytoskeletal interactions, and signal transduction, and mutants with altered receptor function are available. The alpha IIb beta 3 receptor specifically mediates migration of alpha IIb beta 3-transfected CHO cells. The migration of transfected CHO cells was studied on a fibrinogen substrate both by time lapse videomicroscopy and by random and haptotactic transwell assays. Haptotactic and random transwell assays measured distinct aspects of migration, with the random transwell assay correlating most closely with time lapse videomicroscopy. Mutations in the cytoplasmic domains that increase ligand affinity or activation of the alpha IIb beta 3 receptor into a high affinity state by the LIBS6 antibody decreased the migration rate. Likewise, mutations that increase cytoskeletal organization without affecting affinity also decreased the migration rate. In contrast, truncation of the beta chain, which alters cytoskeletal associations as assayed by absence of focal adhesions, decreased haptotactic migration while increasing random migration. These effects on the migration rate were partially compensated for by altering substrate concentration, demonstrating optimum substrate concentrations that supported maximal migration. For example, cells expressing integrins locked in the high affinity state showed maximal migration at lower substrate concentrations than cells expressing low affinity receptor. Together, these results implicate the strength of adhesion between cell and substrate, as modulated by receptor affinity, organization of adhesive complexes, and substrate concentration, as important regulators of cell migration rate. Further, we demonstrate a dominant effect of high affinity integrin in inhibiting migration regardless of the organization of adhesive complexes. These observations have potential implications for tumor metastasis and its therapy.

Amino Acid Sequence↗
PubMed2007

Rho-associated kinase inhibitor reduces tumor recurrence after liver transplantation in a rat hepatoma model.

Tumor recurrence after liver transplantation still remains a significant problem in patients with hepatocellular carcinoma. The small GTPase Rho/Rho-associated kinase (ROCK) pathway is involved in the motility and invasiveness of cancer cells. We investigated whether tacrolimus activated the Rho/ROCK signal pathway to promote the invasiveness of rat hepatocellular carcinoma cells. We also investigated whether the ROCK inhibitor Y-27632 suppressed tumor recurrence after experimental liver transplantation in a rat hepatocellular carcinoma model. Orthotopic liver transplantation was performed in hepatocellular carcinoma cell line McA-RH7777-bearing rats. Tacrolimus was administered to liver transplant rats and these rats were divided into two groups: the Y-27632-treated (10 mg/kg, for 28 days) group and the Y-27632-untreated group. Tacrolimus enhanced the cancer cell migration and stimulated phosphorylation of the myosin light chain (MLC), a downstream effector of Rho/ROCK signaling. Y-27632 suppressed the cancer cell migration and tacrolimus-induced MLC phosphorylation. Suppression of tumor recurrence after liver transplantation and significant prolongation of survival were observed in the Y-27632-treated rats in comparison with theY-27632-untreated rats. Tacrolimus stimulates the Rho/ROCK signal pathway to enhance the invasiveness of hepatocellular carcinoma, and the ROCK inhibitor Y-27632 can be used as a new antimetastatic agent for the prevention of tumor recurrence after liver transplantation.

Amides↗
PubMed1976

Radial migration of DNA molecules in cylindrical flow. III. Circles and the effect of non-gaussian polymer statistics.

We have previously shown that DNA will migrate radially inward in a concentric-cylinder shear flow apparatus. We assumed gaussian chain statistics, and we considered only linear molecules. In this paper, we extend the analysis to closed circular molecules, and we consider non-gaussian statistics for both linears and circles. We find that, in good solvents, the inward radial migration velocity is more sensitive to the molecular weight than M5/2, which we previously reported for gaussian chains. Furthermore, linears migrate radially inward 8 times faster than do circles of the same molecular weight. This suggests the possibility of separating linear from circular DNA in solution.

DNA↗
PubMed1995

Phase transition behavior and molecular structures of monounsaturated phosphatidylcholines. Calorimetric studies and molecular mechanics simulations.

High resolution differential scanning calorimetric studies were performed to investigate the thermotropic phase behavior of 26 molecular species of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines. In parallel with calorimetric studies, the energy-minimized structures and steric energies of the diglyceride moieties of these monoenoic lipids were determined using a molecular mechanics approach. The combined calorimetric and computational studies led to the following results and conclusions. (i) When a single cis-carbon-carbon double bond (delta) is incorporated into a saturated diacylphosphatidylcholine molecule at any position within the central segment of the long sn-2 acyl chain, the resulting monoenoic lipid molecules will, in excess water, exhibit reduced phase transition temperature (Tm) and transition enthalpy (delta H) as they undergo the gel to liquid-crystalline phase transition. The Tm and delta H-lowering effects of the delta bond can be attributed to a decrease in the chain length of the sn-2 acyl chain, a change in the chain length difference between the sn-1 and sn-2 acyl chains, and a local perturbation of the chain-chain van der Waals interaction in the vicinity of the delta bond. (ii) For a series of positional isomers of 1-stearoyl-2-cis-octadecenoylphosphatidylcholine, C(18):C(18:1 delta n)PC, with a delta bond at different positions along the sn-2 acyl chain, the Tm value depends critically on the position of the delta bond. Specifically, the Tm value is minimal as the delta bond is located at the geometric center of the linear segment of the sn-2 acyl chain, and the Tm value is progressively increased as the delta bond migrates toward either end of the sn-2 acyl chain. (iii) The various monoenoic phosphatidylcholines under study can be divided into two groups. The Tm values of most lipids in each group can be correlated in an identical manner with their structural parameters, yielding a common Tm-structure relationship.

Calorimetry, Differential Scanning↗
PubMed2005

Laminin alpha3 LG4 module induces keratinocyte migration: involvement of matrix metalloproteinase-9.

Laminin alpha3 chain, a functionally key subunit of laminin-5, contains a large globular module (G module) which consists of a tandem repeat of five homologous LG modules (LG1-5). We previously demonstrated that the LG4 module of laminin alpha3 chain (alpha3 LG4) induces a matrix metalloproteinase-1 (MMP-1) expression through the interaction with syndecans leading to MAPK activation/IL-1beta expression signaling loop (Utani et al., J. Biol. Chem. 278, 34483-34490, 2003). Here, we show that a recombinant alpha3 LG4 and synthetic peptides containing syndecan binding motif induced a cell motility and a MMP-9 expression in ketarinocytes. The synthetic peptide (A3G756)-induced cell migration and MMP-9 upregulation were inhibited by each application of a heparin and an IL-1 receptor antagonist (IL-1RA), suggesting the involvement of syndecans and IL-1beta autocrine. Furthermore, the A3G756-induced cell motility was inhibited by an MMP-9 inhibitor and a neutralizing antibody of MMP-9, indicating induced cell motility was dependent on an MMP-9 activity. Taken these together, laminin-5 alpha3 LG4 module may play an important role in re-epithelialization at tissue remodeling.

Amino Acid Sequence↗
PubMed2001

New fluorescent probes for the measurement of cell membrane viscosity.

BACKGROUND: Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility. RESULTS: Longer hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses. CONCLUSIONS: The modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

Cell Membrane↗
PubMed2003

Inhibition of myoblast migration via decorin expression is critical for normal skeletal muscle differentiation.

During limb skeletal muscle formation, committed muscle cells proliferate and differentiate in the presence of extracellular signals that stimulate or repress each process. Proteoglycans are extracellular matrix organizers and modulators of growth factor activities, regulating muscle differentiation in vitro. Previously, we characterized proteoglycan expression during early limb muscle formation and showed a spatiotemporal relation between the onset of myogenesis and the expression of decorin, an important muscle extracellular matrix component and potent regulator of TGF-beta activity. To evaluate decorin's role during in vivo differentiation in committed muscle cells, we grafted wild type and decorin-null myoblasts onto chick limb buds. The absence of decorin enhanced the migration and distribution of myoblasts in the limb, correlating with the inhibition of skeletal muscle differentiation. Both phenotypes were reverted by de novo decorin expression. In vitro, we determined that both decorin core protein and its glycosaminoglycan chain were required to reverse the migration phenotype. Results presented here suggest that the enhanced migration observed in decorin-null myoblasts may not be dependent on chemotactic growth factor signaling nor the differentiation status of the cells. Decorin may be involved in the establishment and/or coordination of a critical myoblast density, through inhibition of migration, that permits normal muscle differentiation during embryonic myogenesis.

Animals↗
PubMed1996

A mutation (dosach) in Drosophila which affects aster formation and nuclear migration during cleavage.

Here we describe a new mutant, dosach (dos), in Drosophila melanogaster. In the mutant, centrosomes divide and initiate spindle formation similar to that seen in wild-type embryos. Nevertheless, mutant embryos form cleavage spindles that lack visible asters and display abnormal morphology, including mono- and tri-polar spindles, spindle chains and incorrect alignment. Irregular nuclear migration is also observed in mutant embryos, and this may suggest that astral microtubules are important for spindle spacing during cleavage and also in maintaining the integrity of the mitotic apparatus. Confocal microscopy has been used to correlate organization of microtubules, centrosomal proteins and chromosomes in wild-type and dosach (dos) embryos.

Animals↗
PubMed2002

Identification of neurite outgrowth promoting sites on the laminin alpha 3 chain G domain.

Laminins are expressed in specific tissues and are involved in various biological activities including promoting cell adhesion, growth, migration, neurite outgrowth, and differentiation. The laminin alpha3 chain is mainly located in the skin and is also expressed in the floor plate of the developing neural tube. Previously, we showed that the human laminin alpha3 chain LG4 module binds to syndecan-2/4, a membrane-associated proteoglycan, and promotes human fibroblast adhesion. Here, we have evaluated the neurite outgrowth activity of the laminin alpha3 chain LG4 and LG5 modules. Three overlapping recombinant proteins, which contained LG4 and/or LG5 modules of the human laminin alpha3 chain, were prepared using a mammalian cell expression system. Two proteins, rec-alpha3LG4-5 and rec-alpha3LG4, promoted cell attachment and neurite outgrowth of rat pheochromocytoma PC12 cells, but rec-alpha3LG5 was inactive. Twenty-two peptides covering the entire LG4 module were synthesized and tested for cell attachment and neurite outgrowth activity to identify active sites of the LG4 module. A3G75 (KNSFMALYLSKG, alpha3 chain 1411-1422) and A3G83 (GNSTISIRAPVY, alpha3 chain 1476-1487) promoted PC12 cell attachment and neurite outgrowth. Additionally, A3G75 and A3G83 inhibited PC12 cell attachment to rec-alpha3LG4. These results suggest that the A3G75 and A3G83 sites are important for PC12 cell attachment and neurite outgrowth in the laminin alpha3 chain LG4 module. We also conjugated the A3G75 and A3G83 peptides on chitosan membranes to test their potential as bio-materials. These peptide-conjugated chitosan membranes were more active for neurite outgrowth than the peptide-coated plates. These results suggest that the A3G75- and A3G83-conjugated chitosan membranes are applicable as bio-medical materials for neural tissue repair and engineering.

Amino Acid Sequence↗
PubMed2005

Pathogenic role of the CXCL16-CXCR6 pathway in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive T cell infiltration into the synovium. The accumulated T cells express type 1 cytokines, such as interferon-gamma (IFNgamma) and tumor necrosis factor alpha, and activated markers of inflammation, such as CD154 and inducible costimulator (ICOS). It is thought that chemokines contribute to T cell accumulation in the synovium. In this study, we examined the role of CXCL16 and CXCR6 in T cell migration and stimulation in RA synovium. METHODS: Expression of CXCL16 and CXCR6 was analyzed by immunohistochemistry, reverse transcription-polymerase chain reaction, Western blotting, and/or flow cytometry. Migration activity was assessed using a chemotaxis chamber. IFNgamma production was analyzed by enzyme-linked immunosorbent assay. The effect of anti-CXCL16 monoclonal antibody on murine collagen-induced arthritis (CIA) was evaluated. RESULTS: CXCL16 was expressed in RA synovium. CXCR6 was expressed more frequently on synovial T cells than in peripheral blood. Moreover, CXCR6-positive synovial T cells more frequently expressed CD154 and ICOS than did CXCR6-negative T cells. Stimulation with interleukin-15 (IL-15) up-regulated the expression of CXCR6 on peripheral blood T cells, and then stimulation with CXCL16 induced migration of IL-15-stimulated T cells and enhanced IFNgamma production. Furthermore, anti-CXCL16 monoclonal antibody significantly reduced the clinical arthritis score and reduced infiltration of inflammatory cells and bone destruction in the synovium of mice with CIA. CONCLUSION: Our results indicate that CXCL16 plays an important role in T cell accumulation and stimulation in RA synovium and suggest that CXCL16 could be a target molecule in new therapies for RA.

Aged↗
PubMed1990

Intraspecific myosin light chain polymorphism in the white muscle of herring (Clupea harengus harengus, L.).

The myosin contained in white and red muscles of herring (Clupea harengus harengus) was purified, and its subunit composition analyzed by electrophoretic techniques. The only myosin isoform present in red muscles was made up of one type of heavy chain and two types of light chain. The native myosin from white muscles migrated as one wide band. Analysis of the extracts by SDS/glycerol/PAGE from white muscles revealed one main type of heavy chain. Light chains were identified by SDS-PAGE analysis of electrophoretically purified myosin, and two-dimensional electrophoresis of the extracts demonstrated differences in the light chain composition of white and red muscles. Using this methodology, light chain polymorphism was detected in white muscles among members of the same species.

Animals↗
PubMedDate not supplied

[Analysis of the viral capsid gene of Alfalfa mosaic virus (CP A1MV) cloned to the vector for plant transformation pBI121].

The virus gene CP AlMV cloned to the vector pBI121 (Clontech) was analyzed using the method of restriction analysis and polymerase chain reaction. The restriction analysis with the use of ApaI enzyme has shown presence of the specific fragment with a part of CP gene about 2.5 kb. The same procedure with EcoRI and HindIII enzymes has shown difference in electrophoretic migration of the formed fragments. Polymerase chain reaction analysis has shown the existence of the specific amplification product of the CP gene 666 bp long. Such investigations have proved successful cloning and presence of the CP AlMV gene in vector pBI121 and could afford to make following transformation of the higher plants with attaching genetic tolerance to the virus from Bromoviridae family.

Alfalfa mosaic virus↗
PubMed1999

Turnover rates at regulatory phosphorylation sites on myosin II in endothelial cells.

Assembly and motor activity of non-muscle myosin II can be regulated by phosphorylation. Because myosin II-containing structures undergo continuous assembly, disassembly, and remodeling in living cells, especially during cell migration, myosin II should undergo frequent phosphorylation and dephosphorylation. This study examines the turnover of phosphate on myosin II in stationary and migrating endothelial cells. Cultured bovine aortic endothelial cells were metabolically labeled with (32)P-phosphate, and the incorporation of phosphate into myosin II was assessed by quantitative phosphor imaging of electrophoretic gels of myosin II immunoadsorbed from cell lysates. Likewise, phosphate turnover was measured upon chasing the (32)P with unlabeled phosphate. Phosphate incorporated very slowly into heavy chains, taking >8 h to plateau, and turned over at </=12.7% per hour. Regulatory light chains became completely labeled in </=4 h, and turnover occurred at two rates: 49% turned over at 20% per hour, the remainder at 67% per hour. Peptide mapping showed light chain phosphorylation at serine 19 and threonine 18, but phosphate turnover was the same in mono- and diphosphorylated lights chains, indicating that rates are not different at the two sites. When cells were stimulated to migrate by wounding a confluent monolayer, the rate of light chain dephosphorylation increased and the rate of phosphate incorporation decreased causing a net 10% dephosphorylation of light chains. This process persisted during migration and returned to baseline when the wound was closed. There was no effect on heavy chain phosphates. Light chain dephosphorylation may facilitate migration by mobilizing myosin II during cytoskeletal remodeling.

Animals↗