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At least 721 records · Page 40Linked to original sources

PubMed1981

Apolipoprotein B is structurally and metabolically heterogeneous in the rat.

Electrophoresis of rat apolipoprotein B (apoB) on 5% polyacrylamide gels in the presence of NaDodSO4 separates three major components: PI, which comigrates with human low density lipoprotein (LDL) apoB; PII, a slightly faster-moving satellite band; and PIII, which migrates somewhat more slowly than myosin heavy chain. The proportion of PIII decreases with increasing density of the parent rat lipoprotein, from 90% an 70%, respectively, in chylomicrons and very low density lipoproteins (VLDL), to 7% in the major LDL2 (density 1.038-1.063 g/ml) fraction. A major component that comigrates with rat PIII is a marker for human chylomicron apoB, being absent from human VLDL, intermediate density lipoprotein (IDL), and LDL. Preliminary immunological and peptide mapping data show that rat apoB PI and PIII are closely related structurally, with the latter possibly being a large fragment of the former. Both peptides are synthesized in rat liver and found in Golgi secretory vesicles. Kinetic tracer experiments show that rat PI and PIII are present on separate VLDL particles, both of which are extensively removed from the circulation at the remnant stage, and that the declining PIII-to-PI/II ratios in IDL and LDL may be attributed to the more rapid turnover of PIII-containing lipoproteins at all levels, particularly within the LDL density range.

Animals↗

PubMed2003

Hepatocyte growth factor switches orientation of polarity and mode of movement during morphogenesis of multicellular epithelial structures.

Epithelial cells form monolayers of polarized cells with apical and basolateral surfaces. Madin-Darby canine kidney epithelial cells transiently lose their apico-basolateral polarity and become motile by treatment with hepatocyte growth factor (HGF), which causes the monolayer to remodel into tubules. HGF induces cells to produce basolateral extensions. Cells then migrate out of the monolayer to produce chains of cells, which go on to form tubules. Herein, we have analyzed the molecular mechanisms underlying the production of extensions and chains. We find that cells switch from an apico-basolateral polarization in the extension stage to a migratory cell polarization when in chains. Extension formation requires phosphatidyl-inositol 3-kinase activity, whereas Rho kinase controls their number and length. Microtubule dynamics and cell division are required for the formation of chains, but not for extension formation. Cells in the monolayer divide with their spindle axis parallel to the monolayer. HGF causes the spindle axis to undergo a variable "seesaw" motion, so that a daughter cells can apparently leave the monolayer to initiate a chain. Our results demonstrate the power of direct observation in investigating how individual cell behaviors, such as polarization, movement, and division are coordinated in the very complex process of producing multicellular structures.

Actins↗

PubMed1995

Extragenic suppressors of nudC3, a mutation that blocks nuclear migration in Aspergillus nidulans.

Nuclear migration plays an important role in the growth and development of many organisms including the filamentous fungus Aspergillus nidulans. We have cloned three genes from A. nidulans, nudA, nudC, and nudF, in which mutations affect nuclear migration. The nudA gene encodes the heavy chain of cytoplasmic dynein. The nudC gene encodes a 22-kD protein. The nudF gene was identified as an extra copy suppressor of the temperature sensitive (ts-) nudC3 mutation. The nudC3 mutation substantially decreases the intracellular concentration of the nudF protein at restrictive temperature. This is restored toward the normal level by an extra copy of nudF. To identify other genes whose products interact directly or indirectly with the NUDC protein, we have isolated a set of extragenic suppressors showed them to represent nine different genes, designated sncA-sncI (for suppressor of nudC). sncA-sncH were either dominant or semidominant in diploids homozygous for nudC3 and heterozygous for the snc mutations. All of the suppressors reversed the ts- phenotype of nudC3 by restoring the intracellular concentration of the NUDF protein.

Aspergillus nidulans↗

PubMed2005

Regulation of epithelial tubule formation by Rho family GTPases.

Previous work has established that the integrin signal transduction pathway plays an important role in the regulation of epithelial tubule formation. Furthermore, it has been demonstrated that Rho-kinase, an effector of the Rho signaling pathway, is an important downstream modulator of collagen-mediated renal and mammary epithelial tubule morphogenesis. In the present study, MDCK cells that expressed mutant dominant-negative, constitutively active Rho family GTPases were used to provide further insight into Rho-GTPase signaling and the regulation of epithelial tubule formation. Using collagen gel overlays on MDCK cells as a model system, we observed phosphorylated myosin light chain (pMLC) at the leading edge of migrating lamellipodia. This epithelial remodeling led to the formation of multicellular branching epithelial tubular structures with extensive tight junctions. However, in cells expressing dominant-negative RhoN19, MLC phosphorylation, epithelial remodeling, and tubule formation were inhibited. Instead, only small apical lumens with a solitary tight junctional ring were observed, providing further evidence that Rho signaling through Rho-kinase is important in the regulation of epithelial tubule formation. Because the present model for the Rho signaling pathway proposes that Rac plays a prominent but reciprocal role in cell regulation, experiments were conducted using cells that expressed constitutively active RacV12. When incubated with collagen gels, RacV12-expressing cells formed small apical lumens with simple tight junctions, suggesting that Rac1 signaling also has a prominent role in the regulation of epithelial morphogenesis. Complementary collagen gel overlay experiments with wild-type MDCK cells demonstrated that endogenous Rac1 activation levels decreased over a time course consistent with lamellipodia and tubule formation. Under these conditions, Rac1 was initially localized to the basolateral membrane. However, after epithelial remodeling, activated Rac1 was observed primarily in lamellipodia. These studies support a model in which Rac1 and RhoA are important modulators of epithelial tubule formation.

Animals↗

PubMed1977

Isolation and characterization of myosin from subjects with asymmetric septal hypertrophy.

Human cardiac myosin isolated from operatively obtained samples of ventricular septum and left ventricular free wall of subjects with asymmetric septal hypertrophy (ASH) was compared, with respect to structural and enzymatic properties, to myosin isolated from hearts of subjects without heart disease. The following parameters were studied: (1) activation of myosin ATPase activity by K+-EDTA and Ca2+, (2) molecular weight of the heavy and light chains of myosin as determined by electrophoretic migration in polyacrylamide-sodium dodecyl sulfate (SDS) gels and (3) ability to form bipolar aggregates at low ionic strength, as examined by electron microscopy. No difference was present in any of these parameters between human cardiac myosin from subjects with ASH and from subjects without heart disease. Thus, the genetic defect present in subjects with ASH is not expressed in the particular structural and functional characteristics of myosin evaluated in this study.

Adenosine Triphosphatases↗

PubMedDate not supplied

Characterization of myosin from patients with asymmetric septal hypertrophy.

Human cardiac myosin isolated from operatively obtained samples of ventricular septum and left ventricular free wall of patients with asymmetric septal hypertrophy (ASH) was compared, with respect to structural and enzymatic properties, to myosin isolated from hearts of patients without heart disease. The following parameters were studied: 1) activation of myosin ATPase activity by K+-EDTA and Ca2+,2) molecular weight of the heavy and light chains of myosin as determined by electrophoretic migration in SDS-polyacrylamide gels, and 3) ability to form bipolar aggregates at low ionic strength, as examined by electron microscopy. No difference was present in any of these parameters between human cardiac myosin from patients with ASH and from patients without heart disease. Thus, the genetic defect present in patients with ASH is not expressed in the particular structural and functional characteristics of myosin evaluated in this study.

Actomyosin↗

PubMed1989

The elastin receptor shows structural and functional similarities to the 67-kDa tumor cell laminin receptor.

Laminin- and elastin-binding proteins were isolated by ligand affinity chromatography from plasma membranes of fetal bovine auricular chondroblasts and human A2058 melanoma cells. From both cell types, a 67-kDa protein was identified which bound to either elastin or laminin affinity resins. Structural and functional similarities between the elastin and laminin-binding proteins were suggested by 1) cross-reactivity between antibodies directed against the two proteins; 2) elution of the laminin receptor from laminin columns with soluble elastin peptides; and 3) modulation of substrate binding by galactoside sugars. In addition, extraction properties indicate that both receptors are peripheral membrane proteins whose association with the cell surface is mediated by their lectin properties. Mapping of the binding site on laminin suggests that the 67-kDa chondroblast receptor interacts with a hydrophobic elastin-like sequence in domain V of the B1 chain, and chemotaxis studies indicate that cell migration to elastin peptides and laminin involves the same receptor.

Amino Acid Sequence↗

PubMed1986

Fatty acylation of murine Ia alpha, beta, and invariant chains.

Labeling of murine spleen cells with [3H]palmitate followed by analysis of immunoprecipitated Ia molecules indicated that Ia alpha- and beta-chains and their associated invariant chain contain covalently bound fatty acid. This modification is present in I-A and I-E molecules and has been found in all haplotypes examined. The 3H label was not dissociated from the glycoproteins by detergents or under the denaturing conditions of SDS-polyacrylamide gel electrophoresis. The fatty acid linked to Ii is released by treatment with neutral hydroxylamine, which indicates thioester linkage. The acylation of alpha- and beta-chains appears to involve attachment of palmitoyl groups via an ester linkage sensitive to alkaline hydrolysis. The radioactive species released from the isolated chains by treating with KOH/methanol co-migrated with palmitic acid and palmitic acid methyl ester on thin-layer chromatography.

Acylation↗

PubMed1984

Specific binding of blood coagulation factor XIIIa to thrombin-stimulated platelets.

We studied the binding of 125I-platelet and plasma Factor XIII (125I-Factor XIII) to human platelets. When 125I-Factor XIII was incubated with gel-filtered platelets, calcium chloride (5 mM) and thrombin (1 unit/ml) at 37 degrees C, saturable binding was observed. Half-maximal binding occurred at 1 min. Binding was inhibited 93% by a 100-fold molar excess of unlabeled ligand but not by other purified proteins. Greater than 87% of platelet-bound radioactivity migrated as thrombin-cleaved a-chains (a'-chains) in sodium dodecyl sulfate-polyacrylamide gels indicating that Factor XIIIa but not Factor XIII binds to platelets. 125I-Factor XIIIa does not bind to unstimulated platelets. When platelet secretion was blocked, binding was markedly inhibited. 125I-Factor XIIIa bound minimally to platelets stimulated with agonists other than thrombin. Thus, binding is dependent on platelet activation, as well as modification of platelets by thrombin. 125I-Factor XIIIa bound to gamma-thrombin-stimulated platelets, at concentrations which did not clot fibrinogen. Therefore, Factor XIIIa is not bound to fibrin associated with platelets. Binding was only partially reversible. Approximately 12,000 molecules of Factor XIIIa were bound per platelet. 125I-Factor XIIIa bound normally to platelets from patients with severe Glanzmann's thrombasthenia indicating that 125I-Factor XIIIa does not bind to platelet glycoproteins IIb or IIIa, or platelet-bound fibrinogen. Chymotrypsin treatment of platelets inhibited 125I-Factor XIIIa binding by 78% without inhibiting secretion. Methylamine and putrescine, Factor XIIIa substrates, and N-ethylmaleimide, an active site inhibitor, did not inhibit binding. Factor XIIIa bound to platelets was enzymatically active and catalyzed [3H]putrescine incorporation into platelet proteins. The specific binding of Factor XIIIa to platelets suggests it may play a role in physiologic reactions involving platelets.

Binding Sites↗

PubMed1983

Characterization of membranes removed during open-sky vitrectomy.

Membranes removed during open-sky vitrectomy have been characterized by electron microscopy, reaction with anti-human fibrinogen, susceptibility to enzymatic digestion, amino acid analysis, and electrophoresis in sodium dodecyl sulfate. There were significant differences between longstanding and newly formed membranes. Longstanding membranes contained substantial amounts of hydroxyproline, glycine, and hydroxylysine, were capable of digestion by collagenase but not by plasmin, yielded faint positive results with anti-human fibrinogen, and showed fibrils characteristic of collagen by electron microscopy. After digestion with pepsin, electrophoresis revealed bands that migrated the same distance as vitreous collagen chains. This type of membrane is evidently collagenous in nature. A second type of membrane, which developed in the course of vitrectomy, contained no hydroxyproline, only traces of hydroxylysine, and relatively small amounts of glycine, was digested by plasmin, yielded strong positive results with anti-human fibrinogen, and showed fibers that were not characteristic of collagen by electron microscopy. Electrophoresis demonstrated bands similar to authentic fibrin in these newly formed membranes. These data suggest that this second type of membrane is composed largely of fibrin. Prevention of the formation of this second type of membrane during vitrectomy may require the addition of agents that inhibit fibrin formation.

Amino Acids↗

PubMed1980

Messenger RNA coding for the deleted heavy chain of mouse myeloma MOPC 47A immunoglobulin.

Mouse IgA 47A is an immunoglobulin variant whose heavy (alpha) chain has a molecular weight of 40,000 and lacks the entire CH3 domain. In order to understand better the mechanism by which the CH3 domain is lost, the 47A heavy chain mRNA has been partially purified. The mRNA migrated as a 17 S species on sucrose gradients and coded for the cell-free synthesis of only one serologically reactive protein with an apparent molecular weight of 42,000. Competitive inhibition of immunoprecipitation and tryptic peptide analysis of a cyanogen bromide fragment of the in vitro product revealed significant homology between the authentically secreted heavy chain and the respective cell-free synthesized, immunoprecipitated product. The heavy chain mRNA of 47A possessed a chain length of about 2200 nucleotides which is the same size as a normal alpha heavy chain mRNA, thus demonstrating that the protein deletion is not due to a large deletion at the mRNA level. The deletion in the 47A heavy chain may be due to a frameshift of a single nucleotide followed by premature chain termination.

Animals↗

PubMed2002

Cutting edge: association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes.

The binding of chemokines to their receptors guides lymphocyte migration. However, the precise mechanism that links the chemotactic signals with the energy and traction force generated by the actomyosin complex of the cell has not been elucidated. Using biochemical approaches and mass spectrometry analysis, we found an association between the C-termini of CXCR4 and CCR5 and the motor protein nonmuscle myosin H chain-IIA. Immunoprecipitation experiments revealed that this association also occurs between the endogenous molecules in T lymphocytes. As expected, myosin L chain was also associated with CXCR4. Confocal microscopy analysis showed that CXCR4 and motor protein nonmuscle myosin H chain-IIA colocalize at the leading edge of migrating T lymphocytes, together with filamentous actin and myosin L chain. These results provide the first evidence of a biochemical association between chemokine receptors and motor proteins, a mechanosignaling mechanism that may have a key role in lymphocyte migration.

Actins↗

PubMed2006

Diphosphorylation of the myosin regulatory light chain enhances the tension acting on stress fibers in fibroblasts.

Regulation of the contractile force is crucial for cell migration, cell proliferation, and maintenance of cell morphology. Phosphorylation of the myosin II regulatory light chain (MRLC) is involved in these processes. To show whether the diphosphorylation of MRLC increases the tension acting on stress fibers, changes in the stiffness of fibroblasts expressing wild-type MRLC and a mutant type, which cannot be diphosphorylated, on treatment with lysophosphatidic acid (LPA) were examined by a mechanical-scanning probe microscope (M-SPM). The LPA treatment increased cellular stiffness in the wild-type MRLC expressing cells, while it had no effect on the mutated cells. Immunostaining showed that LPA stimulation induced the diphosphorylation of MRLC. These results suggest that the diphosphorylation of MRLC enhances the tension acting on stress fibers.

Animals↗

PubMed2003

Myosin light chain kinase mediates eosinophil chemotaxis in a mitogen-activated protein kinase-dependent manner.

BACKGROUND: Eosinophil migration in the tissue is one characteristic feature of allergic diseases. The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Myosin light chain kinase (MLCK) is known to regulate cytoskeletal rearrangement and cell motility by means of phosphorylation of myosin light chain (MLC). OBJECTIVE: We have previously shown that mitogen-activated protein (MAP) kinases are important for eosinophil migration. In the present study we hypothesized that MLCK is downstream of MAP kinases, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for eosinophil chemotaxis. METHODS: Blood eosinophils were purified by using Percoll and anti-CD16 antibody-coated magnetic beads. We investigated the phosphorylation of MLCK and MLC by using the phosphorous 32-orthophosphates-labeled eosinophils. The kinase activity of MLCK was determined by measuring the phosphotransferase activity for the MLCK-specific peptide substrate. The chemotaxis assay was performed in a 48-well Boyden microchamber. RESULTS: The phosphotransferase activity of MLCK for a substrate peptide was enhanced in eotaxin-stimulated eosinophils. We also found that eotaxin induced phosphorylation of MLCK in vivo in phosphorous 32-orthophosphate-labeled eosinophils. PD98059 (MAP/extracellular signal-regulated kinase inhibitor) or SB202190 (p38 MAP kinase inhibitor) abrogated the eotaxin-induced phosphorylation of MLCK. The phosphorylation of MLC was upregulated by eotaxin. Eosinophil chemotaxis was inhibited by means of pretreatment of the MLCK inhibitor ML-7. CONCLUSION: These results suggest that eotaxin regulates MLCK through both extracellular signal-regulated kinase 1/2 and p38 MAP kinase. MLCK activation is a critical step in the cytoskeletal rearrangements leading to eosinophil migration.

Chemokine CCL11↗

PubMed2002

The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility.

Integrin affinity is modulated by intracellular signaling cascades, in a process known as "inside-out" signaling, leading to changes in cell adhesion and motility. Protein kinase C (PKC) plays a critical role in integrin-mediated events; however, the mechanism that links PKC to integrins remains unclear. Here, we report that PKCepsilon positively regulates integrin-dependent adhesion, spreading, and motility of human glioma cells. PKCepsilon activation was associated with increased focal adhesion and lamellipodia formation as well as clustering of select integrins, and it is required for phorbol 12-myristate 13-acetate-induced adhesion and motility. We provide novel evidence that the scaffolding protein RACK1 mediates the interaction between integrin beta chain and activated PKCepsilon. Both depletion of RACK1 by antisense strategy and overexpression of a truncated form of RACK1 which lacks the integrin binding region resulted in decreased PKCepsilon-induced adhesion and migration, suggesting that RACK1 links PKCepsilon to integrin beta chains. Altogether, these results provide a novel mechanistic link between PKC activation and integrin-mediated adhesion and motility.

Animals↗

PubMedDate not supplied

[Isolation of a gamma heavy chain fragment from normal human serum].

Several components of catodic electrophoretic migration in serum and urine are present in normal individuals and in rabbit serum. There also exists in man and in some animals, serum fractions of low molecular weight. These types of serum components may be or may not be related with the IgG. In a previous study we have isolated two components in the slow catodic electrophoretic area of the normal human serum (NHS). One of them was identified as an IgG subclass and the other component presented a clear line of precipitation to gamma heavy chain specific immuno-serum. This latter component was found in the post gamma-globulin area crossing the IgG arc in the I.E. analysis. Its molecular weight was variable from 3700 to 9500. In this paper a differential analysis of the gamma fragment isolated for us, is made and its relationship with Fc subfragments of pepsin-digested IgG is studied. In order to obtain this comparative study, the electrophoresis, gel diffusion immunoelectrophoresis gel chromatography and analytic ultracentrifugation techniques are employed. The post-gammaglobulin fraction has been isolated from total normal human serum without previous manipulation, or with the gammaglobulin fraction precipitated with saturated ammonium sulphate, in Sephadex G-200 chromatography. These two fractions present similar immunelectrophoretical characteristics. The constant sedimentation is 0.90 S and the approximated molecular weight is 7000. Since the pepsin digestion of IgG produced Fc subfragments of low molecular weight, we have isolated and submitted this immunoglobulin to peptic digestion. The G-75 Sephadex filtration shows an isolated post-gamma-globulin of I.E. sedimentation constant and molecular weight whose characteristics are similar to the isolated serum post-gammaglobulin fraction. The antigenical analysis in I.D. shows a total identity between the pepsin digested post-gammaglobulin and the fragment obtained for us from the human serum to an anti-heavy gamma chain immuno-serum and to a rabbit serum anti-fraction produced for us. These events suggest that this isolation post-gamma-globulin fraction corresponds to a Fc subfragment of the IgG.

Animals↗

PubMed1993

Differential expression of the common beta and specific alpha chains of the receptors for GM-CSF, IL-3, and IL-5 in endothelial cells.

The present study was designed to reexamine the interaction of granulocyte-macrophage colony-stimulating factor (GM-CSF) with endothelial cells (EC) and to investigate the expression of CSF receptor chains in these cells. In agreement with previous data, GM-CSF induced directional migration and, to a lesser degree, proliferation of human umbilical vein EC. When compared to basic fibroblast growth factor, GM-CSF was comparable in terms of chemotactic activity and was substantially less active in terms of proliferation. Binding studies confirmed the presence of receptors for GM-CSF (GM-CSFR) on EC. The expression of the beta chain common to the GM-CSFR, IL-3 receptor, and IL-5 receptor, as well as of the individual alpha chains, was studied by Northern analysis and/or reverse transcription and polymerase chain reaction. EC expressed high levels of the common beta chain transcripts. Expression of the alpha(GM) and alpha(IL-5) chain mRNA was minimal or absent in normal EC, though the transformed ECV304 endothelial cell line had substantial amounts of alpha(GM) chain mRNA. Unexpectedly, EC expressed alpha(IL-3) chain transcripts. IL-3 induced migration of EC across polycarbonate filters, whereas IL-5 was inactive.

Animals↗

PubMed1995

Expression of kinesin heavy chain isoforms in retinal pigment epithelial cells.

To examine the possible role of kinesin in pigment granule migration in the retinal pigment epithelium (RPE) of teleosts, we investigated the expression and distribution of kinesin heavy chain (KHC) in RPE. Blots of fish RPE lysates probed with two well-characterized antibodies to KHC (H2 and HD) displayed a prominent band at 120 kD. A third KHC antibody (SUK4) recognized a band at 118 kD. The 118 kD band was also occasionally present in blots probed with H2, suggesting the presence of two KHC isoforms in teleost RPE. Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from RPE using primers homologous to conserved regions of the KHC motor domain resulted in the identification of two putative KHC genes (FKIF1 and FKIF5) based on partial amino acid sequences. Previous studies had demonstrated a requirement for microtubules in pigment granule aggregation in RPE. In addition, the reported microtubule polarity orientation in RPE apical projections is consistent with a role for kinesin in pigment granule aggregation. Immunofluorescent localization of KHC in isolated RPE cells using H2 revealed a mottled distribution over the entire cell body, with no detectable selective association with pigment granules, even in cells fixed while aggregating pigment granules. Microinjected KHC antibodies had no effect on pigment granule aggregation or dispersion, although each of the three antibodies has been shown to block kinesin function in other systems. Thus we found no evidence for KHC function in RPE pigment granule aggregation. However, the two KHC isoforms may participate in other microtubule-dependent processes in RPE.

Amino Acid Sequence↗