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Results for “Chain Migration”

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PubMed1977

Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C.

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.

Animals↗

PubMed2005

The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities.

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.

Animals↗

PubMed1999

Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

Actins↗

PubMed2006

Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement.

Embryonic cell movement is essential for morphogenesis and the establishment of body shapes, but little is known about its mechanism. Here we report that pregnenolone, which is produced from cholesterol by the steroidogenic enzyme Cyp11a1 (cholesterol side-chain cleavage enzyme, P450scc), functions in promoting cell migration during epiboly. Epiboly is a process in which embryonic cells spread from the animal pole to cover the underlying yolk. During epiboly, cyp11a1 is expressed in an extra-embryonic yolk syncytial layer. Reducing cyp11a1 expression in zebrafish using antisense morpholino oligonucleotides did not perturb cell fates, but caused epibolic delay. This epibolic defect was partially rescued by the injection of cyp11a1 RNA or the supplementation of pregnenolone. We show that the epibolic delay is accompanied by a decrease in the level of polymerized microtubules, and that pregnenolone can rescue this microtubule defect. Our results indicate that pregnenolone preserves microtubule abundance and promotes cell movement during epiboly.

3-Hydroxysteroid Dehydrogenases↗

PubMed1996

Regulation of the beta 2 subunit chain of laminin in developing rabbit fetal lung tissue.

Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The beta 2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the beta 2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin beta 2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin beta 2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin beta 2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin beta 2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin beta 2 protein induction observed in fetal lung tissue, laminin beta 2 mRNA levels were maximal late in gestation. Utilizing a laminin beta 2 chain cRNA probe and in situ hybridization, we detected laminin beta 2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin beta 2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin beta 2 chain may be involved in alveolar epithelial cell differentiation.

Amino Acid Sequence↗

PubMed1978

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus.

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the identity of the fatty acyl chains can uniquely specify or determine which side of the membrane individual phosphatidylethanolamine molecules come to occupy. Although fatty acyl chain asymmetry and phosphatidylethanolamine asymmetry are correlated in VSV, no simple rules can be discerned which uniquely relate the two paramaters.

Chemical Phenomena↗

PubMed1979

Two spontaneous BALB/c lymphomas synthesize IgM: monomers and half molecules are isolated and characterized whereas another molecule resembles IgD.

Spontaneous lymphomas of BALB/c mice, both in vivo tumors and cell lines established in long term tissue cultures, were investigated for their ability to synthesize IgM by using radiolabeled amino acid precursors. Immunoglobulins manufactured by lymphomas K46 and L10A had the m.w. of monomeric IgM and IgM half molecule. Both of these molecules could be immunoprecipitated with class-specific anti-IgM but not anti-IgA or anti-IgG. When precipitated with polyvalent anti-Ig L10A synthesized monomeric immunoglobulins that migrated as two peaks in contrast to their single counterpart precipitated with anti-IgM. The second peak migrated in the region expected for IgD. Monomer and half molecules were composed of similar ratios of mu-chains to light chains linked by disulfide bonds. The mu2L2 monomer of these B cell lines migrated slightly slower in SDS PAGE than a mu2L2 secreted by a myeloma. Thus, these lymphomas synthesize immunoglobulins with the chemical and antigenic characteristics typical of monomeric membrane-attached IgM and IgM half molecules, plus a molecule resembling IgD on L10A only. Lymphoma assembly of monomeric IgM may follow the same initial biosynthetic sequence as myeloma assembly.

Animals↗

PubMed1977

Radioimmunoassay for the antigenic determinants of cholera toxin and its components.

A radioimmunoassay procedure is described for the detection of cholera toxin and its component polypeptide chains. Cholera toxin, A subunit, B subunit, alpha chain, and gamma chain were iodinated by the chloramine T procedure. Radiolabeling did not significantly alter the polyacrylamide electrophoretic migration patterns of the toxin or its components. Moreover, radiolabeled toxin, B subunit, and alpha chain preparations retained substantial ability to bind to intestinal mucosal homogenates. The minimal amount of antitoxin detectable with radiolabeled toxin was 0.04 antitoxin units/ml. Substitution of radiolabeled B subunit, A subunit, and alpha chain for radiolabeled toxin decreased the sensitivity of the test. Radiolabeled gamma chain did not bind to the antitoxin preparation. Competitive inhibition studies, with titrated anti-choleragen serum and radiolabeled toxin or components, indicated that the minimum concentration of toxin detectable was 7.0 x 10(-8) mumol/ml at a 90% inhibition level. The A subunit and alpha chain preparations inhibited the binding of the radiolabeled B subunit to antitoxin sites. Conversely, B subunit inhibited the binding of radiolabeled A subunit and alpha chain to antitoxin. The gamma chain did not show any reaction with antitoxin or cross-reaction with either whole toxin or its components. These results strongly suggest that the A subunit and the alpha chain contain antigenic determinant(s) that are common to the B subunit. The B subunit (beta chain) and the alpha chain of cholera toxin may therefore contain region(s) of chemical similarity.

Antigens, Bacterial↗

PubMed2001

Tumour necrosis factor-alpha provokes upregulation of alpha2beta1 and alpha5beta1 integrins, and cell migration in OST osteosarcoma cells.

OST cells enhance the induction of matrix metalloproteinase (MMP)-9 by tumour necrosis factor (TNF)-alpha and the corresponding metastasis to lungs in vivo (Kawashima et al., 1994). We focused on the adhesive and migratory properties of OST cells, and investigated the expression of integrins in OST cells stimulated by TNFalpha in vitro. OST cells potentiated not only adhesion to the extracellular matrix (ECM) but also the migration on ECM. On competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses, the amounts of alpha2 (4.9-fold), alpha5 (1.2-fold) and alpha(v) (4.9-fold) were upregulated by TNFalpha at the transcriptional level. Alpha-5 showed a slight increase by flow cytometry; however, alpha2 and alphav integrins remained unchanged at the protein level. Immunofluorescence study disclosed integrins of alpha2beta1 and alpha5beta1 were much clustered at cell processes by TNFalpha stimulation, probably related to increased cell adhesion and migration. Therefore, the upregulation of alpha2beta1 and alpha5beta1 integrins seems to contribute to tumour invasion and metastatic potential.

Cell Adhesion↗

PubMed2004

CD98hc (SLC3A2) mediates integrin signaling.

Integrins regulate cellular behaviors through signaling pathways, including Rho GTPases and kinases. CD98 heterodimers, comprised of a heavy chain (CD98hc, SLC3A2) and one of several light chains, interact with integrins through CD98hc. CD98hc overexpression leads to anchorage-independent cell growth and tumorigenesis in 3T3 fibroblasts and activates certain integrin-regulated signaling pathways. To establish the biological function of CD98hc, we disrupted the gene and analyzed CD98hc-null cells. Here we report that CD98hc contributes to integrin-dependent cell spreading, cell migration, and protection from apoptosis. Furthermore, CD98hc is required for efficient adhesion-induced activation of Akt and Rac GTPase, major contributors to the integrin-dependent signals involved in cell survival and cell migration. CD98 promotes amino acid transport through its light chains; however, a CD98hc mutant that interacts with beta1 integrins, but not CD98 light chains, restored integrin-dependent signaling and protection from apoptosis. beta1 integrins are involved in the pathogenesis of certain cancers. CD98hc deletion markedly impaired the ability of embryonic stem cells to form teratocarcinomas in mice; teratocarcinoma formation was reconstituted by reexpression of CD98hc or of the mutant that interacts exclusively with integrins. Thus, CD98hc is an integrin-associated protein that mediates integrin-dependent signals, which promote tumorigenesis.

Animals↗

PubMed2001

Ligand migration in human myoglobin: steric effects of isoleucine 107(G8) on O(2) and CO binding.

To investigate the ligand pathway in myoglobin, some mutant myoglobins, in which one of the amino acid residues constituting a putative ligand-docking site, Ile107, is replaced by Ala, Val, Leu, or Phe, were prepared and their structural and ligand binding properties were characterized. The kinetic barrier for the ligand entry to protein inside was lowered by decreasing the side-chain volume at position 107, indicating that the bulky side chain interferes with the formation of the activation state for the ligand migration and the free space near position 107 would be filled with the ligand in the activation state. Another prominent effect of the reduced side-chain volume at position 107 is to stabilize the ligand-binding intermediate state. Because the stabilization can be ascribed to decrease of the positive enthalpy, the enlarged free space near position 107 would relieve unfavorable steric interactions between the ligand and nearby amino acid residues. The side-chain volume at position 107, therefore, is crucial for the kinetic barrier for the ligand migration and free energy of the ligand-binding intermediate state, which allows us to propose that some photodissociated O(2) moves toward position 107 to be trapped and then expelled to the solvent.

Alanine↗

PubMed1983

Protease production by Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The temporal appearance of extracellular proteases produced by Pseudomonas aeruginosa was analyzed by pH 9 and pH 4 polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Ammonium sulfate precipitates of culture supernatants from various stages of growth revealed a time-dependent increase in number and amount of proteolytically active proteins. One mucoid P. aeruginosa clinical isolate and its derived nonmucoid variant, as well as two other nonmucoid variant P. aeruginosa strains (all from cystic fibrosis patients), showed similar production of five differently migrating proteases (P1 to P5, numbered according to increasing net negative charge) in pH 9 PAGE and one protease in pH 4 PAGE. P2, P3, and P5 increased to maximum concentrations at 24 to 48 h, decreasing thereafter, whereas P4 continued increasing even at 83 h, and P1 fluctuated. P3 was identified as an elastase. P2 was possibly composed of polypeptide chains bridged by disulfide bonds, since without reduction it migrated in sodium dodecyl sulfate-PAGE as a single protein, and with reduction it migrated as three protein bands. Two-dimensional PAGE revealed multiple molecular weight species within protease-positive bands in pH 9 gel strips. Isoelectric focusing gave a pattern of protein separation that correlated with two-dimensional PAGE analysis. Thus, greater heterogeneity of active proteases than previously reported has been demonstrated in all P. aeruginosa clinical isolates studied by sensitive two-dimensional PAGE analysis.

Cystic Fibrosis↗

PubMed2026

Quantum Threats to Bitcoin, Cryptocurrency and Blockchain.

OBJECTIVE: The authors evaluated how quantum computing threatens the cryptographic primitives used in Bitcoin and other blockchain systems. These findings were translated into a standards-aligned post-quantum migration profile for healthcare ledgers, including consent, identity, provenance, audit, and encrypted off-chain data exchange. METHODOLOGY: Narrative analysis, theoretical security analysis, and healthcare-oriented deployment mapping of classical public-key and hash primitives used in blockchain protocols were paired with an implementation-oriented migration profile based on finalized National Institute of Standards and Technology (NIST) post-quantum standards. We summarize the mathematical assumptions underlying RSA, Elliptic Curve Cryptography/Elliptic Curve Digital Signature Algorithm (ECC/ECDSA), and Secure Hash Algorithm (SHA-2/SHA-3-family) hash functions; analyze their susceptibility to Shor's and Grover's quantum algorithms; compare Federal Information Processing Standards (FIPS) 203 Module-Lattice-Based Key-Encapsulation Mechanism Standard (ML-KEM), FIPS 204 Module-Lattice-Based Digital Signature Standard (ML-DSA), and FIPS 205 Stateless Hash-Based Digital Signature Standard (SLH-DSA); and map their distinct roles to healthcare-ledger authorization, auditability, identity, and encrypted off-chain exchange. The term Advanced Hybrid Module-LWE & Code-Based (AHMC) is used only as shorthand for a standards-aligned hybrid post-quantum cryptography (PQC) migration profile and does not denote a proprietary product, a novel algorithm, or a new cryptographic primitive. Proposed adoption of hybrid, quantum-resistant cryptographic primitives for cryptocurrency wallets, transaction signatures, and ledger security during an interim migration period (hybrid classical + PQC, followed by PQC-only). Qualitative and implementation-oriented assessment of (1) break feasibility of RSA/ECC under Shor's algorithm, (2) effective security reduction for hash functions under Grover's algorithm, (3) security assumptions and composition requirements of a standards-aligned hybrid migration profile, (4) transaction-size and verification-cost impact, and (5) healthcare-specific implications for long-retention consent, identity, provenance, and audit records. RESULTS: Shor's algorithm reduces integer factorization and discrete logarithms to polynomial time, directly compromising Rivest-Shamir-Adleman (RSA) and ECC/ECDSA once fault-tolerant, large-scale quantum computers exist. Grover's algorithm yields a quadratic speedup for brute-force search, effectively halving the security margin of symmetric keys and hash functions at fixed output sizes. The AHMC-L1/L3/L5 profiles use ML-KEM-512/768/1024 for key establishment and ML-DSA-44/65/87 for transaction authentication, with SLH-DSA as a hash-based fallback. During a hybrid ECDSA+PQC migration, verification requires one classical and one PQC verification per authorization; transaction-size overhead is dominated by PQC signatures, approximately 2.4 KB for ML-DSA-44, 3.3 KB for ML-DSA-65, and 4.6 KB for ML-DSA-87 before script and encoding overhead. For healthcare ledgers, these findings support selective use of post-quantum signatures for:high-value state transitions,one-time public-key registration where possible, andcontinued off-chain storage of protected health information.Deployment suitability remains contingent on workflow-specific latency, storage, availability, key lifecycle, and side-channel testing. CONCLUSIONS: Quantum risk to blockchain signatures has direct implications for healthcare systems that depend on long-lived consent, identity, provenance, and audit records. A staged, standards-aligned migration profile can preserve authorization and ledger verifiability while keeping protected health information off-chain. The AHMC label refers only to this migration profile, not to a new cryptographic primitive; healthcare adoption requires open implementations, empirical benchmarking, crypto-agile key governance, and side-channel-resistant engineering.

ML-DSA↗

PubMed2005

A new charge-associated mechanism to account for the production of fragment ions in the high-energy CID spectra of fatty acids.

A new mechanism, termed a charge-assisted process, is proposed as an additional mechanism to the charge-remote process to account for ions of the [M - CnH2n+2] series found in the positive and negative high energy CID spectra of fatty acids and related compounds when ionized as closed-shell ([M - H]- or [M + X]+) species. The new mechanism is based on that commonly invoked to account for similar ions in the electron-impact spectra of derivatized fatty acids whereby the positive charge on the derivative abstracts a hydrogen atom from various positions of the alkyl chain to leave a radical that initiates a radical-induced cleavage of the chain. It is proposed that in the high energy CID spectra of closed-shell ions, similar hydrogen migrations occur but unpairing of electrons is avoided by charge transfer to the alkyl chain. This charge then initiates a concerted cleavage of the chain to give an allylic carbonium (positive ion spectrum) or carbanion (negative ion spectrum). The mechanism avoids the need to involve radicals or loss of hydrogen atoms from even-electron (closed shell) ions and provides a driving force for the reaction, namely, the formation of ions with a stabilized charge. An extension of the mechanism is also proposed to account for the formation of odd-electron ions from these compounds. The charge-assisted mechanism does not rule out the occurrence of other mechanisms that have been accepted for many years but provides an alternative process that can account for some spectral features which were difficult to explain earlier.

Fatty Acids↗

PubMed2006

HIV-1 gp120 compromises blood-brain barrier integrity and enhances monocyte migration across blood-brain barrier: implication for viral neuropathogenesis.

Human immunodeficiency virus-1 (HIV-1) encephalitis is characterized by brain infiltration of virus-infected monocytes and macrophages. Cellular products and viral proteins secreted by infected cells likely play an important role in blood-brain barrier (BBB) impairment and the development of HIV-1-associated dementia (HAD). We previously demonstrated that HIV-1 envelope glycoprotein gp120 induces toxicity and alters expression of tight junction proteins in human brain microvascular endothelial cells (HBMECs). Here, we delineate the mechanisms of gp120-induced BBB dysfunction. Human brain microvascular endothelial cells expressed HIV-1 co-receptors (CCR5 and CXCR4). Exposure of HBMECs to gp120 derived from macrophage (CCR5) or lymphocyte (CXCR4)-tropic viruses decreased BBB tightness, increased permeability, and enhanced monocyte migration across in vitro BBB models. Blood-brain barrier integrity was restored after gp120 removal. CCR5 antibodies and inhibitors of myosin light chain kinase or protein kinase C (PKC) blocked gp120-enhanced monocyte migration and permeability of BBB in vitro. Exposure of HBMECs to gp120 induced release of intracellular calcium ([Ca(2+)](i)) that was prevented by CCR5 antibody and partially blocked by CXCR4 antagonist. Human immunodeficiency virus-1 gp120 activated three PKC isoforms in HBMECs [PKC-alpha/betaII, PKC(pan)-betaII and PKC-zeta/lambda]. Furthermore, specific PKC inhibitors (acting at the ATP-binding and calcium release site) blocked gp120-induced PKC activation and prevented increase in BBB permeability, supporting the biologic significance of these results. Thus, gp120 can cause dysfunction of BBB via PKC pathways and receptor mediated [Ca(2+)](i) release leading to cytoskeletal alterations and increased monocyte migration.

Blood-Brain Barrier↗

PubMed2003

Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration.

Biglycan and decorin are small chondroitin/dermatan sulphate proteoglycans in the extracellular matrix of connective tissue that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. We show for the first time that biglycan and decorin induce morphological and cytoskeletal changes in fibroblasts, resulting in an increase in migration. Biglycan changed the cell shape of fibroblasts with formation of long protruding filamentous processes. This was also seen for decorin but to a lesser extent. Using fluorescence staining of F-actin fibres it was possible to show that these long filamentous processes were supported by long thick bundles of actin, together with an induced formation of stress fibres after stimulation with biglycan and decorin. Moreover, a reorganisation of alpha-smooth muscle actin was clearly seen in these cultures. Decorin also stimulated alpha-smooth muscle actin expression in the cells. Using cDNA Atlas Arrays we were also able to show that the mRNA level of a number of the intracellular regulators and effectors involved in cell migration were increased. For example, the focal adhesion proteins paxillin and zyxin, and some of the small Rho GTPases such as RhoA, Rac1 and Cdc42 were upregulated. After treatment with biglycan or decorin, additional results showed an increased activation of RhoA (1.8- and 1.5-fold, respectively) and Rac1 (1.8- and 1.5-fold, respectively) after 15 minutes. These factors are known to be involved in fibroblast migration, and as expected a 1.3- to 1.6-fold increase in migration could be observed after stimulation with biglycan or decorin. This induced migration was caused by the core protein, as treatment with glycosaminoglycan chains alone did not have any effect. In summary, these data indicate that biglycan- and decorin-induced fibroblast cytoskeletal and signalling changes result in an increased cell migration, and demonstrate their potential role in the remodelling process.

Actins↗

PubMed2000

Unusual pressure effects on ligand rebinding to the human myoglobin Leucine 29 mutants.

Using high pressure flash photolysis, we revealed that the side chain of Leu(29) controls the reaction volume of the ligand migration process in myoglobin, which is the primary factor for the unusual activation volume of ligand binding in some Leu(29) mutants. As we previously reported (Adachi, S., Sunohara, N., Ishimori, K., and Morishima, I. (1992) J. Biol. Chem. 267, 12614-12621), CO bimolecular rebinding in the L29A mutant was unexpectedly decelerated by pressurization, suggesting that the rate-determining step is switched to ligand migration. However, very slow CO bimolecular rebinding of the mutants implies that bond formation is still the rate-determining step. To gain further insights into effects of the side chain on ligand binding, we prepared some new Leu(29) mutants to measure the CO and O(2) rebinding reaction rates under high hydrostatic pressure. CO bimolecular rebinding in the mutants bearing Gly or Ser at position 29 was also decelerated upon pressurization, resulting in apparent positive activation volumes (DeltaV), as observed for O(2) binding. Based on the three-state model, we concluded that the increased space available to ligands in these mutants enhances the volume difference between the geminate and deoxy states (DeltaV(32)), which shifts the apparent activation volume to the positive side, and that the apparent positive activation volume is not due to contribution of the ligand migration process to the rate-determining step.

Carbon Monoxide↗

PubMed2003

LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment.

This study analyzes signaling events initiated through binding of the leukocyte integrin LFA-1 to ICAM-1, which leads to T cell attachment, polarization and random migration. These events are critically dependent on dynamic changes in the acto-myosin cytoskeleton under the regulation of myosin light chain kinase and ROCK (Rho kinase). A key finding is that the activity of these two kinases is spatially segregated. Myosin light chain kinase (MLCK) must operate at the leading edge of the T cell because blocking its activity causes the polarized T cell to retract from the front of the cell. These activities are mirrored by inhibiting calmodulin, the activator of MLCK. In contrast inhibition of ROCK (and RhoA) has the effect of preventing detachment of the T cell trailing edge, showing that this kinase operates at the rear of the cell. This compartmentalized activity of the two kinases is reflected in their localization within the T cell. Myosin light chain kinase is concentrated at the leading edge, overlapping F-actin, whereas ROCK is more widely distributed in the trailing edge of the T cell. Thus these two kinases perform two different functions in the migrating T cell, with myosin light chain kinase activity important for attachment and movement at the leading edge and ROCK activity required for the detachment of the trailing edge. These two actomyosin-dependent processes operate coordinately to cause forward migration of a T cell.

Cell Adhesion↗