Profiles of Physarum Microplasmodial Phosphatase Activity Crucial to Cytoplasmic Streaming and Spherule Formation.

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme.

Inhibitory effect of recombinant human CXCL8(3-72)K11R/G31P on atherosclerotic plaques in a mouse model of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease in which the plaques were built up inside of the artery. Interleukin-8 (IL-8, CXCL8) is an inflammatory factor, known to play an important role in the development of atherosclerosis. G31P is an antagonist of the IL-8 receptor, which plays roles in vascular smooth muscle cell (VSMC) proliferation and migration.

Calcium inhibition as an intracellular signal for actin-myosin interaction.

Intracellular signaling pathways include both the activation and the inhibition of biological processes. The activation of Ca regulation of actin-myosin interactions was examined first, whereas it took 20 years for the author to clarify the inhibitory mode by using Physarum polycephalum, a lower eukaryote. This review describes the investigation of the inhibitory mode since 1980.

Calcium-mediated regulation of recombinant hybrids of full-length Physarum myosin heavy chain with Physarum/scallop myosin light chains.

Physarum myosin is a Ca(2+)-binding protein and its activity is inhibited by Ca(2+) In the present study, to clarify the light chains (LCs) from the different species (Physarum and scallop) and to determine the specific Ca(2+)-regulated effects, we constructed hybrid myosins with a Physarum myosin heavy chain (Ph·HC) and Physarum and/or scallop myosin LCs, and examined Ca(2+)-mediated regulation of ATPases and motor activities. In these experiments, it was found that Ca(2+) inhibited motilities and ATPase activities of Physarum hybrid myosin with scallop regulatory light chain (ScRLC) and Physarum essential light chain (PhELC) but could not inhibit those of the Physarum hybrid myosin mutant Ph·HC/ScRLC/PhELC-3A which lacks Ca(2+)-binding ability, indicating that PhELC plays a critical role in Ca(2+)-mediated regulation of Physarum myosin. Furthermore, the effects of Ca(2+) on ATPase activities of Physarum myosin constructs are in the following order: Ph·HC/PhRLC/PhELC > Ph·HC/ScRLC/PhELC > Ph·HC/PhRLC/ScELC > Ph·HC/ScRLC/ScELC, suggesting that the presence of PhRLC and PhELC leads to the greatest Ca(2+) sensitivity of Physarum myosin.

Heat shock protein 60 stimulates the migration of vascular smooth muscle cells via Toll-like receptor 4 and ERK MAPK activation.

Accumulating evidence indicates that heat shock protein (HSP) 60 is strongly associated with the pathology of atherosclerosis (AS). However, the precise mechanisms by which HSP60 promotes atherosclerosis remain unclear. In the present study, we found that HSP60 mRNA and protein expression levels in the thoracic aorta are enhanced not only in a mouse model of AS but also in high-fat diet (HFD) mice.

Electron microscopic examination of podosomes induced by phorbol 12, 13 dibutyrate on the surface of A7r5 cells.

The role of myosin light chain kinase (MLCK) in inducing podosomes was examined by confocal and electron microscopy. Removal of myosin from the actin core of podosomes using blebbistatin, a myosin inhibitor, resulted in the formation of smaller podosomes. Downregulation of MLCK by the transfection of MLCK small interfering RNA (siRNA) led to the failure of podosome formation.

Nicotine exposure alters human vascular smooth muscle cell phenotype from a contractile to a synthetic type.

Objective: Cigarette smoking is a known risk factor for arteriosclerosis. In atheromatous plaques, vascular smooth muscle cells (VSMCs) display a phenotype that is different from the contractile type under normal conditions. Nicotine is the major pharmacological agent in cigarette smoke.

Platelet-derived growth factor-BB induces matrix metalloproteinase-2 expression and rat vascular smooth muscle cell migration via ROCK and ERK/p38 MAPK pathways.

Matrix metalloproteinases (MMP) play a pivotal role in the pathogenesis of cardiovascular diseases. Their expressions are altered in response to a variety of stimuli, including growth factors, inflammatory markers, and cytokines. In this study, we demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces a dose- and time-dependent increase in MMP-2 expression in rat vascular smooth muscle cells (VSMC).

Inverse interaction between tropomyosin and phosphorylated myosin in the presence or absence of caldesmon.

In the present study, co-sedimentation assay, intrinsic fluorescence intensity measurement, and Mg²⁺-ATPase activity analysis were carried out to investigate the direct effect of tropomyosin (TM) on unphosphorylated myosin (UM) or phosphorylated myosin (PM) in the presence or absence of caldesmon (CaD). Results showed that TM significantly decreased the sedimentation, intrinsic fluorescence intensity, and the Mg²⁺-ATPase activity of PM, but not UM. In the presence of CaD, TM also significantly decreased these parameters irrespective of myosin phosphorylation, suggesting that the interaction between TM and CaD abolished the effects of TM on PM or UM and that there was an inverse interaction between TM and PM, characterized by the decreased PM sedimentation and intrinsic fluorescence intensity.

A maternal diet rich in fish oil may improve cardiac Akt-related signaling in the offspring of diabetic mother rats.

Objective: Newborns of diabetic mothers have abnormal circulatory organs, so in this study, we explore insulin signaling in the newborn rat heart.

Methods: Pregnant rats were divided into streptozotocin-induced diabetic groups (DM) and control groups (CM). Rats were fed lard (21% fat), fish oil (21% fat), or a control diet (7% fat).

Rho-associated protein kinase isoforms stimulate proliferation of vascular smooth muscle cells through ERK and induction of cyclin D1 and PCNA.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) plays an important role in vascular diseases. The Rho-associated protein kinase (ROCK) signaling pathway is now well recognized for its role in VSMC migration and proliferation. Recently, a number of studies revealed that different isoforms of ROCK have distinct functions in VSMCs.

Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis.

In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis.

Recombinant human CXCL8(3-72)K11R/G31P regulates smooth muscle cell proliferation and migration through blockage of interleukin-8 receptor.

Atherosclerosis is a chronic inflammatory disease with multiple contributing factors. Hyperlipidemia is one of the major independent risks, and interleukin-8 (IL-8), as an inflammatory factor, plays an important role in the development of atherosclerosis. The aims of the study were to examine the therapeutic efficacy of G31P, an antagonist of IL-8 receptor, with a mouse model of hyperlipidemia and the potential mechanisms of G31P through the vascular smooth muscle cell (VSMC) proliferation and migration in a cell line.

Calcium-dependent regulation of the motor activity of recombinant full-length Physarum myosin.

We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin.

Down-regulation of myosin light chain kinase expression in vascular smooth muscle cells accelerates cell proliferation: requirement of its actin-binding domain for reversion to normal rates.

Myosin light-chain kinase (MLCK) is a multi-domain protein with kinase and actin-binding domains, among others. Deficiency of MLCK expression in GBaSM-4 vascular smooth muscle cells enhanced cell proliferation rate and shortened cell doubling time. Transient transfection of the MLCK-deficient cells with cDNA constructs of either wild-type MLCK or its mutant lacking the kinase activity reverted the cell proliferation rate to that of wild-type cells, whereas that of MLCK lacking the actin-binding domain maintained cell proliferation at an elevated rate similar to the MLCK-deficient cells.

Interaction between myosin and a trace amount of caldesmon.

Caldesmon (CaD) is known as an actin binding protein. In this study, we proposed that a trace amount of caldesmon (TACD) could highly, efficiently, interact with myosin by producing a 'domino-like cascade' and characterized that TACD (lowest caldesmon/myosin molar ratio: 1/10,000) significantly increased precipitations and intrinsic tryptophan fluorescence intensity of myosin in both phosphorylated and unphosphorylated states compared to the base controls (P < 0.01).

A galactosamine-mediated drug delivery carrier for targeted liver cancer therapy.

In order to minimize the side effect of cancer chemotherapy, a novel galactosamine-mediated drug delivery carrier, galactosamine-conjugated albumin nanoparticles (GAL-AN), was developed for targeted liver cancer therapy. The albumin nanoparticles (AN) and doxorubicin-loaded AN (DOX-AN) were prepared by the desolvation of albumin in the presence of glutaraldehyde crosslinker. Morphological study indicated the spherical structure of these synthesized particles with an average diameter of around 200 nm.

Myosin light chain kinase/actin interaction in phorbol dibutyrate-stimulated smooth muscle cells.

Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun.

Transport of single cells using an actin bundle-myosin bionanomotor transport system.

The potential of using actin bundles for the transport of liposomes and single cells across myosin-coated surfaces is investigated. Compared to that observed with filamentous actin, the liposome transport using actin bundles was more linear in nature and able to occur over longer distances. Bundles, but not filamentous actin, were capable of moving single cells.

Effect of cigarette smoke components on vascular smooth muscle cell migration toward platelet-derived growth factor BB.

Cigarette smoking is one of the factors causing accumulation of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques. Changes in cell migration toward platelet-derived growth factor BB were investigated using a Boyden chamber after 48-h preincubation of GBaSM-4 VSMCs with nicotine or nicotine-free cigarette smoke extract (CSE). A nicotine concentration of 0.

Improved drug targeting of cancer cells by utilizing actively targetable folic acid-conjugated albumin nanospheres.

Folic acid-conjugated albumin nanospheres (FA-AN) have been developed to provide an actively targetable drug delivery system for improved drug targeting of cancer cells with reduced side effects. The nanospheres were prepared by conjugating folic acid onto the surface of albumin nanospheres using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as a catalyst. To test the efficacy of these nanospheres as a potential delivery platform, doxorubicin-loaded albumin nanospheres (DOX-AN) and doxorubicin-loaded FA-AN (FA-DOX-AN) were prepared by entrapping DOX (an anthracycline, antibiotic drug widely used in cancer chemotherapy that works by intercalating DNA) into AN and FA-AN nanoparticles.

Calcium regulation of the ATPase activity of Physarum and scallop myosins using hybrid smooth muscle myosin: the role of the essential light chain.

To examine the role of two light chains (LCs) of the myosin II on Ca2+ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca2+ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca2+ even though they did not support motility.

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum.

A phosphatase was purified through a combination of ion-exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6.

Application of poly(amidoamine) dendrimers for use in bionanomotor systems.

The study and utilization of bionanomotors represents a rapid and progressing field of nanobiotechnology. Here, we demonstrate that poly(amidoamine) (PAMAM) dendrimers are capable of supporting heavy meromyosin dependent actin motility of similar quality to that observed using nitrocellulose, and that microcontact printing of PAMAM dendrimers can be exploited to produce tracks of active myosin motors leading to the restricted motion of actin filaments across a patterned surface. These data suggest that the use of dendrimer surfaces will increase the applicability of using protein biomolecular motors for nanotechnological applications.

Blebbistatin, a myosin II inhibitor, suppresses contraction and disrupts contractile filaments organization of skinned taenia cecum from guinea pig.

To explore the precise mechanisms of the inhibitory effects of blebbistatin, a potent inhibitor of myosin II, on smooth muscle contraction, we studied the blebbistatin effects on the mechanical properties and the structure of contractile filaments of skinned (cell membrane permeabilized) preparations from guinea pig taenia cecum. Blebbistatin at 10 microM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration but had little effects on the Ca(2+)-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.