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Electrospun drug-eluting fibers have demonstrated potentials in topical drug delivery applications, where drug releases can be modulated by polymer fiber compositions. In this study, blend fibers of polycaprolactone (PCL) and polyethylene oxide (PEO) at various compositions were electrospun from 10 wt% of polymer solutions to encapsulate a model drug of ibuprofen (IBP). The results showed that the average polymer solution viscosities determined the electrospinning parameters and the resulting average fiber diameters. Increasing PEO contents in the blend PCL/PEO fibers decreased the average elastic moduli, the average tensile strength, and the average fracture strains, where IBP exhibited a plasticizing effect in the blend PCL/PEO fibers. Increasing PEO contents in the blend PCL/PEO fibers promoted the surface wettability of the fibers. The in vitro release of IBP suggested a transition from a gradual release to a fast release when increasing PEO contents in the blend PCL/PEO fibers up to 120 min. The in vitro viability of blend PCL/PEO fibers using MTT assays showed that the fibers were compatible with MEF-3T3 fibroblasts. In conclusion, our results explained the scientific correlations between the solution properties and the physicomechanical properties of electrospun fibers. These blend PCL/PEO fibers, having the ability to modulate IBP release, are suitable for topical drug delivery applications. [ABSTRACT FROM AUTHOR]

Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape.sup.1. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients.sup.2,3. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail.sup.3-7, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited.sup.7,8. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells.sup.9-12, which can be activated to form functional filaments as needed.sup.13--but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction. High-resolution cryo-electron microscopy structure of smooth muscle myosin II in the inhibited state enables increased understanding of the functions of the head and tail regions in regulation of myosin activity and the pathological mechanisms of disease mutations., Author(s): Shixin Yang [sup.1] [sup.3] , Prince Tiwari [sup.1] , Kyoung Hwan Lee [sup.1] [sup.4] , Osamu Sato [sup.2] , Mitsuo Ikebe [sup.2] , Raúl Padrón [sup.1] , Roger Craig [...]

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Adenosine protects the heart from adrenergic overstimulation. This adeno-protection includes the direct anti-adrenergic action via adenosine [A.sub.1] receptors ([A.sub.1]R) on the adrenergic signaling pathway. An indirect [A.sub.1]R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-[epsilon] to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether [A.sub.1]R activation of PKC-[epsilon] induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the [A.sub.1]R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-[epsilon] and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-[epsilon] to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate [A.sub.1]R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-[epsilon] co-IP with RACK2 by 186, 49, and > 1,000%, respectively. The [A.sub.1]R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-[epsilon] with RACK2 by 61%. With rat cardiomyocytes, the [beta]-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, [A.sub.1]R stimulation of the heart is associated with PLC-initiated PKC-[epsilon] translocation and association with RACK2. adenosine [A.sub.1] receptor; protein kinase C-[epsilon]; receptor for activated C kinase 2; rodent

Dramatic and vascular bed-specific hemodynamic changes occur in pregnancy and hypertension of pregnancy (HtP). Because myosin phosphatase (MP) is the primary effector of smooth muscle relaxation and a key target of signaling pathways that regulate vascular tone, we hypothesized that MP expression would be altered in these conditions. The abundance of the targeting/regulatory subunit of MP (MYPT1) mRNA and protein was increased 1.7- to 2.0-fold specifically in the uterine arteries (UAs) of late-pregnant rats without isoform switching. In a model of HtP in which nitric oxide (NO) synthesis is blocked by the chronic administration of [N.sup.[omega]]-nitro-L-arginine methyl ester, MYPT1 was downregulated and switched to the splice variant isoform that codes for the COOH-terminal leucine zipper motif. This was associated with increased sensitivity of the main UA and its subbranches to the vasorelaxant effects of the NO donor drug sodium nitroprusside. This difference was abolished by pretreatment with the phosphatase inhibitor tautomycetin. The sensitivity of relaxation to the NO second messenger cGMP was also increased under calciumclamp conditions in permeabilized UAs, indicating heightened activation of MP. The changes in MP expression in HtP were largely prevented by treatment with the antihypertensive medicine hydralazine. We propose that MYPT1 isoform switching is an adaptive response to reduce vascular resistance and maintain uterine blood flow in the setting of hypertension-triggered inward remodeling of the UAs in hypertension of pregnancy. smooth muscle; gene expression; guanosine 3',5'-cyclic monophosphate; contractility; signaling; [N.sup.[omega]]-nitro-L-arginine methyl ester; sodium nitroprusside

The mechanoenzymatic function of human myosin Vb (HuM5B) if reported for the first time. The results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle and the actin filament assay reveals that a single HuM5B molecule is enough to move the actin filament continuously, indicating that HuM5b is a processive motor.

Protein kinase C[member of] and the antiadrenergic action of adenosine in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 287: H1721-H1729, 2004. First published June 17, 2004; 10.1152/ajpheart.00224.2004.--Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine At receptors ([A.sub.1]R). The role of PKC[member of] in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKC[member of] was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKC[member of] construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKC[member of] was found localized primarily in transverse tubules. The [A.sub.1]R agonist chlorocyclo-pentyladenosine (CCPA) at 1 [micro]M rendered an enhanced localization of PKC[member of] in the t-tubular system. The [beta]-adrenergic agonist isoproterenol (Iso; 0.4 [micro]M) elicited a 29-36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKC[member of] overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKC[member of] inhibitory peptide. These results suggest that the translocation of PKC[member of] to the t-tubular system plays an important role in [A.sub.1]R-mediated antiadrenergic actions in the heart. [beta]-adrenergic; cardiomyocyte shortening; transfection; t tubules; PKC[member of] translocation

Class V myosin (myosin-V) was first found as a processive motor that moves along an actin filament with large ([approximately equal to] 36-nm) successive steps and plays an important role in cargo transport in cells. Subsequently, several other myosins have also been found to move processively. Because myosin-V has two heads with ATP- and actin-binding sites, the mechanism of successive movement has been generally explained based on the two-headed structure. However, the fundamental problem of whether the two-headed structure is essential for the successive movement has not been solved. Here, we measure motility of engineered myosin-V having only one head by optical trapping nanometry. The results show that a single one-headed myosin-V undergoes multiple successive large ([approximately equal to] 32-nm) steps, suggesting that a novel mechanism is operating for successive myosin movement.

Myosin phosphatase is a target for signaling pathways that modulate calcium sensitivity of force production in smooth muscle. Myosin phosphatase targeting subunit 1 (MYPT1) isoforms are generated by cassette-type alternative splicing of exons in the central and 3' portion of the transcript. Exclusion of the 3' alternative exon, coding for the leucine zipper (LZ)-positive MYPT1 isoform, is associated with the ability to desensitize to calcium (relax) in response to NO/cGMP-dependent signaling. We examined expression of MYPT1 isoforms and smooth muscle phenotype in normal rat vessels and in a prehepatic model of portal hypertension characterized by arteriolar dilation. The large capacitance vessels, aorta, pulmonary artery, and inferior vena cava expressed predominantly the 3' exonout/LZ-positive MYPT1 isoform. The first-order mesenteric resistance artery (MA1) and portal vein (PV) expressed severalfold higher levels of MYPT1 with predominance of the 3' exon-included/LZ-negative isoform. There was minor variation in the presence of the MYPT1 central alternative exons. Myosin heavy and light chain splice variants in part cosegregated with MYPT1 isoforms. In response to portal hypertension induced by PV ligature, abundance of MYPT1 in PV and MA1 was significantly reduced and switched to the LZ-positive isoform. These changes were evident within 1 day of PV ligature and were maintained for up to 10 days before reverting to control values at day 14. Alteration of MYPT1 expression was part of a complex change in protein expression that can be generalized as a modulation from a phasic (fast) to a tonic (slow) contractile phenotype. Implications of vascular smooth muscle phenotypic diversity and reversible phenotypic modulation in portal hypertension with regards to regulation of blood flow are discussed. hypertrophy; phenotype; cGMP signaling

Myosin light chain phosphatase (PP1M) is composed of three subunits, i.e., M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosin binding subunit, the function of M20 is unknown. In the present study, we found that M20 binds to microtubules. The binding activity was revealed by cosedimentation of M20 with microtubules and binding of tubulin to M20 affinity resin. Green fluorescent protein (GFP)-tagged M20 (M20-GFP) was expressed in chicken primary smooth muscle cells and COS-7 cells and was used as a probe for studying the association between M20 and microtubules in living cells. M20-GFP was localized on filamentous structures in both cell types. Colocalization analysis revealed that M20-GFP colocalized with tubulin. Treatment with nocodazole, but not cytochalasin B, abolished the filamentous structure of M20-GFP. These results indicate that M20-GFP associates with microtubules in cells. Microinjection of rhodamine-tubulin into the M20-expressing cells revealed that incorporation of rhodamine-tubulin into microtubules was significantly facilitated by microtubule-associated M20. Consistent with this result, M20 enhanced the rate of tubulin polymerization in vitro and produced elongated microtubules. These results suggest that M20 has a microtubule binding activity and plays a role in regulating microtubule dynamics. M20; tubulin; myosin light chain phosphatase; protein phosphatase; green fluorescent protein