Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle

Displacement of the contractile protein tropomyosin from actin filament exposes the myosin-binding sites on actin, resulting in actin-myosin interaction and muscle contraction. The objective of the present study was to better understand the interaction of tropomyosin with heat shock protein (HSP)27 in contraction of smooth muscle cells of the colon. We investigated the possibility of a direct protein-protein interaction of tropomyosin with HSP27 and the role of phosphorylated HSP27 in this interaction. Immunoprecipitation studies on rabbit smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that the association of tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)-tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to phosphomimic HSP27 mutant compared with wild-type and nonphosphomimic HSP27. Our data suggest that, in response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin. acetylcholine; fusion proteins; serine     

A minimally invasive approach for correction of chin ptosis

Although ptosis of the tip of the chin is common and can be seen in persons of any age, it is frequently seen in older patients seeking facial rejuvenation. A variety of techniques have been described to correct ptosis of the chin. The authors describe a minimally invasive method that can be used correct chin ptosis. This technique uses a small intraoral incision to place a U-shaped Prolene suture that gathers the soft tissue of the chin and elevates it above the lower border of the mandibular symphysis. A retrospective review of 314 cases performed in conjunction with face lifts between January of 1994 and January of 2000 was performed to evaluate this technique. There were no significant complications, and long-term results have been very satisfactory and lasting.     

Neuronal and muscular alterations caused by two wheat endosperm proteins,puroindoline-a and alpha1-purothionin,are due to ion pore formation

Using the patch-clamp technique it was found that the toxicity of the two wheat endosperm proteins puroindoline-a and alpha1-purothionin probably results from the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.Abbreviations PIN-a puroindoline-a - PTH alpha1-purothionin Presented at the Biophysical Society Meeting on Ion Channels—from structure to disease held in May 2003, Rennes, France     

Role of oxidative stress in age-related reduction of NO-cGMP-mediated vascular relaxation in SHR

The incidence of hypertension increases during the late stages of aging; however, the vascular mechanisms involved are unclear. We investigated whether the late stages of aging are associated with impaired nitric oxide (NO)-mediated vascular relaxation and enhanced vascular contraction and whether oxidative stress plays a role in the age-related vascular changes. Aging (16 mo) male spontaneously hypertensive rats (SHR) nontreated or treated for 8 mo with the antioxidant tempol (1 mM in drinking water) or vitamin E (E; 5,000 IU/kg chow) and vitamin C (C; 100 mg. kg-1. day-1 in drinking water) and adult (12 wk) male SHR were used. After the arterial pressure was measured, aortic strips were isolated from the rats for measurement of isometric contraction. The arterial pressure and phenylephrine (Phe)-induced vascular contraction were enhanced, and the ACh-induced vascular relaxation and nitrite/nitrate production were reduced in aging compared with adult rats. In aging rats, the arterial pressure was nontreated (188 +/- 4), tempol-treated (161 +/- 6), and E + C-treated (187 +/- 1 mmHg). Phe (10-5 M) caused an increase in active stress in nontreated aging rats (14.3 +/- 1.0) that was significantly (P < 0.05) reduced in tempol-treated (9.0 +/- 0.7) and E + C-treated rats (9.8 +/- 0.6 x 104 N/m2). ACh produced a small relaxation of Phe contraction in nontreated aging rats that was enhanced (P < 0.05) in tempol- and E + C-treated rats. l-NAME (10-4 M), inhibitor of NO synthase, or ODQ (10-5 M), inhibitor of cGMP production in smooth muscle, inhibited ACh relaxation and enhanced Phe contraction in tempol- and E + C-treated but not the nontreated aging rats. ACh-induced vascular nitrite/nitrate production was not different in nontreated, tempol- and E + C-treated aging rats. Relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was smaller in aging than adult rats but was not different between nontreated, tempol- and E + C-treated aging rats. Thus, during the late stages of aging in SHR rats, an age-related inhibition of a vascular relaxation pathway involving not only NO production by endothelial cells but also the bioavailability of NO and the smooth muscle response to NO is partially reversed during chronic treatment with the antioxidants tempol and vitamins E and C. The data suggest a role for oxidative stress in the reduction of vascular relaxation and thereby the promotion of vascular contraction and hypertension during the late stages of aging.     

GM-CSF increases airway smooth muscle cell connective tissue expression by inducing TGF-beta receptors

Fibrosis around the smooth muscle of asthmatic airway walls leads to irreversible airway obstruction. Bronchial epithelial cells release granulocyte/macrophage colony-stimulating factor (GM-CSF) in asthmatics and are in close proximity to airway smooth muscle cells (ASMC). The findings in this study demonstrate that GM-CSF induces confluent, prolonged, serum-deprived cultures of ASMC to increase expression of collagen I and fibronectin. GM-CSF also induced ASMC to increase the expression of transforming growth factor (TGF)-beta receptors type I, II, and III (TbetaR-I, TbetaR-II, TbetaR-III), but had no detectable effect on the release of TGF-beta1 by the same ASMC. The presence of GM-CSF also induced the association of TGF-beta1 with TbetaR-III, which enhances binding of TGF-beta1 to TbetaR-II. The induction of TbetaRs was parallel to the increased induction of phosphorylated Smad2 (pSmad2) and connective tissue growth factor (CTGF), indicative of TGF-beta-mediated connective tissue synthesis. Dexamethasone decreased GM-CSF-induced TbetaR-I, TbetaR-II, TbetaR-III, pSmad2, CTGF, collagen I, and fibronectin. In conclusion, GM-CSF increases the responsiveness of ASMC to TGF-beta1-mediated connective tissue expression by induction of TbetaRs, which is inhibited by corticosteroids.     

The Slc11a1 (Nramp1) gene controls efficacy of mycobacterial treatment of allergic asthma

Genes controlling antibacterial resistance may be important in the hygiene hypothesis, which states that lack of bacterial infections during childhood would favor development of allergic disease. We, therefore, studied whether Nramp1 (Slc11a1) alleles, which determine susceptibility (Nramp1(s)) or resistance (Nramp1(r)) to intracellular bacteria, affect the efficacy of heat-killed Mycobacterium vaccae in the treatment of allergic asthma in a mouse model. Treatment of OVA-sensitized Nramp1(s) mice with M. vaccae suppressed airway hyperresponsiveness, airway eosinophilia, Ag-specific IgE, and IL-4 and IL-5 production after OVA aerosol challenge. In contrast, M. vaccae hardly affected these parameters in Nramp1(r) mice. In addition, The Nramp1 gene affected both T cell-mediated responses to M. vaccae in vivo and the level of macrophage activation after stimulation with M. vaccae in vitro. In conclusion, the efficacy of M. vaccae in preventing allergic and asthmatic manifestations in a mouse model is strongly affected by Nramp1 alleles. These findings could have important implications for the future use of mycobacteria and their components in the prevention or treatment of allergic asthma. A new link is described between genes, the environment, and the development of allergy, in which the Nramp1 gene fine tunes the hygiene hypothesis.     

Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament

Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.