To produce an immune reaction against a foreign protein usually requires purification of that protein, which is then injected into an animal. The isolation of enough pure protein is time-consuming and sometimes difficult. Here we report that such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. This is achieved using a hand-held form of the biolistic system which can propel DNA-coated gold microprojectiles directly into cells in the living animal. Genetic immunization may be time- and labour-saving in producing antibodies and may offer a unique method for vaccination.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma.As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling.Anti-inflammatory therapy, however, does not ''cure'' asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM.In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

The intermediate filament framework is one of the three cytoskeletal systems in mammalian cells. Its well spread filamentous structure from the nucleus to the plasma membrane is believed to provide protection against various mechanical stresses (1, 2). Intermediate filaments also undergo disassembly/assembly and spatial reorganization in cells in response to external stimulation and during mitosis. The dynamic property of the intermediate filament system plays a fundamental role in mediating changes in cell shape, division, and migration; signaling molecule distribution; and smooth muscle force development (1, 3-7).The dynamic characteristics of the intermediate filament network may be regulated by protein phosphorylation. Vimentin is the most abundant intermediate filament protein in various cell types, including smooth muscle cells (2,5,6). Vimentin phosphorylation in association with vimentin disassembly and spatial reorganization occurs during mitosis or in response to extracellular stimulation (8, 9). In cultured smooth muscle cells, contractile stimulation triggers vimentin phosphorylation at Ser-56 concurrently with vimentin partial disassembly and spatial reorientation (6).The disassembly and spatial reorganization of the vimentin network may regulate the translocation of certain molecules (7, 10, 11). The adapter protein p130 Crk-associated substrate (CAS) 2 has been shown to participate in the signaling processes that regulate smooth muscle contraction and cell migration (12-14). Our recent study has suggested that vimentin phosphorylation and disassembly are related to CAS redistribution during contractile activation of smooth muscle (10). In addition, external stress initiates Rho kinase redistribution associated with vimentin depolymerization in fibroblasts and the translocation of Ca 2ϩ /calmodulin-dependent protein kinase II in differentiated smooth muscle cells, which may be an important event for cell signaling (7, 11).p21-activated kinase (PAK) may be an upstream regulator of the vimentin network (6, 9). In cultured smooth muscle cells, agonist-mediated vimentin phosphorylation at Ser-56 and spatial reorientation of the vimentin network are inhibited by silencing of PAK1, a dominant isoform in smooth muscle (6,15). Additionally, PAK has been implicated in modulating smooth muscle contraction; introduction of an active PAK isoform into smooth muscle potentiates force development at constant intracellular calcium (16). Expression of an inactive PAK1 mutant attenuates migration of cultured smooth muscle cells in response to platelet-derived growth factor (15).In response to external stimulation, PAK undergoes autophosphorylation at Thr-423, which increases PAK activity for substrates (17,18). In addition to the small GTPases Cdc42 and Rac1, the activity of PAK may be regulated by the paxillin kinase linker/PIX (PAK-interacting exchange factor; guanine nucleotide exchange factor) (19 -21). CAS has been shown to interact with the paxillin kinase linker/PIX via CrkII and paxillin (21-* This work was ...

Contractile stimulation has been shown to initiate actin polymerization in smooth muscle tissues, and this actin polymerization is required for active tension development. We evaluated whether neuronal Wiskott-Aldrich syndrome protein (N-WASp)-mediated activation of the actin-related proteins 2 and 3 (Arp2/3) complex regulates actin polymerization and tension development initiated by muscarinic stimulation in canine tracheal smooth muscle tissues. In vitro, the COOH-terminal CA domain of N-WASp acts as an inhibitor of N-WASp-mediated actin polymerization; whereas the COOH-terminal VCA domain of N-WASp is constitutively active and is sufficient by itself to catalyze actin polymerization. Plasmids encoding EGFP-tagged wild-type N-WASp, the N-WASp VCA and CA domains, or enhanced green fluorescent protein (EGFP) were introduced into tracheal smooth muscle strips by reversible permeabilization, and the tissues were incubated for 2 days to allow for expression of the proteins. Expression of the CA domain inhibited actin polymerization and tension development in response to ACh, whereas expression of the wild-type N-WASp, the VCA domain, or EGFP did not. The increase in myosin light-chain (MLC) phosphorylation in response to contractile stimulation was not affected by expression of either the CA or VCA domain of N-WASp. Stimulation of the tissues with ACh increased the association of the Arp2/3 complex with N-WASp, and this association was inhibited by expression of the CA domain. The results demonstrate that 1) N-WASp-mediated activation of the Arp2/3 complex is necessary for actin polymerization and tension development in response to muscarinic stimulation in tracheal smooth muscle and 2) these effects are independent of the regulation of MLC phosphorylation.

Smooth muscle cell migration has been implicated in the development of respiratory and cardiovascular systems; and airway/vascular remodeling. Cell migration is a polarized cellular process involving a protrusive cell front and a retracting trailing rear. There are three cytoskeletal systems in mammalian cells: the actin cytoskeleton, the intermediate filament network, and microtubules; all of which regulate all or part of the migrated process. The dynamic actin cytoskeleton spatially and temporally regulates protrusion, adhesions, contraction, and retraction from the cell front to the rear. c-Abl tyrosine kinase plays a critical role in regulating actin dynamics and migration of airway smooth muscle cells and nonmuscle cells. Recent studies suggest that intermediate filaments undergo reorganization during migration, which coordinates focal adhesion dynamics, cell contraction, and nucleus rigidity. In particular, vimentin intermediate filaments undergo phosphorylation and reorientation in smooth muscle cells, which may regulate cell contraction and focal adhesion assembly/disassembly. Motile cells are characterized by a front-rear polarization of the microtubule framework, which regulates all essential processes leading to cell migration through its role in cell mechanics, intracellular trafficking, and signaling. This review recapitulates our current knowledge how the three cytoskeletal systems spatially and temporally modulate the migratory properties of cells. We also summarize the potential role of migration-associated biomolecules in lung and vascular diseases.