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Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research. [ABSTRACT FROM AUTHOR]

Interest in mesenchymal stem cell derived extracellular vesicles (MSC‐EVs) as therapeutic agents has dramatically increased over the last decade. Current approaches to the characterization and quality control of EV‐based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label‐free immunosensing technique for characterizing clinically approved umbilical cord MSC‐EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers. Furthermore, analysis of QCM dissipation versus frequency allowed us to quantitatively determine the ratio of marker‐specific EVs versus non‐vesicular particles (NVPs) – a parameter that cannot be obtained by any other technique so far. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and NVPs in our EV preparations. This measurement modality makes it possible to identify EV sub‐fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label‐free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM‐I) and AFM measurements provides a robust multi‐marker approach to the characterization of clinically approved EV therapeutics and opens the door to improved quality control. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) derived from the secretome of human mesenchymal stromal cells (MSC) contain numerous factors that are known to exert anti‐inflammatory effects. MSC‐EVs may serve as promising cell‐based therapeutics for the inner ear to attenuate inflammation‐based side effects from cochlear implantation which represents an unmet clinical need. In an individual treatment performed on a 'named patient basis', we intraoperatively applied allogeneic umbilical cord‐derived MSC‐EVs (UC‐MSC‐EVs) produced according to good manufacturing practice. A 55‐year‐old patient suffering from Menière's disease was treated with intracochlear delivery of EVs prior to the insertion of a cochlear implant. This first‐in‐human use of UC‐MSC‐EVs demonstrates the feasibility of this novel adjuvant therapeutic approach. The safety and efficacy of intracochlear EV‐application to attenuate side effects of cochlea implants have to be determined in controlled clinical trials. [ABSTRACT FROM AUTHOR]

Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC-sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC-sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC-sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid-membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC-sEV preparations might also be measured against a well-characterized MSC-sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC-sEV biology and therapeutic potency onto quantifiable features of each preparation. [ABSTRACT FROM AUTHOR]

Abstract: Assisted colonisation, one form of species translocation, has been proposed as a tool for helping species to track suitable conditions in a changing climate. There are considerable practical challenges associated with it, including predicting where to place translocated individuals. This problem may be particularly big for small and immobile species, where small‐scale microenvironmental conditions de‐couple them from environmental conditions as projected in large‐scale climate models. To investigate this problem, we developed a survey‐based model to predict the occurrence of our target species, the fruticose terricolous arctic‐alpine lichen, Flavocetraria nivalis, within the Cairngorm Mountains. We then undertook an experimental translocation of this species. A second model, using variables that were significant in the survey‐based model, was only fair at predicting the initial pattern of survival at the recipient site. However, model fit of the translocation survival model improved over time as the distribution of surviving individuals more accurately reflected the distribution of suitable environmental conditions. In addition, model predictive power increased with the addition of data on microclimatic conditions at recipient plots. Synthesis and applications. Our results demonstrate that, for species which respond strongly to local environmental conditions, are immobile and, to some extent, decoupled from larger scale climates, it may be difficult to build a priori accurate predictive models of habitat suitability. In these cases, a combination of modelling and expert judgement, along with the movement of substantial numbers of transplants, may be the appropriate options for maximising the success of assisted colonisation. [ABSTRACT FROM AUTHOR]

Question We compared active and abandoned beech coppices in terms of: (1) structural features, (2) total, understorey and overstorey plant diversity across spatial scales, and (3) species richness of beech forest specialist species. Location Central Apennines, Italy. Methods We applied a multi-scale approach, working at fine scale (management unit) and at a coarser scale (forest patch). Two forest patches were selected according to management regimes (active coppicing, 1-40 yrs; abandoned coppicing, >40 yrs) in each of the two study areas (Bolognola and Ussita). Within each forest patch, 20 stands were sampled in 20 m × 20 m plots, for a total of 80 sampling units. Results We found that the height of standards and number of dominant trees were negatively correlated with the total number of species. At the plot scale, the number of beech specialist species was significantly higher in abandoned plots. At the forest patch scale, the number of species in Bolognola was markedly higher in the actively coppiced forest than in the abandoned one, while the opposite result was found at Ussita. Regarding the beech specialist species richness, the abandoned forest at Ussita showed higher richness than the coppiced forest, while in Bolognola we found the opposite. The managed forest hosted more overstorey species than the abandoned one in both areas. Surprisingly, at forest patch scale in Bolognola, the total beech specialist species richness was higher in the coppiced plots than in the abandoned ones. The species assemblages were more similar between patches having different management regimes within the same area, than between patches having the same management regime across different areas. Conclusions This material provides a novel contribution to the study of species diversity patterns in this forest system, suggesting the importance of a multiple scale approach in forest diversity studies. The beech specialist species can largely persist in a heterogeneous coppice landscape, where abandoned stands are mixed with stands under regular coppicing. The results can link current knowledge about beech coppice diversity on the landscape scale with that on the plot scale, and thus help guide new conservation planning. [ABSTRACT FROM AUTHOR]

Habitat associations of farmland birds are well studied, yet few have considered relationships between species distribution and soil properties. Charadriiform waders (shorebirds) depend upon penetrable soils, rich in invertebrate prey. Many species, such as the Northern Lapwing Vanellus vanellus, have undergone severe declines across Europe, despite being targeted by agri-environment measures. This study assessed whether there were additive effects of soil variables (depth, pH and organic matter content) in explaining Lapwing distribution, after controlling for known habitat relationships, at 89 farmland sites across Scotland. The addition of these soil variables and their association with elevation improved model fit by 55%, in comparison with models containing only previously established habitat relationships. Lapwing density was greatest at sites at higher elevation, but only those with less peaty and less acidic soil. Lapwing distribution is being constrained between intensively managed lowland farmland with favourable soil conditions and upland sites where lower management intensity favours Lapwings but edaphic conditions limit their distribution. Trials of soil amendments such as liming are needed on higher elevation grassland sites to test whether they could contribute to conservation management for breeding Lapwings and other species of conservation concern that depend upon soil-dwelling invertebrates in grassland soils, such as Eurasian Curlew Numenius arquata, Common Starling Sturnus vulgaris and Ring Ouzel Turdus torquatus. Results from such trials could support improvement and targeting of agri-environment schemes and other conservation measures in upland grassland systems. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed. [ABSTRACT FROM AUTHOR]

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system. [ABSTRACT FROM AUTHOR]

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

1. The study of the spatial pattern of species abundance is complicated by statistical problems, such as spatial autocorrelation of the abundance data, which lead to the confusion of environmental effects and dispersal. 2. Atlas-derived data for the rook in Scotland are used as a case study to propose an approach for assessing the likely contribution of dispersal and local environmental effects, based on a Bayesian Conditional Autoregressive (CAR) approach. 3. The availability of moist grasslands is a key factor explaining the spatial pattern of abundance. This is influenced by a combination of climatic and soil-related factors. A direct link to soil properties is for the first time reported for the wide-scale distribution of a bird species. In addition, for this species, dispersal seems to contribute significantly to the spatial pattern and produces a smoother than expected decline in abundance at the north-western edge of its distribution range. Areas where dispersal is most likely to be important are highlighted. 4. The approach described can help ecologists make more efficient use of atlas data for the investigation of the structure of species abundance, and can highlight potential sink areas at the landscape and regional scale. 5. Bayesian spatial models can deal with data autocorrelation in atlas-type data, while clearly communicating uncertainty through the estimation of the full posterior probability distribution of all parameters. [ABSTRACT FROM AUTHOR]

Regional-scale ecological processes are mediated by processes occurring at local scales. Spatially explicit models are needed to understand the broad-scale consequences of a large number of local processes, driven by factors which are heterogeneous at a broad scale. A Geographic Information System (GIS)-based mechanistic model, which can be used as a flexible tool to investigate the regional-scale effects of changes in environmental factors on herbage production from a Lolium perenne sward, was developed and used to investigate the consequences of aggregating driving variables at different resolutions. The model allows rigorous scaling up of previously existing field-based modelling approaches within a GIS context. The model's mechanistic approach allows flexibility in the simulation of the separate effects of environmental driving variables and of cutting regimes. The driving variables (temperature, solar radiation, available soil moisture and soil nitrogen status) are scaled up using geostatistical techniques. The model is used to evaluate weekly changes in herbage production under environmental conditions, variable both in space and time, under different cutting regimes. It is shown that, in this model, aggregation at 1 km2 is a good compromise between accuracy and practical feasibility, and that, while ignoring heterogeneity over many square kilometres can induce large errors, their magnitude and direction also depend on the model response curve to an input variable. The results obtained were consistent with the known trends in influential environmental factors. The programming within a GIS makes the model flexible in its application and, therefore, makes it easy to apply at a variety of scales. [ABSTRACT FROM AUTHOR]

The phosphorylation-dephosphorylation of serine and threonine residues of calponin is known to modulate in vitro its interaction with F-actin and is thought to regulate several biological processes in cells, involving either of the calponin isoforms. Here, we identify, for the first time, tyrosine-phosphorylated calponin h3 within COS 7 cells, before and after their transfection with the pSV vector containing cDNA encoding the cytoplasmic, Src-related, tyrosine kinase, Fyn. We then describe the specific tyrosine phosphorylation in vitro of calponin h1 and calponin h3 by this kinase. 32P-labeling of tyrosine residues was monitored by combined autoradiography, immunoblotting with a specific phosphotyrosine monoclonal antibody and dephosphorylation with the phosphotyrosine-specific protein phosphatase, YOP. PhosphorImager analyses showed the incorporation of maximally 1.4 and 2.0 mol of 32P per mol of calponin h3 and calponin h1, respectively. As a result, 75% and 68%, respectively, of binding to F-actin was lost by the phosphorylated calponins. Furthermore, F-actin, added at a two- or 10-fold molar excess, did not protect, but rather increased, the extent of 32P-labeling in both calponins. Structural analysis of the tryptic phosphopeptides from each 32P-labeled calponin revealed a single, major 32P-peptide in calponin h3, with Tyr261 as the phosphorylation site. Tyr261 was also phosphorylated in calponin h1, together with Tyr182. Collectively, the data point to the potential involvement, at least in living nonmuscle cells, of tyrosine protein kinases and the conserved Tyr261, located in the third repeat motif of the calponin molecule, in a new level of regulation of the actin–calponin interaction. [ABSTRACT FROM AUTHOR]

: Lumiracoxib (Prexige; Novartis Pharma AG, Basel, Switzerland) is a cyclooxygenase-2 selective inhibitor associated with improved gastrointestinal safety compared with nonsteroidal anti-inflammatory drugs, in patients with osteoarthritis. : To compare the gastroduodenal safety of lumiracoxib with ibuprofen and celecoxib in patients with rheumatoid arthritis. : A total of 893 patients with rheumatoid arthritis were randomized to lumiracoxib 400 mg once daily, lumiracoxib 800 mg once daily, ibuprofen 800 mg three times daily or celecoxib 200 mg twice daily for 13 weeks, in a double-blind randomised controlled clinical trial. The primary endpoint was the cumulative incidence of gastroduodenal ulcers over 13 weeks. : The incidence of gastroduodenal ulcers ≥3 mm with lumiracoxib 400 mg once daily (2.8%) or lumiracoxib 800 mg once daily (4.3%) was significantly lower than with ibuprofen (13.6%, all P < 0.01) and not different from celecoxib (1.9%). The incidence of adverse events was similar for lumiracoxib 400, 800 mg and celecoxib (78, 75 and 77%, respectively) and higher with ibuprofen (86%). Discontinuation for adverse events was highest for ibuprofen (12.5% vs. 7.9–8.8% for the other groups). : Lumiracoxib demonstrated gastroduodenal safety superior to ibuprofen and similar to celecoxib in patients with rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Cysteine-rich proteins (CRPs) have been shown to be involved in cell differentiation, transcriptional regulation and the organisation of the actin cytoskeleton. Thus far, the latter function has been inferred solely from the in vitro interaction of CRP1, CRP2, and CRP3 with α-actinin and zyxin. We show here that purified, recombinant CRP2 binds directly to F-actin in vitro in co-sedimentation assays. Using a green fluorescent protein (GFP)-tagged construct of CRP2 we analysed its localisation and dynamics in A7r5 rat smooth muscle cells. CRP2 was associated with the actin cytoskeleton and decorated actin stress fibres in a continuous fashion, unlike the periodic labelling pattern observed for α-actinin and zyxin, which also accumulate in focal adhesions. Using live video fluorescence microscopy we observed the behaviour of GFP-CRP2 during the dynamic rearrangement of the actin cytoskeleton in phorbol 12,13-dibutyrate-treated A7r5 cells. In contrast to the actin-binding proteins SM22α and α-actinin, GFP-CRP2 did not translocate into the podosomes induced by this treatment, but remained preferentially bound to the stress fibres, suggesting an actin filament-stabilising role for CRP2. When fused to the mitochondrial targeting sequence from the Listeria protein ActA, GFP-CRP2 was almost completely localised to mitochondria, but no significant recruitment of either α-actinin or zyxin could be observed. Taken together, our results demonstrate that CRP2 can bind to F-actin directly and that the association with the actin cytoskeleton is independent of α-actinin or zyxin localisation in the cell, thus questioning the role of CRP2 as a regulator of α-actinin function in vivo. [Copyright &y& Elsevier]

Metastasis of diseased cells is the basic event leading to death in individuals with cancer. Establishment of metastasis requires that tumour cells migrate from the site of the primary tumour into the circulation system, escape from the vasculature and form secondary tumours at novel sites. These processes depend to a large degree on cytoskeletal remodeling. We show here that multiple copies of the short actin-binding module CLIK23 from human or Caenorhabditis elegans calponin proteins effectively inhibit cell motility on two dimensional matrices and suppress soft agar colony formation of metastatic melanoma and adenocarcinoma cells of murine and human origin. Ectopic expression of CLIK23 modules for 30 days results in the formation of multinucleated cells. The repeat displays true modular behaviour, resulting in increased cytoskeletal effects in direct correlation with the increase in number of modules. Our results demonstrate that the role of calponin in the signature profile of metastasising cells is that of a mechanical stabiliser of the actin cytoskeleton, which interferes with actin turnover by binding at a unique interface along the actin filament. [Copyright &y& Elsevier]

With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross-linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties. [Copyright &y& Elsevier]

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer-readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes. [Copyright &y& Elsevier]

Smooth muscle cells possess a structural lattice composed of two primary parts: the `cytoskeleton' that pervades the cytoplasm and the `membrane skeleton' that provides anchorage for the cytoskeleton and contractile apparatus at the cell surface. The cytoskeleton contains two major components: first, a complement of actin filaments that links the cytoplasmic dense bodies at equispaced intervals in longitudinal fibrils; and second, a network of desmin intermediate filaments that co-distributes with the cytoskeletal actin. The actin filaments of the contractile apparatus are presumed to interface with the cytoskeleton at the cytoplasmic dense bodies and with the longitudinal rib-like arrays of dense plaques of the membrane skeleton that couple to the extracellular matrix. The present report focuses attention on the functional role of intermediate filaments and on the molecular domain structure of the protein calponin, which is found both in the cytoskeleton and the contractile apparatus. New information about the role of intermediate filaments in smooth muscle has come from studies of transgenic mice in which desmin expression has been ablated. These have shown that while desmin is dispensable for normal development and viability its absence has significant consequences for the mechanical properties of muscle tissue. Thus, the visceral smooth muscles develop only 40% of the normal contractile force and the maximal shortening velocity is reduced by 25–40%. Intermediate filaments therefore play an active role in force transmission and do not contribute solely to cell shape maintenance, as has hitherto been presumed. Recent studies on calponin have revealed a second actin binding domain at the C-terminus of the molecule and have also pinpointed an N-terminal domain that shares homology with a growing family of actin binding and signalling molecules. How these newly identified features of calponin relate to its function in vivo remains to be established. [ABSTRACT FROM AUTHOR]

The cytoskeletal component vinculin has been demonstrated by hydrophobic photoradiolabelling, to insert into bilayers containing acidic phospholipids and trace amounts of a photoactivatable analogue of lecithin. It is shown in this study that the higher-molecular-mass variant metavinculin and α-actinin, also share this property. α-Actinin and vinculin were also shown to associate with phosphatidylserine liposomes by chromatography of protein/lipid mixtures on a Bio-Gel A-5m column. Furthermore, interesting differences in the behaviour of binary mixtures of these proteins, in the presence of phosphatidylserine liposomes, are shown. Thus, incubation of α-actinin with vinculin or metavinculin, prior to the addition of liposomes, strongly inhibited the photoradiolabelling of α-actinin under conditions in which the liposome surface was non-limiting, but enhanced the labelling of vinculin. In contrast, vinculin and metavinculin did not mutually influence their labelling. Using gel-filtration chromatography, it was shown that α-actinin still bound to the vinculin-liposome complex, under conditions similar to those used for hydrophobic photolabelling with a non-limiting lipid surface. In the presence of limiting amounts of liposomes, the α-actinin/vinculin ratio was markedly decreased in the liposome fractions. Our results suggest the formation of a ternary complex consisting of vinculin, α-actinin and phospholipids. In this complex, both proteins interact at the bilayer, resulting in an altered conformation of the two proteins and, as a consequence, in modified bilayer interactions. [ABSTRACT FROM AUTHOR]

Calponin is a basic smooth-muscle-specific protein capable of binding to F-actin, tropomyosin and calmodulin in vitro. Using two-dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a pi identical to the most basic adult a variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees-Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985–5991], which has sequence motifs related to calponin, displays a similar isoform pattern during development; one isoform (a) is present in the embryo and three in the adult. In living smooth-muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32PO4, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two-dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. These results support the claim [Barany, M., Rokolya, A. & Barany, K. (1991) FEBS Lett. 279, 65–68] that calponin phosphorylation is not involved in smooth muscle regulation in vivo, as has been suggested from in vitro studies [Winder, S. J. & Walsh, M. J. (1990) J. Biol. Chem. 265, 10148–10155]. In vitro translation of porcine and chicken smooth-muscle mRNA produced only a single (a) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the a isoform and a second more basic spot, not observed in tissue samples. Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down-regulated like calponin, metavinculin and heavy-caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the a SM 22 isoform during smooth-muscle phenotype modulation in vitro. [ABSTRACT FROM AUTHOR]

We have analyzed the structure, origin and expression of the high-molecular-mass muscle-specific variant of vinculin, called meta-vinculin. The meta-vinculin-specific inserts from the human and avian molecules have been isolated and sequenced and the sequences confirmed via cloning of the corresponding cDNA. Comparison of the human, avian and determined porcine sequences revealed cross-species identity in the C-terminal half of the insert. Human and porcine meta-vinculin were highly similar in the insert region, showing only five amino acid exchanges; avian meta-vinculin showed 22 exchanges in the same region compared to human meta-vinculin and exhibited, in addition, one extra amino acid, making 69 in all. Each insert was flanked by characteristic KWSSK motifs. Evidence for two vinculin mRNA species in human uterus smooth muscle was provided by reverse transcription combined with the polymerase chain reaction, as well as by ribonuclease-mapping analysis of cDNA/mRNA hybrids. One of the mRNA species contained an additional 204-nucleotide insert that precisely encoded the meta-vinculin-specific peptide. Sequence analysis of the appropriate portion of the human vinculin gene showed that the section coding for the meta-vinculin-specific insert is present as a discrete exon. Thus, meta-vinculin and vinculin mRNA are generated by alternative splicing. [ABSTRACT FROM AUTHOR]