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Unusual symptoms were observed in summer 1997 in field zucchini of several cultivars grown in central Italy. Symptoms included reduction in growth, severe mosaic, blistering and deformation of leaves, and malformation on fruits. Plants gave negative results in enzyme-linked immunosorbent assay (ELISA) for cucumber mosaic cucumovirus, squash mosaic comovirus, papaya ringspot, zucchini yellow fleck, zucchini yellow mosaic, and watermelon mosaic 2 potyviruses. Positive reactions were obtained in ELISA with a monoclonal antibody that reacts with many potyviruses (from J. Vetten, Braunschweig) and with polyclonal antibodies (Sanofi, France) to Moroccan watermelon mosaic potyvirus (MWMV). Field symptoms were reproduced in zucchini cvs. Genovese and Striato d'Italia by mechanical inoculation of samples from symptomatic field plants. Chenopodium amaranticolor, C. quinoa, and Gomphrena globosa gave local lesions, while Citrullus lanatus cv. Crimson Sweet, Cucumis melo cv. Top Mark, C. metuliferus, C. sativus cvs. Marketer, MM76, and Sweet Slice, and Cucurbita maxima reacted with systemic mosaic. C. melo cv. Doublon formed necrotic local lesions followed by systemic necrosis. No infection occurred in Nicotiana benthamiana, N. clevelandii, N. glutinosa, Phaseolus vulgaris cv. Black Turtle, Pisum sativum cv. Alaska, tobacco White Burley, and Vigna unguiculata. These data are in agreement with the known host range of MWMV. MWMV is a tentative species in the genus potyvirus, widely present in Africa and occasionally found in Spain (1,2). Further spread of this virus in the Mediterranean area will create new problems for commercial cucurbit production and breeding, and for diagnosis. References: (1) E. Kabelka and R. Grumet. Euphytica 95:237, 1997. (2) N. M. McKern et al. Arch. Virol. 131:467, 1993.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system – such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1β, chemokine IL-8 and enzyme caspase-1 – was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and β-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1β, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

The necrotic disorder of freesia (Freesia refracta hyb., Family Iridaceae) was first described in The Netherlands before 1970. In following years, the disorder was widely reported in other European countries and more recently also in the United States and in New Zealand. The presence of the Freesia sneak virus (FreSV, Ophiovirus genus) has been widely associated with the necrotic disease, but some uncertainty remains. Freesia leaves showing necrotic disease were subjected to Ophiovirus purification and the product obtained was analyzed. Several differently shaped virus particles were visualized by TEM and a new virus-like agent, with a ca. 30 kDa coat protein was detected by western blotting. Previously uncharacterized viruses, possibly transmitted by the same vector, might have a role in the disease, at least in mixed infection, and are now thickening the plot about the necrotic disease causal agent.

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.

Matrix metalloproteinases (MMPs) is a family of proteolytic enzymes involved in remodeling of extracellular matrix. Although proteolytic enzymes are produced by many cell types, mast cells seem to be more important than other types in remodeling of pulmonary arteries during hypoxia. Therefore, we tested in vitro production of MMPs and serine proteases in four cell types (mast cells, fibroblasts, vascular smooth muscle cells and endothelial cells) cultivated for 48 h under normoxic or hypoxic (3 % O2) conditions. MMP-13 was visualized by immunohistochemistry, MMP-2 and MMP-9 were detected by zymography in cell lysates. Enzymatic activities (MMPs, tryptase and chymase) were estimated in the cultivation media. Hypoxia had a minimal effect on total MMP activity in the cultivation media of all types of cells, but immunofluorescence revealed higher intensity of MMP-13 in the cells exposed to hypoxia except of fibroblasts. Tryptase activity was three times higher and chymase activity twice higher in mast cells cultivated in hypoxia than in those cultured in normoxia. Among all cell types studied here, mast cells are the most abundant source of proteolytic enzymes under normoxic and hypoxic conditions. Moreover, in these cells hypoxia increases the production of both specific serine proteases tryptase and chymase, which can act as MMPs activators.

Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma x glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.

Various techniques for coating synthetic surfaces with fibrin structures were tested for seeding bovine pulmonary artery endothelial cells (EC). Two-dimensional fibrin (Fb) structures (2D Fb) were obtained by successively repeating adsorption of fibrinogen (Fbg), incubating the surface with thrombin (Thr) solution, and inhibiting surface-attached Thr. Three-dimensional fibrin networks immobilized at the surface (3D Fb) were formed by catalytic action of surface-attached thrombin on an ambient Fbg solution. Ultra-thin 3D Fbs were obtained if thrombin inhibitors antithrombin III and heparin were added into an Fbg solution. The formation of surface fibrin structures was observed in situ using surface plasmon resonance. The morphology of the structures was studied by transmission and scanning electron microscopy. A polylactide fibrous scaffold was modified with a surface fibrin film without filling the inner pores with a bulk gel. The growth of EC seeded on a polystyrene surface coated with the Fb films was evaluated by the number and morphology of the adhering ECs and the concentration of beta-actin, vinculin, alphav-intergrin, and von Willebrand factor (vWF). The best initial cell spreading after 1 day was observed on 2D Fb and ultra-thin 3D Fb. The highest concentration of vWF, a marker of EC differentiation, was observed after 3 days on thick 3D Fbs. The highest EC population densities after 7 days were observed on 2D Fb and thick 3D Fb. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

Degradable three-dimensional porous scaffolds applicable as cell carriers for bone tissue engineering were developed by an innovative solvent casting/particulate leaching technique from poly(L-lactide-co-glycolide) (PLG). Three types of PLG scaffolds were prepared, and these had the same high porosity (83%) but increasing diameter of the pores (180-200 microm, 250-320 microm, and 400-600 microm) and increasing pore interconnectivity. The colonization of the scaffolds with human osteoblast-like MG 63 cells was then studied in vitro in a conventional static cell culture system. The number of cells growing on the scaffolds on days 1 and 7 after seeding was highest in the material with the largest pore diameter, but on day 15, the differences among the scaffolds disappeared. Confocal microscopy revealed that on day 1 after seeding, the cells penetrated to a depth of 490 +/- 100 microm, 720 +/- 170 microm, and 720 +/- 120 microm into the scaffolds of small, medium, and large pore size, respectively. Incorporation of bromodeoxyuridine into newly synthesized DNA and the concentration of vinculin, beta-actin, osteopontin, and osteocalcin in cells on the scaffolds of all pore sizes were similar to the values obtained on standard tissue culture polystyrene, which indicated good biocompatibility of the scaffolds. These results suggest that all scaffolds could serve as good carriers for bone cells, although the quickest colonization with cells was found in the scaffolds with the largest pore diameter from 400 to 600 microm.

11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.

Summary C6 glioma cells, treated with a cytostatic dose of cisplatin (1.66×10 −5 M) ceased dividing by 24 h and, most of them had undergone apoptosis by 72 – 96 h. The reactive cells were classified into 5 types (T-I to V), according to the ultrastructure of nuclei. At 4 h, 20.4% of cells (T-I) showed minute condensation and margination of chromatin. The nuclear envelope (NE) formed slim and deep invaginations consisting of the inner or both membranes. The later kind of NE invaginations often extended to the enlarged nucleoli and contained nucleolus-like material at its cytoplasmic side. Some nuclear pores were covered with a dome-shaped “cap” formed by fine filamentous material. The number of T-I cells increased to 53.3% by 72 h. In T-II cells, which appeared at 24 h, the chromatin was condensed into dense irregular masses separated from the NE by a lucent space with filamentous structures preventing complete margination of chromatin. Nucleoli of T-II cells were small and showed partial segregation of their components. The “capped” pores were absent in these apparently more damaged cells. From 24 h, cells with large and lobulated nuclei (T-III) started to increase in number and peaked at 72 h (6.6%). Except for some small lobules, the chromatin of T-III cells was moderately aggregated and the NE was well preserved. Typical apoptotic cells with highly condensed and marginated chromatin (T-IV) peaked at 48 – 72 h (2.4 – 4.8%). They appeared in 2 varieties, including cells with wrinkled nuclei with less condensed and incompletely marginated chromatin or more lobulated forms with highly condensed marginated chromatin suggesting their origin from T-II or T-III cells. T-IV cells, as well as their fragments, underwent phagocytosis and secondary necrosis (T-V cells, 48.6% at 96 h). Two alternative routes of nuclear changes leading to cisplatin-triggered apoptosis, as represented by the sequence T-I→T-III→T-IV/V or T-I→T-II→T-IV/V, may explain the initially less or more damaged cells. These alternatives, together with progressive recruitment of reactive cells, suggest intrapopulation differences in the sensitivity of cells or in the cell cycle perturbations induced by cisplatin. Except for the T-IV and T-V cells, observed alterations of cytoplasmic organelles, including mitochondria, were fewer than reported in previous studies on cisplatin.

We have previously demonstrated the developmental increase of the activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD) in the rat ileum which correlated with the developmental surge of plasma concentrations of corticosteroids, thyroid hormones and insulin. To ascertain whether these hormones directly stimulate 11betaHSD activity we used explant cultures of ileum and distal colon. The intestinal segments of young, 7-day-old rats, were cultured 48 hours in the presence of aldosterone (10[-7] M), dexamethasone (10[-7] M), triiodothyronine (10[-7] M) or insulin (10[-7] M) and 11betaHSD activity was evaluated by measuring the conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone. The activity of 11betaHSD was significantly increased following 48 h treatment with dexamethasone and aldosterone, whereas insulin and triiodothyronine were without any effect. Corticosterone oxidation was inhibited by carbenoxolone and progesterone. It is being concluded, that both glucocorticoids and mineralocorticoids but not insulin or triiodothyronine induce intestinal 11betaHSD activity.

Impatiens necrotic spot tospovirus (INSV) virions were purified using a procedure devised for tomato spotted wilt tospovirus (TSWV) from systemically infectedNicotiana benthamiana plants grown at 33 °C day/26 °C night and a photoperiod of 14 hours. With plants grown at 24/18 ° C purification was unsuccessful. In SDS-PAGE the protein pattern of INSV was similar to that reported for TSWV, except the appearance of a single G2 protein band. A polyclonal antiserum, prepared against virions, reacted in Western blots with INSV nucleoprotein and glycoproteins but only with TSWV glycoproteins. In DAS ELISA the antiserum reacted with both INSV and TSWV infected plant sap and, after absorption with TSWV, only with INSV. In TAS ELISA the antiserum trapped both INSV and TSWV nucleoproteins and glycoproteins as detected by specific monoclonal antibodies, and, after absorption with TSWV, only the homologous proteins. This appears to be the first report of the purification of INSV virions and the production of an antiserum reacting with both nucleoprotein and glycoprotein antigens.

Tobacco necrosis virus (TNV) was isolated from glasshouse zucchini plants in Liguria, northern Italy, showing yellow spots on young leaves and necrotic symptoms on older leaves, petioles and stems. No other viruses were present in these plants. Experimentally, the virus systemically infected zucchini seedlings grown in naturally infected soil but only local infections were induced when leaves and roots were mechanically inoculated. The virus was identified as serotype D, devoid of satellite particles. This appears to be the first report of TNV in zucchini. Three ELISA procedures for field detection of TNV using polyclonal antibodies for serotype D were compared. Antibodies were conjugated with I) alkaline phosphatase (AP-AB);2) N-Hydroxy-succinimid obiotin (BNHS-Ab); 3) N-biotinyl-6-aminocaproic hydrazide (BACH-Ab). The procedure using BNHS-Ab detected the lowest concentration of the homologous virus, followed by BACH-Ab and AP-Ab. Only with biotinylated antibodies. TNV serotype A was also detected. Zusammenfassung Charakterisierung eines Isolats des Tobacco necrosis virus aus Zucchini Das Tobacco necrosis virus (TNV) wurde in Ligurien (Norditalien) aus Gewachshaus-Zucchinipflanzen isoliert, deren junge Blatter gelbe Flecken aufwiesen und deren altere Blatter, Blattstiele und Stengel Nekrosen zeigten. Die Pflanzen enthielten keine anderen Viren. Zucchinisamlinge, die in naturbch TNV-verseuchtem Boden wuchsen, wurden im Versuch systemisch von dem Virus befallen. Nach mechanischer Inokulation von Blattern und Wurzeln induzierte TNV dagegen nur lokale Infektionen. Das Virus besas keine Satellitenpartikel und wurde als Serotyp D identifiziert. Dies ist scheinbar der erste Bericht von TNV-Befall bei Zucchini. Drei ELISA-Verfahren zum Nachweis von TNV in Feldmaterial, die polyklonale Antikorper fur Serotyp D verwendeten, wurden verglichen. Die Antikorper wurden konjugiert mit: 1) alkalischer Phosphatase (AP-Ab), 2) N-Hydroxy-succinimidobiotin (BNHS-Ab), und 3) N-biotinyl-6-aminocapronhydrazid (BACH-Ab). Bei Verwendung von BNHS-Ab wurde die niedrigste Konzentration des homologen Virus erfast, gefolgt von BACH-Ab und AP-Ab. Nur mit biotinylierten Antikorpem konnte auch der TNV Serotyp A nachgewiesen werden.

Tospovirus serogroups I and III have recently been designated as species, tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), while the species status of serogroup II isolates remains undefined. Fifteen Tospovirus isolates from ornamental and vegetable crops in Liguria, Italy, were found to belong either to TSWV (seven isolates) or to INSV (eight isolates) on the basis of test-plant reactions, serological techniques using DAS ELISA kits raised against the nucleoproteins of the type members of the two species, and cytopathology. None of them could be assigned to serogroup II using DAS ELISA kits raised against nucleoproteins of this serogroup. Italian isolates representative of the two species reacted in indirect ELISA using a polyclonal antiserum against the entire particle of a TSWV isolate, but with higher intensity for our TSWV isolates than for the INSV isolates. Western blots and dot immunobinding assays confirmed that the nucleoproteins of the two species are unrelated whereas the glycoproteins are related. The cytopathology was similar for two isolates representative of TSWV and INSV, except that the type of filaments encountered was different, and appeared to be characteristic of the species.

A virus was isolated in Italy from Tamus communis in which it caused symptoms of red mosaic and leaf distortion. The virus, for which the name tamus red mosaic virus (TRMV) is suggested, was differentiated from other potexviruses or possible potexviruses on the basis of host range, particle properties and serology. In crude preparations, TRMV had elongated particles of modal length 550–560 nm and diameter of 13 nm. TRMV was easily purified from Chenopodium quinoa and had a buoyant density of 1·34 g/cm3 in CsCl and 1·27 g/cm-3 in Cs2SO4. An antiserum to TRMV was obtained with a titre of 1/1024 in slide precipitin tests. TRMV was related to, but distinct from, tamus latent virus, a potexvirus isolated from Tamus communis in the UK, and more distantly related to potato virus X, cactus virus X and commelina virus X. Polyacrylamide gel electrophoresis (PAGE) indicated that the virus particles contained a single protein species of estimated molecular weight 25·2 kDa. Enzymatic digestions, agarose gel electrophoresis and infectivity of the isolated nucleic acid indicated that the virus has a single major molecule of single-stranded positive-sense RNA of estimated molecular weight 2120 kDa (ca. 6·4 kb).

Systemic and pulmonary hypertension is characterised by structural reconstruction of the vascular wall which includes hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs) and fibroproduction. We hypothesise that these changes are stimulated by non-enzymatic modification of collagen molecules in the injured vascular wall by radicals. We exposed collagen III to ultraviolet (UV) light irradiation which, as indicated by fluorescence and electrophoretic analyses, resulted in its fragmentation. Both irradiated and control unmodified collagen were adsorbed on culture dishes and seeded with VSMCs derived from the rat thoracic aorta. During the first week after seeding, the cells on the modified collagen attained significantly higher population density (by 15-83%), higher mitotic index (by 31-135%) and higher BrdU labelling index (by 32%). However, these cells were less resistant to spontaneous and trypsin-mediated detachment from the growth support. As revealed using enzyme-linked immunosorbent assay in 3-day-old cultures, the cells growing on the irradiated collagen exhibited a lower concentration of beta-1 integrins (-10%, measured per milligram of protein), vinculin (-18%), talin (-6%) and vimentin (-15%). Immunofluorescence staining showed that these molecules were distributed more diffusely and less organised into focal adhesion plaques or cytoskeletal fibres. The concentration of two adhesion molecules of immunoglobulin type, ICAM-1 and VCAM-1, was increased by 11% and 16%, respectively. The concentration of alpha-v integrins and alpha-actin was unchanged; the latter, however, formed fewer distinct microfilament bundles in cells on the modified collagen. Our results suggest that the VSMCs growing on UV-modified collagen are more prone to escape the growth control mediated by cell-extracellular matrix contact and can bind the cells of the immune system.

Elena Filová,1 Tomáš Suchý,2,3 Zbynek Sucharda,2 Monika Šupová,2 Margit Žaloudková,2 Karel Balík,2 Vera Lisá,1 Miroslav Šlouf,4 Lucie Bacáková11Department of Biomaterials and Tissue Engineering, Institute of Physiology, 2Department of Composite and Carbon Materials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, 3Laboratory of Biomechanics, Department of Mechanics, Biomechanics and Mechatronics, Faculty of Mechanical Engineering, CTU in Prague, 4Department of Morphology and Rheology of Polymer Materials, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech RepublicAbstract: Hydroxyapatite (HA) is considered to be a bioactive material that favorably influences the adhesion, growth, and osteogenic differentiation of osteoblasts. To optimize the cell response on the hydroxyapatite composite, it is desirable to assess the optimum concentration and also the optimum particle size. The aim of our study was to prepare composite materials made of polydimethylsiloxane, polyamide, and nano-sized (N) or micro-sized (M) HA, with an HA content of 0%, 2%, 5%, 10%, 15%, 20%, 25% (v/v) (referred to as N0–N25 or M0–M25), and to evaluate them in vitro in cultures with human osteoblast-like MG-63 cells. For clinical applications, fast osseointegration of the implant into the bone is essential. We observed the greatest initial cell adhesion on composites M10 and N5. Nano-sized HA supported cell growth, especially during the first 3 days of culture. On composites with micro-size HA (2%–15%), MG-63 cells reached the highest densities on day 7. Samples M20 and M25, however, were toxic for MG-63 cells, although these composites supported the production of osteocalcin in these cells. On N2, a higher concentration of osteopontin was found in MG-63 cells. For biomedical applications, the concentration range of 5%–15% (v/v) nano-size or micro-size HA seems to be optimum.Keywords: nanoparticles, hydroxyapatite, osteoblasts, composite materials

Endothelial cells derived from the bovine pulmonary artery (line CPAE, CCL 209, American Tissue Culture Collection, Rockville, MD, USA) were cultured on pristine or fluorine ion-irradiated polystyrene (5 x 10(12) or 5 x 10(14) F ions/cm2, 150 keV). At 24-h post-seeding interval, the number of cells which adhered to the ion-modified polystyrene was significantly higher than on the unmodified material (+20 and +58% in cultures with the polystyrene irradiated by lower and higher ion doses, respectively). On day 7, the populations cultured on the irradiated substrates grew to higher densities, exceeding the controls at the lower and higher ion doses by 69 and 180%, respectively. The cells on ion-implanted samples were also larger (+70-95% and +90-99% at the lower and higher ion doses, respectively) and contained more protein (+16% at both ion doses). As was shown by ELISA, the polystyrene irradiated by the higher ion dose enhanced the expression of a cytoskeletal protein, vimentin (+65%) and protein of focal adhesion plaques, talin (+15%). The content of integrin alpha5beta1 (VLA-5), receptor for fibronectin, was increased at both lower and higher ion doses (+22 and +57%). In contrast to this, the content of ICAM-1 and vinculin was similar in cells grown on both pristine and ion-irradiated growth substrates. Moreover, the expression of VCAM-1 and ELAM-1 was lower by 11-14% in both ion dose groups. The present study has shown that ion implantation of polymers improves the adhesion and growth of endothelial cells without elevating the expression of immunoglobulin and selectin types of adhesion molecules. This surface modification should promote colonization of an artificial vascular prosthesis by endothelial cells and make it less vulnerable by immune system cells of the recipient.

In 1-day cultures with 10% serum, the number of rat aortic smooth muscle cells (VSMC) adhering to the growth support was similar in cells from both sexes, whereas in 1% serum, the number of VSMC from male donors was lower. In 10% serum medium, the doubling time was significantly shorter and the number of [3H]thymidine-labelled nuclei was higher in cells of high passage from male rats. In serum-free medium, these differences increased and were also seen in cells of low passage number. Morphologically, the cells in male-derived cultures at higher passage number were mainly spindle-shaped, formed well-developed 'hills and valleys' and possessed longitudinally oriented bundles of alpha-actin-containing microfilaments. Most cells from female rats were flat, polygonal, the multilayered 'hills' were less prominent, with alpha-actin microfilaments forming a mesh-like network.

Vascular smooth muscle cells derived from the rat aorta were cultured on unmodified or F+ ion-implanted polystyrene (5 ×1012 or 5 ×1014 ions/cm2, energy 150 keV). In 1-day-old cultures, the cells adhered to the modified polystyrene in higher numbers and over larger contact areas. Increased resistance of the cells to trypsin-mediated detachment from the growth support indicated an improved adhesion of cells to the modified polymer at later culture intervals. The cells cultured on ion-modified polymers also were larger and had a higher total protein content. By use of immunocytochemistry, several specific protein species were increased, including the cytoskeletal alpha-actin and vimentin and the plasma membrane-associated vinculin, talin, alpha-v integrins, ICAM-1, and VCAM-1, which account for stronger cell–cell and cell–extracellular matrix adhesion. The lower number of cells found floating in the medium suggests that the spontaneous detachment of cells from the modified polystyrene was lower and that the viability of the adhered cell population was higher. As was shown by the two-parameter flow-cytometric measurements of BrdU incorporation and DNA content, as well as by 3H-thymidine autoradiography, the cell proliferation on samples modified by the dose of 5 ×1012 ions/cm2 was similar to that in controls; and at the dose of 5 ×1014 ions/cm2, it tended to be even lower. The cells grown on the polymer implanted with the dose of 5 ×1012 ions/cm2 responded to a new artificially created cell-free area in a confluent cell layer by more intense migration whereas at the dose of 5 ×1014 ions/cm2, the migration ability of cells was similar to that on the unmodified polymer. The data revealed a higher biocompatibility of ion-implanted polystyrene with vascular smooth muscle cells in culture. There was better adhesion, differentiation, and survival, and there was neither excessive migration nor proliferation. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 49, 369–379, 2000.

An undescribed virus, here named ranunculus white mottle virus, was isolated in Italy from cultivated ranunculus showing mottle and distortion of leaves. The virus was mechanically transmissible to several herbaceous hosts. In negative stain, the particles appeared as circularised supercoiled threads 3 nm in diameter of different contour lengths; in some conditions the circles collapsed to form linear pseudobranched structures 9 nm in diameter. Immunolabeling of thin sections showed that viral antigen was widely distributed in the cytoplasm of parenchyma cells. The virus was not serologically related to the morphologically similar tenuiviruses, citrus psorosis-ringspot virus and tulip mild mottle mosaic virus. A major 43 kDa protein was present in purified preparations and in infected plant tissue, as also was a minor 28 kDa protein, serologically related to the major one. Nucleic acids extracted from purified particles consisted of at least three RNAs, of approximately 7.5, 1.8 and 1.5 kb, which appeared partly in single- and partly in double-stranded form. Purified preparations, but not viral RNAs, when mechanically inoculated, were infectious. Host range, tissue tropism, particle morphology and coat protein size place the virus closest to citrus psorosis-ringspot and tulip mild mottle mosaic viruses. These three viruses in turn show similarities with the Tenuiviruses and Bunyaviridae.

We compared responses of Chinese hamster ovary (CHO) cell lines stably transfected with human genes for the M1-M5 muscarinic receptor subtypes to several stimuli. While ATP brought about similar increases in the concentration of intracellular Ca2+ ions ([Ca2+]i) in the cell lines expressing all individual receptor subtypes, carbachol acted with much higher potency and efficacy on the cells expressing the M1, M3, and M5 receptor subtypes than on those expressing the M2 and M4 subtypes. The maximum [Ca2+]i responses to ATP corresponded to 41-75% of the maximum responses to carbachol in the cells expressing the M1, M3, and M5 receptor subtypes. The responses to ATP were strongly suppressed (> 75% decrease) by a preliminary administration of a maximally active concentration of carbachol in these three cell lines, whereas the responses to carbachol were less sensitive to the preliminary administration of a maximally active concentration of ATP (< 25% decrease). It appears likely that carbachol and ATP release Ca2+ ions from identical intracellular stores. Tetradecanoylphorbol acetate (TPA) strongly inhibited the responses of [Ca2+]i to both carbachol and ATP and enhanced the incorporation of [14C] choline into lipids in all five CHO cell lines investigated. On the other hand, the incorporation of [14C] choline into lipids was diminished by carbachol in the cell line expressing the M3 receptor subtype and unchanged in the other cell lines. This effect of carbachol was not dependent on the presence of extracellular Ca2+ ions and was not affected by TPA, which diminished the response of [Ca2+]i to muscarinic stimulation. It is suggested that it was due to muscarinic receptor-mediated activation of phospholipase D.

Broad bean wilt virus (BBWV), first isolated from broad bean (Vicia faba) in Australia (Stubbs, 1947), is the type member of the genus Fabavirus. Fabaviruses are aphid-transmitted viruses infecting a wide range of plants worldwide. Cooper (1994) noted that this genus includes a number of poorly characterized isolates with different common names and from many different plant species. We shall try to clarify this rather confused situation. BBWV isolates were divided by Uyemoto and Provvidenti (1974) into two serotypes (I and II). The type strain belongs to serotype I. Nasturtium ringspot virus (NRSV) (Smith, 1950), petunia ringspot virus (PeRSV) (Rubio-Huertos, 1959), parsley virus 3 (PV3) (Frowd and Tomlinson, 1970, 1972), and P.O. pea streak virus (Kim and Hagedorn, 1959) are isolates or strains closely related to BBWV serotype I. BBWV serotype II was first detected in Plantago lanceolata in the United States (Uyemoto and Provvidenti, 1974), and later found in several other plant species. A possible third serotype was reported from Australia (Shukla and Gough, 1983), and an isolate from Cynara scolymus was reported to differ from both recognized serotypes: Migliori (1993) named it artichoke French latent virus (AFLV). Lamium mild mosaic virus (LMMV) (Lovisolo, 1957) was isolated in the United Kingdom from Lamium album and a few other species of Labiatae (Lovisolo, 1957; Lisa et al., 1982), but has not been reported since. It is distantly serologically related to BBWV, but distinct from it, and is regarded as another fabavirus.

Nicotiana benthamiana was transformed with the nucleoprotein (N) gene of an Italian isolate of tomato spotted wilt virus (TSWV). Forty-five T1 primary transformant lines were analyzed for the expression of N protein and for resistance to TSWV and three other tospoviruses: impatiens necrotic spot virus (INSV), groundnut bud necrosis virus (GBNV), and groundnut ringspot virus (GRSV). Thirteen of these lines were further characterized. Resistance to all TSWV isolates tested was found in two lines. The expression of the transgene (N mRNA) was lower in these resistant lines than in any of the susceptible lines, and the transgene N protein was either absent or present below detectable levels. These lines were susceptible to the other tospoviruses tested, but they developed symptoms milder than controls when inoculated with GRSV. Some of the lines producing high levels of N protein showed delays (of 2-3 weeks) in symptom expression with at least one of the TSWV isolates tested and symptom delay or attenuation with INSV or GRSV (or both). From our results it appears that high expression of TSWV N protein retards, in some cases, disease development by TSWV and INSV. In contrast, the lack of detectable expression of the transgenic N protein, accompanied by limited production of N transcripts, conferred TSWV-specific resistance.

Lily mottle virus (LMoV), genus Potyvirus, an important virus of lily that also causes flower-breaking in tulip (1), is considered to have a natural host range restricted to the family Liliaceae. In 1996, escarole (Cichorium endivia L. var. latifolium LAM) plants growing in fields close to Torino, Italy, and showing mosaic and necrotic spots on outer leaves were infected by a potyvirus related to LMoV. The virus was identified by immunoelectron microscopy (IEM) done on experimentally infected Nicotiana benthamiana and Chenopodium quinoa. The virus isolated from escarole (LMoV-E) had an experimental host range similar to that of lily isolates of LMoV, although species within the Liliaceae were not tested. LMoV-E systemically infected all nine escarole cultivars and six of seven endive cultivars (C. endivia L. var. crispum LAM) but did not infect any of six lettuce and two chicory cultivars (C. intybus L. var. foliosum HEGI). Symptoms ranged from mild to severe mosaic and were generally more severe on escarole than on endive. Some of the same escarole, endive, and lettuce cultivars were inoculated with a typical LMoV isolate from lily (Le97/49, from A. F. L. M. Derks, the Netherlands), which induced mild systemic infections in only one escarole and one endive cultivar. Both cultivars were also susceptible to LMoV-E. LMoV-E was purified from N. benthamiana, and an antiserum was prepared. IEM decoration titer experiments were done with LMoV-E and four other LMoV isolates from Liliaceae and their homologous antisera. Heterologous titers ranging from identity to serological differentiation index values of 2 to 4 were obtained, confirming the identity of the escarole isolate as a LMoV strain and indicating considerable serological variability among LMoV isolates. In a field survey of endive and escarole crops in 1998, in the area where LMoV-E was first identified, the virus was again detected by enzyme-linked immunosorbent assay in 4 of 80 escarole plants tested. LMoV-E appears to be a LMoV strain particularly adapted to escarole. To our knowledge, this is the first report of LMoV identified in a naturally infected host outside monocotyledonous plants. Reference: (1) E. L. Dekker et al. J. Gen. Virol. 74:881, 1993.

SUMMARY A virus associated with a mosaic disease in melons in the Ourmia district in northern Iran was mechanically transmissible to a wide range of test plants but was not transmitted using aphids, the whitefly Trialeurodes vaporariorum or the mite Tetranychus urticae. Its rather stable particles somewhat resembled those of both geminiviruses and alfalfa mosaic virus; they were 18·5 nm in mean width, parallel-sided, and of several discrete lengths, 30 nm and 37 nm being the shortest and commonest lengths. Both ends of all particles were sharply triangular in profile. The particles contained linear single-stranded RNA of three sizes, estimated as 0·91,0·35 and 0·32, all × 106 molecular weight, and two coat proteins of 26·3 and 23·3 × 103 estimated molecular weight. The virus, named Ourmia melon virus, appears to be the first described representative of a new virus group.

White clover cryptic viruses (WCCV) 1, 2, and 3 were purified from apparently healthy plants with yields of about 200 μg of virus/kg of tissue. After isopycnic centrifugation in CsCl, WCCV 1 and 2 formed homogeneous bands at densities of 1.392 and 1.375 g/ml, respectively, whereas WCCV 3 was not recovered. Particles of WCCV 1 and 3 were 34 nm in diameter, without obvious morphological subunits, whereas WCCV 2 particles were 38 nm in diameter, with prominent morphological subunits. Antisera with titers up to 1:2048 were obtained against WCCV 1 and 2. Each of the three viruses contained two segments of linear dsRNA ( M r from 1.03 to 1.49 X 10 −6 . Virus-like particles, probably those of WCCV 1 and 2 were detected in low numbers in the cytoplasm of about 1/10 of the parenchyma cells of carrier plants examined by thin sectioning. The particles of WCCV 1 and 2 were not transmitted by grafting or mechanical inoculation, but were seed transmitted. Evidence is given that WCCV 1 and 2 multiply in their carrier plants, and are not viruses of associated parasitic or commensal organisms.

The nucleic acid of carnation cryptic virus has been identified as double-stranded RNA on the basis of (i) susceptibility to RNase in 0.1 × SSC (SSC = 0.15 M NaCl-0.015 M sodium citrate buffer, pH 7.0) and resistance to RNase in 2 × SSC and to DNase, (ii) reaction with an antiserum containing antibodies to dsRNA, and (iii) electron microscopic appearance in nondenaturing media similar to that of the dsRNA of maize rough dwarf virus. Upon electrophoresis in 5% polyacrylamide gels carnation cryptic virus RNA separated into three major segments plus a fourth minor component, and had a total molecular weight of about 4 × 106.

SUMMARY Small isometric virus-like particles were found in low concentration in apparently healthy carnations of the Mediterranean, miniature and Chinese type but not in eleven Sim cultivars tested. Most carnations containing these particles were from Italy but some were from France and the USA. The particles were not transmitted by grafting or by mechanical inoculation but were seed-transmitted to a large proportion of seedlings. Antisera to partially purified particles were obtained. The particles did not react with antisera to twenty-eight isometric plant viruses or virus-like particles but were serologically related to similar particles found in carnations in England, Holland and Israel. When negatively stained, the particles were isometric with a diameter of about 29 nm and a rounded rather than angular profile, but without clear substructure; some particles were penetrated by the stain. The particles remained intact in neutral sodium phosphotungstate. After isopycnic centrifugation in CsCl solution, preparations of particles formed a main band of mean density 1.377 g/ml and other fainter bands that varied in intensity and position in different preparations. In thin sections of carnations, no virus-like particles or cytological abnormalities were observed.

La microscopie électronique de particules purifiées d'un virus trouvé sur D. sanguinalis au Vanuatu a permis de déterminer son appartenance au groupe des geminivirus. Les préparations de particules virales contiennent une protéine de poids moléculaire égal à 27500 et des ADN monocaténaires circulaire et linéaire de 2350 nucléotides. Des particules géminées ont été trouvées dans les coupes fines de cellules infectées à l'intérieur de structures cristallines à la fois dans le noyau et dans le cytoplasme. Ce virus est sérologiquement apparenté au virus de la striure du maïs

Publisher Summary It is interesting that at this relatively late stage in virus research, new kinds of viruses continue to come to light. Cryptic viruses successfully evaded detection until recently because they induce no or, perhaps in some cases, very slight disease symptoms, are not transmissible in the ordinary way, and have particles present in such low concentrations as to escape casual discovery. The fact that cryptic viruses appear to cause no economically important disease has tended to give them low research priority. Nevertheless they are of interest as they contain double-stranded (ds) RNA, present only in another very different group of plant viruses, and appear unable to pass from carrier to non-carrier cells across a graft union. The latter is a decidedly non-virus like property. They are also of practical importance as they may be responsible for misleading results with methods for the detection of single-stranded RNA viruses based on the presence in plants of the double-stranded replicative forms.

Maize rough dwarf virus, a reolike virus, was purified from roots of maize by clarification with the fluorocarbon 1,1,2-trifluoro-1,2,2-trichloroethane (Freon 113) or with carbon tetrachloride followed by sucrose density gradient centrifugation. The product consisted mostly of complete particles, but some subviral particles were present. The virus was spherical, varying from 63 to 70 nm in diameter under different staining conditions; it had a double capsid and typical reovirus structure with, probably, 92 morphological units in the outer capsid. In addition, each particle possessed 12 projecting spikes (A spikes) each some 11 nm long, one at each 5-fold symmetry axis. Various physical or chemical treatments stripped off the A spikes and part of the outer capsid to give a spherical 50–57 nm subviral particle possessing 12 previously hidden spikes (B spikes) each about 8 nm long, coaxial with the A spikes. Each B spike was implanted on a differentiated part of the inner capsid here called a baseplate. Treatment of whole virus or subviral particles with aqueous neutral potassium phosphotungstate produced smooth subviral particles without B spikes, and longer treatment with phosphotungstate or brief treatment with n-butanol also removed the nucleic acid to give ghosts exhibiting internal structures. Detached B spikes were composed of 5 morphological subunits surrounding a central hole. Infectivity of preparations was tested by injecting them into adult females of the planthopper vector Laodelphax striatellus, which were later screened for production duction of virus symptoms on maize and barley. Crude and purified preparations containing the large virus particles were infectious; Freon-treated preparations possessed the highest infectivity. Subviral preparations made by chloroform treatment were not or very slightly infectious and smooth subviral particles made by treatment with phosphotungstate were not infectious.