Transcriptional landscapes of floral meristems in barley.

Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

Synergistic roles of neuregulin-1 and insulin-like growth factor-I in activation of the phosphatidylinositol 3-kinase pathway and cardiac chamber morphogenesis.

Cardiac chamber morphogenesis requires the coordinated growth of both cardiac muscle and endocardial cell lineages. Paracrine growth factors may modulate the coordinated cellular specification and differentiation during cardiac chamber morphogenesis, as suggested by the essential role of endothelial-derived growth factors, neuregulin-1, and insulin-like growth factor-I. Using the whole mouse embryo culture system for delivery of diffusible factors into the cardiac chamber, neuregulin-1 was shown to promote trabeculation of the ventricular wall. Another factor, insulin-like growth factor-I, had no apparent effect by itself. Combined treatment with neuregulin-1 and insulin-like growth factor-I strongly induced DNA synthesis of cardiomyocytes and expansion of both the ventricular compact zone and the atrioventricular cushions leading to chamber growth and maturation. In cultured cardiomyocytes, combined neuregulin-1 and insulin-like growth factor-I also had a synergistic effect to promote DNA synthesis and cellular growth, which were prevented by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Adenoviral delivery of dominant negative Rac1, which acts downstream of phosphatidylinositol 3-kinase, blocked the effect of combined neuregulin-1/insulin-like growth factor-I treatment. These studies support the concept that the interaction of neuregulin-1 and insulin-like growth factor-I pathways plays an important role in coordinating cardiac chamber morphogenesis and may occur through convergent activation of phosphatidylinositol 3-kinase.

Detection of antibodies to bovine viral diarrhoea virus (BVDV) and characterization of genomes of BVDV from Brazil.

An ELISA for the detection of antibodies to bovine viral diarrhea virus (BVDV) was developed based on antigens derived from a genotype I BVDV strain isolated in Switzerland. Using monoclonal antibodies we showed that this antigen contained the conserved non-structural protein NS3 whereas it essentially lacked the more strain-specific E2 surface glycoprotein. This ELISA has a sensitivity of 97.5% and a specificity of 99.2% as compared to the serum neutralization test (SNT). Preliminary experiments showed that this ELISA reliably detects antibodies to BVDV strains circulating in Brazil. Serum samples obtained from 430 adult cattle on 19 farms of the State of Rio Grande do Sul (Brazil) and one farm from Corrientes (Argentina) were tested for antibodies by means of this ELISA. We found antibodies in 56% +/- 15.1% of the cattle sera tested, which indicates that, in Brazil, the prevalence of infection with BVDV is similar to that found in Europe and the USA. Our sequence analysis of two BVDV isolates showed that BVDV of both genotypes I and II circulate in Brazil.

Field and vaccine strains of fowlpox virus carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus.

For baculoviruses and herpesviruses, integration of transposons or retroviruses into the virus genome has been documented. We report here that field and vaccine strains of fowlpox virus (FPV) carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus (REV). Using PCR and hybridization analysis we observed that vaccine and field strains of FPV carry REV sequences integrated into a previously uncharacterized region of the right 1/3 of the FPV genome. Long-range PCR, hybridization, and nucleotide sequence determination demonstrated that one vaccine strain (FPV S) and recently isolated field strains carry a near-full-length REV provirus. For another vaccine strain (FPV M) a rearranged remnant of the LTR was found at the same insertion site. By Western blotting and reverse transcriptase assays we were unable to demonstrate free REV in supernatants of FPV S cultures. The near-full-length REV provirus integrated into the FPV genome is infectious since FPV S DNA gave rise to REV upon transfection into chicken embryo fibroblasts. Upon infection of chickens with FPV S, all chickens developed high-titered antibodies to REV, and REV was isolated from the blood of half of the inoculated chickens. Our observations add to the list of targets for retrovirus integration into DNA virus genomes. The integration of a near-full-length, and apparently infectious, REV provirus into FPV provides additional transmission routes for the retrovirus by way of the infectious cycle of FPV, including the possibility of mechanical transmission by biting insects since FPV is believed to be transmitted by this route. For large DNA viruses, including the poxviruses, retrovirus integration with attendant possibilities of gene transduction may be an important mechanism for virus evolution, including the acquisition of cellular genes with the potential to modify virus virulence and pathogenicity.

Vaccinia virus-expressed bovine ephemeral fever virus G but not G(NS) glycoprotein induces neutralizing antibodies and protects against experimental infection.

Two related glycoproteins (G and G(NS)) encoded in the bovine ephemeral fever virus (BEFV) genome were expressed from recombinant vaccinia viruses (rVV). Both proteins were detected in lysates of rVV-infected cells by labelling with D-[6-3H]glucosamine or by immuno-blotting. The recombinant G protein (mol. mass 79 kDa) appeared slightly smaller than the native G protein but reacted with monoclonal antibodies directed against all defined neutralizing antigenic sites (G1, G2, G3a, G3b and G4). The recombinant G(NS) protein (mol. mass 90kDa) was identical in size to the native G(NS) protein and failed to react by immuno-fluorescence with anti-G protein monoclonal or poly-clonal antibodies. Antisera raised in rabbits against rVV-G or rVV-G(NS) both reacted strongly by immuno-fluorescence and immuno-electron microscopy with BEFV-infected cells. The G protein was localized intracellularly in the endoplasmic reticulum/Golgi complex and at the cell surface associated with budding and mature virus particles. The G(NS) protein also localized intracellularly in the endoplasmic reticulum/Golgi complex; however, at the cell surface it was associated with amorphous structures and not with budding or mature virions. Rabbits vaccinated with rVV-G developed high levels of antibodies which neutralized BEFV grown in either mammalian or insect cells. Cattle vaccinated with rVV-G also produced neutralizing antibodies and were protected against experimental BEFV infection. In contrast, rVV-G(NS) vaccinated rabbits and cattle failed to produce neutralizing antibodies and, after challenge, BEFV was isolated from two-thirds of the vaccinated cattle.

N-cadherin in adult rat cardiomyocytes in culture. I. Functional role of N-cadherin and impairment of cell-cell contact by a truncated N-cadherin mutant.

N-cadherin is a transmembrane Ca(2+)-dependent glycoprotein that is part of adherens junctions. It functions with the cell adhesion N-terminal extracellular domain as a site of homophilic cell-cell contacts. The intracellular C-terminal domain provides via a catenin complex the interaction with the cytoskeleton. Ectopic expression of chicken N-cadherin in adult rat cardiomyocytes (ARC) in culture was obtained after microinjection into non-dividing cardiomyocytes; it was demonstrated that the exogenous protein colocalized with the endogenous N-cadherin at the plasma membrane of the cell and formed contact sites. A dominant negative chicken N-cadherin mutant was constructed by a large deletion of the extracellular domain. This mutant was expressed and inhibited the function of the endogenous rat N-cadherin probably by competing for the catenin complex binding domain, which is essential for the formation of a stable cell-cell contact of ARC. The injected cells lost contact with neighbouring cells and retracted; the connexons of the gap junctions were pulled out as well. This could be avoided by another N-cadherin mutation, which, in addition to the N-terminal truncation, contained a deletion of the catenin binding domain. In the case of the truncated N-cadherin at the N terminus, the sarcomeric structure of the myofibrils of ARC was also affected. Myofibrils were the most vulnerable cytoskeletal structures affected by the overexpressed dominant negative N-cadherin mutation. Similar behaviour was shown when cardiomyocytes separated following Ca2+ depletion and when new cell-cell contacts were formed after Ca2+ replenishment. N-cadherin is thought to be the essential component for establishing new cell-cell contacts which eventually led to a new formation of intercalated disc-like structures in the cardiac cell culture.

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes.

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The 'redifferentiation model' of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

Genetic heterogeneity within the coding regions of E2 and NS3 in strains of bovine viral diarrhea virus.

We have amplified and sequenced parts of the genomes of eleven laboratory strains of bovine viral diarrhea (BVD) virus originating from North America, New Zealand and Europe. The cumulative nucleotide (nt) sequence heterogeneity of the amplified fragments located in the analysed region of the gene encoding the nonstructural protein NS3 (P80) was 24% as compared to 47% for E2 (Gp53). The nt substitutions in the E2 region resulted in replacements in 42% of amino acid (aa) positions, while the deduced aa sequence of all BVD virus strains remained identical in NS3 and differed from the corresponding region of classical swine fever viruses. This makes possible the differentiation of bovine and porcine pestiviruses. It is suggested that genetic heterogeneity results from passage in transiently infected animals.

Retrovirus-like particles produced by vaccinia viruses expressing gag-pro-pol region genes of bovine leukaemia virus.

Processing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rVVs) expressing regions of the bovine leukaemia virus genome. Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rVV carrying the gag and truncated protease (pro) gene. Processing of Pr44 was observed after infection of cells with a rVV carrying the gag and pro gene or a rVV expressing the gag, pro and polymerase (pol) gene. Reverse transcriptase activity was detected only in association with particles produced by gag-, pro- and pol-expressing recombinants. Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane. Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rVVs expressing gag and pro or gag, pro and pol formed mature particles.

[Bovine virus diarrhea/mucosal disease: a review]. / Bovine Virusdiarrhoe/Mucosal Disease: eine Ubersicht.

Infections with the Bovine Viral Diarrhea/Mucosal Disease Virus (BVDV) are widespread and cause a variety of diseases including reproductive disorders, abortion and malformation, pneumoenteritis, thrombocytopenia and mucosal disease. Together with the closely related border disease virus of sheep (BDV) and European Swine fever virus (CSFV), also referred to as Hog Cholera virus, BVDV is now classified in the genus pestivirus of the Flaviviridae family. The BVDV exists in two biotypes, noncytopathic and cytopathic, the latter differing in structural proteins from the noncytopathic biotypes. In virus-free animals infection is transient and mostly subclinical or mild but may also lead to an array of diverse symptoms such as pneumoenteritis (often in combination with other microorganisms). Infection of the developing fetus early in gestation with a noncytopathic biotype of BVDV may result in persistent infection and birth of apparently healthy calves. Such calves may later in their lives develop Mucosal Disease, a lethal course of infection associated with a mutation to the cytopathic biotype or superinfection with a cytopathic BVDV antigenically similar to the non-cytopathic virus already present in these animals. Diagnosis of infections with BVDV is based on the clinical symptoms and demonstration of virus. Paired serum samples allow the detection of seroconversion in acute infections while persistently infected animals are immunotolerant and generally lack antiviral antibody. Although generally found in their respective host species, pestiviruses of cattle, sheep and pigs are capable of crossing the species barrier into the other species. The existence of pestiviruses in wild ruminants and boars may complicate control strategies that are aimed at removing virus carriers and the control of animal movements.

Adult rat cardiomyocytes in culture A model system to study the plasticity of the differentiated cardiac phenotype at the molecular and cellular levels.

Adult rat cardiomyocytes (ARCs) in long-term culture, which show a distinct adaptive flexibility, are presented as a system to study cardiac cell hypertrophy in vitro. In the first 1-2 weeks after isolation, ARCs undergo a process of de- and redifferentiation during which the cell morphology is remodeled and the myofibrillar apparatus is restructured, accompanied by a cell enlargement. The growing cells spread and eventually establish new cell-cell contacts, which display newly formed intercalated discs; synchronous cell beating is resumed in the resulting tissuelike sheet. During myofibrillogenesis, the early fetal program of gene expression is reactivated for several genes, as is observed during hemodynamic overload hypertrophy. The cells resume hormonal activity and express atrial natriuretic factor (ANF); the expression pattern of ANF is also reminiscent of that seen in hypertrophy. In cells grown in a medium conditioned by 12-day ARCs, though, myofibrillogenesis is accelerated and accompanied by a downregulation of ANF. In a creatine-deficient medium, on the other hand, the ARCs display giant mitochondria with paracrystalline inclusions imitating a situation found, for example, in mitochondrial myopathies.

Cloning and expression in mammalian cells of porcine tumor necrosis factor alpha: examination of biological properties.

We have cloned a full length complementary DNA (cDNA) of the porcine tumor necrosis factor alpha (pTNF-alpha) gene and expressed it in porcine and murine cells. Total RNA obtained from lipopolysaccharide (LPS) stimulated porcine peripheral blood mononuclear cells was reverse transcribed with a specific antisense pTNF-alpha primer to generate a single stranded cDNA which was subsequently amplified by the polymerase chain reaction utilizing an additional pTNF-alpha specific sense primer. The resulting double stranded cDNA was introduced into the pBMGNeo expression vector and transfected by electroporation in porcine (PK(15)) and murine (L929) cell lines. TNF-alpha bioactivity was detected in the supernatant of the transfected cells using a standard L929 bioassay or a PK(15) bioassay. The activity was zinc inducible as expected for a gene controlled by a metallothionein promoter. The bioactivity was not lowered by an anti-mouse TNF-alpha antiserum neutralizing murine, but not human TNF-alpha and a broad immunoreactive band of 17-19 kD was detected using an anti-mouse TNF-alpha serum suitable for immunoblotting. This newly developed tool will allow us to investigate the role of TNF-alpha in pathogenesis of viral infections and gram-negative sepsis.

The utilization of learner-centered development in a proprietary security setting.

The author discusses the present role of proprietary security officers, their learning needs, and ways to train them through the application of learner-centered development.

Sequence and expression of a wheat gene that encodes a novel protein associated with pathogen defense.

Wheat (Triticum aestivum) exhibits local acquired resistance to the powdery mildew pathogen Erysiphe graminis f. sp. tritici. The resistant state can be induced by a preinoculation with the nonhost pathogen E. g.f. sp. hordei, the barley powdery mildew, and is accompanied by the activation of putative defense genes. Here, we report the sequence of a pathogen-induced gene, WIR1a, and a corresponding cDNA, WIR1, that encode novel defense-related proteins of 88 and 85 amino acids, respectively. Analysis of the primary structure of these proteins predicts them to be integral membrane proteins with extracytoplasmic C-terminal domains rich in proline and glycine, through which the proteins possibly interact with the cell wall.

Structure of an mdr-like gene from Arabidopsis thaliana. Evolutionary implications.

Multidrug resistance of mammalian tumor cells is caused by the enhanced expression of P-glycoproteins. These proteins are encoded by mdr genes and mediate the energy-dependent efflux of a variety of lipophilic drugs from cells. To test whether in plants mdr-like genes might be involved in certain cases of cross-resistance to different herbicides, we have cloned and characterized a gene from Arabidopsis thaliana, atpgp1, encoding a putative P-glycoprotein homologue. Like the mammalian P-glycoproteins, with which it shares extensive sequence homology and a similar organization in structural domains, this protein is internally duplicated. Seven of the nine introns in the atpgp1 gene match introns in the mammalian mdr genes to within a few nucleotides, and the positions of these suggest that P-glycoprotein genes evolved by duplication and subsequent fusion of an intron-containing primordial gene prior to the evolutionary separation of plants and mammals. The atpgp1 gene gives rise to transcripts present in all plant parts but particularly abundant in inflorescence axes.

Cloning and sequencing of cDNAs encoding a pathogen-induced putative peroxidase of wheat (Triticum aestivum L.).

We report here the complete amino acid sequence of a pathogen-induced putative peroxidase from wheat (Triticum aestivum L.) as deduced from cDNA clones representing mRNA from leaves infected with the powdery mildew fungus Erysiphe graminis. The protein consists of 312 amino acids, of which the first 22 form a putative signal sequence, and has a calculated pI of 5.7. Sequence comparison revealed that the putative wheat peroxidase is most similar to the turnip (Brassica rapa) peroxidase, with which it shares 57% identical and 13% conserved amino acids.

[Veterinary medicine and molecular biology: possibilities and limits of virus detection]. / Veterinärmedizin und Molekularbiologie: Möglichkeiten und Grenzen der Virusdetektion.

In the last years molecular biology has gained more and more influence in veterinary medicine, e.g. in animal breeding. In addition, methods of molecular biology may open new areas in the diagnosis of viral diseases, because they are fast and very sensitive. The following article displays an overview of molecular biological techniques that are currently in use for virus detection. In addition, the limits of these methods are discussed. In particular, the polymerase chain reaction (PCR) is described in some detail. Since this method has a considerable impact on the progress in molecular biology, it may as well be of importance for the detection of viruses. The potential of PCR in virus detection is illustrated using sheep and goat lentivirus and Bovine Viral Diarrhea/Mucosal Disease virus.

Detection of bovine viral diarrhea (BVD) virus using the polymerase chain reaction.

The approach of reverse transcription (RT) followed by the polymerase chain reaction (PCR) was used to amplify three different fragments of the bovine viral diarrhea virus (BVDV) genome. Two sets of primers framed two different regions within the gene coding for protein p80, the third set of primers was selected to amplify cDNA within the envelope glycoprotein (gp53) region. All three sequences could be detected in the homologous strain (NADL), whereas only some of the fragments could be amplified in heterologous strains of BVDV. RNA extracted from infected cells as well as RNA extracted from viral particles could be detected using RT-PCR. The detection limit was 10(-1)-10(-2) TCID50 in ethidium bromide stained gels and could be further enhanced to 10(-2)-10(-4) TCID50 by hybridization after Southern transfer. The speed and the sensitivity of this method might be of relevance for diagnostic purposes as well as for studies on epidemiology and pathogenesis of infection with BVD virus.