Monoclonal antibodies against transferrin. Precipitating mixtures and lack of inter-species cross-reactivity

Five stable hybridoma lines were prepared using the myeloma cell line P3-X63-Ag.653 and spleen cells of mice hyperimmunized by pig transferrin. All hybridomas grew well in mouse peritoneal cavity and produced antibodies of the IgG₁ subclass. Antibody preparations obtained from ascitic fluids tested for their capacity of antigen precipitation. No precipitation was obtained with single antibodies and with pairs of antibodies. Three out of 10 possible triads gave clear and sharp precipitation zones and rings in immunodiffusion tests performed in agar gel. All 5 antibodies were shown by quantitative enzyme-immunoassay to be specific for pig transferrin: no cross-reaction was obtained with mouse, human, horse and sheep transferrins.     

Induction of NO synthesis in macrophages by Newcastle disease virus is associated with activation of nuclear factor-{kappa}B

Newcastle disease virus (NDV) has received much attention recentlybecause of its non-specific immune stimulating potential andits various anti-tumor activities. Here we describe that NDVinduces synthesis of NO and causes an activation of nuclearfactor-kB (NF-kB) In murine macrophages. These reactions werepart of an activation process which included also stimulationof adenosine deaminase and inhibition of 5'-nucleotidase. NDV-mediatedNO synthesis and NF-kB activation were blocked by an antioxidant(butylated hydroxyanisole), by an inhibitor of protein tyrosinekinase (genistein) and of protein kinase A (H-89), but not byan inhibitor of protein kinase C (staurosporin). These datasuggest that signalling requirements of NF-kB activation andNO production in NDV-treated macrophages are similar.     

Mutational pattern of influenza B viruses adapted to high growth replication in embryonated eggs

Improved replication of influenza viruses in embryonated chicken eggs (CE) permits increased vaccine production and availability. We investigated the growth properties of influenza B viruses in relation to specific mutations occurring after serial passage in CE. In serial passage experiments yielding high growth variants of B/Victoria/504/2000, mutations predicted to alter amino acid (AA) composition occurred only near the receptor-binding pocket of the hemagglutinins (HA) and in no other genes. Two B/Victoria/504/2000 high growth variants had the same AA substitutions in HA (R162M and D196Y), but the higher yield variant had a third substitution (G141E), which also altered antigenic characteristics. In a serial passage experiment yielding a high growth variant of B/Hong Kong/330/2001, mutations predicted to alter AA composition occurred only in PB2 and NP in domains predicted to relate to RNP formation and function. Our results indicate that adaptation of influenza B viruses to high-yield replication by serial passage in CE requires few mutations either in internal or external genes. Specific modifications of genes or a combination of genes could be used to optimize or create influenza B viruses for specific growth substrates.     

Temperature compensation of neuromuscular modulation in aplysia

Physiological systems that must operate over a range of temperatures often incorporate temperature-compensatory mechanisms to maintain their output within a relatively narrow, functional range of values. We analyze here an example in the accessory radula closer (ARC) neuromuscular system, a representative part of the feeding neuromusculature of the sea slug Aplysia. The ARC muscle's two motor neurons, B15 and B16, release, in addition to ACh that contracts the muscle, modulatory peptide cotransmitters that, through a complex network of effects in the muscle, shape the ACh-induced contractions. It is believed that this modulation is critical in optimizing the performance of the muscle for successful, efficient feeding behavior. However, previous work has shown that the release of the modulatory peptides from the motor neurons decreases dramatically with increasing temperature. From 15 to 25 degrees C, for example, release decreases 20-fold. Yet Aplysia live and feed successfully not only at 15 degrees C, but at 25 degrees C and probably at higher temperatures. Here, working with reduced B15/B16-ARC preparations in vitro as well as a mathematical model of the system, we have found a resolution of this apparent paradox. Although modulator release decreases 20-fold when the temperature is raised from 15 to 25 degrees C, the observed modulation of contraction shape does not decrease at all. Two mechanisms are responsible. First, further downstream within the modulatory network, the modulatory effects themselves-experimentally dissected by exogenous modulator application-have temperature dependencies opposite to that of modulator release, increasing with temperature. Second, the saturating curvature of the dose-response relations within the network diminishes the downstream impact of the decrease of modulator release. Thus two quite distinct mechanisms, one depending on the characteristics of the individual components of the network and the other emerging from the network's structure, combine to compensate for temperature changes to maintain the output of this physiological system.     

Observation of a nematic phase displayed by a polysiloxane with trinitrofluorenones as side groups

The synthesis and the results of the structural study of two copolysiloxanes with laterally fixed trinitrofluorenone (TNF) units is reported. The two copolysiloxanes having 2,4 ( 1a ) and 5,3 ( 1b ) dimethylsiloxane comonomer units per TNF side group differ significantly in their phase behaviour as evident from optical microscopy, differential scanning calorimetry and X-ray scattering: 1b shows a nematic mesophase whereas 1a is an amorphous material. The different phase behaviour is discussed in terms of microphase separation between the siloxane backbone and TNF side groups.     

Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel

Comprehensive genome scans involving many thousands of SNP assays will require significant amounts of genomic DNA from each sample. We report two successful methods for amplifying whole-genomic DNA prior to SNP analysis, multiple displacement amplification, and OmniPlex technology. We determined the coverage of amplification by analyzing a SNP linkage marker set that contained 2320 SNP markers spread across the genome at an average distance of 2.5 cM. We observed a concordance of >99.8% in genotyping results from genomic DNA and amplified DNA, strongly indicating the ability of both methods used to amplify genomic DNA in a highly representative manner. Furthermore, we were able to achieve a SNP call rate of >98% in both genomic and amplified DNA. The combination of whole-genome amplification and comprehensive SNP linkage analysis offers new opportunities for genetic analysis in clinical trials, disease association studies, and archiving of DNA samples.     

Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.     

Structural variations of DNA + Mn2+ complexes during helix-coil transition

The helix-coil transition in Phage T2 DNA in the presence of 6,4 · 10^?3 mol/l Mn²⁺ is studied using light scattering and UV spectroscopy. The transition range is about 0,5°C. Near the temperature of the end of melting T_f the molecular weight M_w and the radius of gyration R_z of the complex are observed to decrease to about one half. At a temperature 0,1–0,25°C higher than T_f, M_w and R_z pass through a minimum, which implies that aggregation is preceded by unwinding of DNA strands. Thus, rise in temperature rather than Mn²⁺ -induced aggregation causes DNA + Mn²⁺ melting.