Regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase from Sorghum: An immunological study using specific anti-phosphorylation site-antibodies

A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C₄-phospho enolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C₄ PEPCs mutated in the N-terminal phosphorylation domain and a C₃-like PEPC isozyme from Sorghum behaved similarly to their C₄ counterpart with respect to these phosphorylation-site antibodies.Abbreviations PEPC phospho enolpyruvate carboxylase - PKA catalytic subunit of the cAMP-dependent protein kinase - KLH Keyhole Limpet Haemocyanin - IgG immunoglobulin G - PEP phospho enolpyruvate - SDS-PAGE sodium dodecyl sulfate, polyacrylamide gel electrophoresis - MDH malate deshydrogenase     

Development of a Monoclonal Antibody for the Detection and Quantification of Predation on Slugs within the Arion hortensis agg. (Mollusca: Pulmonata)

The Arion hortensis agg. (Mollusca: Pulmonata) are major slug pests of agriculture and horticulture throughout much of the temperate World. A monoclonal antibody was created that could specifically identify and quantify the remains of these slugs within the carabid predator Pterostichus melanarius Illiger, a polyphagous species found in arable crops in Europe and North America. The antibody was shown to be specific for the two species of pest slug within this species aggregate, Arion distinctus Mabille and A. hortensis Férussac, and did not cross-react with any other invertebrates tested, including other slugs in the same genus. It proved to be highly sensitive in an enzyme-linked immunosorbent assay. The mean detection period for the remains of A. distinctus in predator foregut samples was 3.28 days at 16°C. However, A. distinctus remains could be identified in male carabids for 30% longer than in females, emphasizing the importance of separate analyses. The value of the antigen half-life as a measure of the detection period was questioned because analysis of covariance showed that the regression slopes for antigen decay were not significantly different between the two sexes, giving very similar half-lives (approx 18.5 h for females and 19 h for males). Calculated quantities of soluble slug proteins in predator foreguts declined more rapidly over time than concentrations, allowing separate assessment of the importance of these two variables on detection periods. It was concluded that a valuable new antibody had been created and characterized, which can be used for the analysis of predation in the field.     

Antibodies to Aeromonas salmonicida Lipopolysaccharide and cell wall antigens in rabbit and salmon immune sera

Atlantic salmon (Salmo salar L.) immunised with A-layer positive or A-layer negative strains ofAeromonas salmonicida did not produce antibodies reactive with proteinase K-digested LPS in the low molecular weight area corresponding to the core-region of LPS. The salmon produced antibody titres as high as those produced by rabbit when assayed against whole bacteria or LPS in ELISA. The salmon antibodies against the A-layer positive strain of A. salmonicida lysed rabbit erythrocytes sensitised with LPS from the A-layer positive strain of A. salmonicida. This was in contrast to the non-haemolytic activity of the salmon antibodies against the A-layer negative strain, indicating differences in epitopes between the two strains.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

丙型肝炎病毒免疫电镜的初步研究

采用组织匀浆免疫沉淀后负染、免疫组化块染后包埋、原位包埋等免疫电镜技术,研究丙型肝炎病毒（ＨＣＶ）。组织匀浆、免疫沉定、负染后在电镜下观察到与ＨＣＶ相关的类病毒颗粒,形态与披膜病毒相似,大小多在５５～６５ｎｍ,圆形,有包膜,边缘略有突起或比较平滑,有胶体金结合在此种颗粒上及其周围。无关单抗阴性对照无类似颗粒及胶体金。免疫酶染电镜下还见到成堆可疑颗粒。此外,ＨＣＶ－Ｅ区抗原染色后原位包埋,尚发现胶体金大多结合于大小５０ｎｍ左右圆形结构的内部,表明Ｅ区单抗针对的特异性抗原位点位于这种结构的内侧。     

Subcellular Localisation of 14-3-3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.     

Serological characterisation of citrus tristeza virus isolates from Israel

Seven monoclonal antibodies (MAbs) showing homologous reactions with the VT strain of citrus tristeza virus (CTV) were tested against 21 CTV strains or isolates, representing the range of biological diversity and the geographical distribution of CTV in Israel. All the CTV strains gave positive reactions in ELISA with polyclonal antibodies and with one MAb (#25#2). Two MAbs; #13#21 and #3/7 reacted with 19 and 12 out of the 21 CTV strains, respectively. Seventeen CTV strains, including all those previously assigned by sequencing their coat protein gene (CPG) to the CPG-VT group (Mawassi, Gafny & Bar-Joseph, 1993), reacted with five or more MAbs. Four CTV strains of minor epidemiological importance, including two members of the CPG-MT group, did not react with five or more of the MAbs. These results indicated the existence of two serogroups of Israeli CTV strains that can be differentiated by MAbs and which closely correlate with and extend the previous CPG grouping. The extensive biological variation within each CPG group confirms recent analyses suggesting that the CTV pathogenic traits are not necessarily associated with a sequence or antigenic variation of the CTV-CPG.     

Hyperosmotic hbridoma cell cultures: Increased monoclonal antibody production with addition of glycine betaine

When mouse hybridoma cells were grown in culture media which were made hyperosmotic through the addition of NaCl or sucrose, the specific rate of antibody production increased with medium osmolality, reaching approx. 1.9 times the level obtained at physiological osmolality. However, due to a simultaneous reduction of the maximal cell density in the hyperosmotic media, the effect of the increased production rate did not give significant increases in the maximum antibody titer obtained in the cultures. When the osmoprotective compound, glycine betaine, was included in the NaCl- or sucrose-stressed cultures, the specific antibody production rate wasincreased up to 2.6-fold and maximum antibody titer up to twofold over that obtained in the control culture (physiological osmolality). A similar pattern of response was observed when other osmoprotective compounds (sarcosine, proline, glycine) were added to NaCl-stressed hybridoma cell cultures. For the present experiments, the results suggest that medium osmolality, rather than growth rate, will determine the specific antibody production rate by hybridoma cell line 6H11 growing in hyperosmotic culture media. (c) 1994 John Wiley & Sons, Inc.     

The Super-Spinner: A low cost animal cell culture bioreactor for the CO2 incubator

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran^® flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO₂ conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTR_max) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTR_max was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.