Purification and immunological properties of an NAD(H) dependent glutamate dehydrogenase from soybean cells (Glycine max L.)

Studies were carried out on glutamate dehydrogenase (GDH, EC 1.4.1.2) isolated from the SB1 and SB3 soybean (Glyciene max L. cv. Mandarin) cell cultures. The NAD(H) dependent enzyme from SB1 and SB3 cells was purified to homogeneity, and that from the SB3 cells studied in detail. It was shown to be activated by calcium. The molecular weight of the native enzyme was found to be 263 000 ± 12 000. The molecular weight of the subunits was shown to be 41 000 ± 2000, which indicates that the enzyme has a hexameric structure. Anti-GDH antibodies were produced in rabbits, to GDH purified to homogeneity from both cell cultures. Each antibody preparation reacted with the purified enzyme produced from either cell culture. Antibodies to GDH from SB3 cells were utilized to study the apparent induction of GDH, which occurs when these cells are grown in a medium with ammonium ions as the sole nitrogen source. The increase in GDH activity was shown to be due to de-novo protein synthesis. The anti-SB3-GDH antibody preparation was also tested for cross reactivity with crude GDH preparations from a number of plant sources, and purified GDH from a number of other organisms. The antibody was shown to cross react with a number of the GDH preparations.     

Antibodies to pollen exines

A polyclonal antiserum and monoclonal antibodies have been prepared to purified pollen exines of Calocedrus decurrens Florin. The location of the antigen is in the exine, as shown by light-and electron-microscopic immunocytochemistry. The greatest reduction in antibody binding follows treatment of the exine with chemicals known to alter sporopollenin. These results provide evidence that sporopollenin is antigenic. Exines of ten species of gymnosperms and angiosperms also bound the polyclonal antiserum, indicating similarity of sporopollenin structure.     

Conservative and variable regions of homologous snake phospholipases A2 sequences: Prediction of the taxonspecific peptides structure

Homologous amino acid sequences of phospholipases A₂ (PLA₂) of snakes belonging to the families Elapidae, Viperidae, and Colubridae were considered in order to study the conservative and variable regions location. The PLA₂ sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair similarity matrix. Results of the intergroup comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown that the results of the comparison between two phylogenetic groups of snake PLA₂ did not much depend on the number of each group representatives and did not markedly change if one of the groups was represented by the single sequence. The knowledge of the number and location of conservative and variable regions and their dependence on the phylogenetic relations between compared taxa may be used to predict a synthetic peptide structure to obtain specific antibodies against PLA₂ of one of these taxons. Such prediction is possible if there is a specific region conservative for one taxon but variable for two of them.     

Cytofluorometric analysis of anti-lymphocyte antibodies in AIDS

Abstract Anti-lymphocyte antibodies (ALA) have been detected in the plasma of 53.8% of HIV-positive patients tested (CD4/CD8 ratios: mean 0.265; range 0.01 to 0.5) using analytical continuous-flow cytofluorometry. IgG from the AIDS plasma was seen to bind to normal PBL in 53.8% of cases (14/26). In double labelling experiments CD4 + lymphocytes, CD8 + lymphocytes, and B lymphocytes were all bound by the ALA, but monocytes were not bound. Pre-adsorption of the diluted AIDS plasma onto an excess of mouse spleen cells did not remove lymphocyte binding activity. No evidence was found for preferential binding to phytohaemagglutinin-stimulated lymphocytes.
ALA could not be detected in the plasma of normal subjects, patients with acute renal failure undergoing renal dialysis, or patients with high levels of circulating immune complexes.     

A rat oocyte differentiation antigen (OA-1) detected by a monoclonal antibody

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti-OA-1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four-cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.     

Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery

The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth. (c) 1993 John Wiley & Sons, Inc.     

Multiple xenobiotic-inducible P450s are involved in alkoxycoumarin and alkoxyresorufin metabolism in higher plants

O-Dealkylation of two series of fluorescent 7-alkoxy-coumarins and 7-alkoxyphenoxazones by plant cytochrome P450s was investigated in Helianthus tuberosus tuber tissues treated with prototype P450 inducers, environmental pollutants or agrochemicals. Methoxy-, ethoxy-, propoxy-and butoxycoumarins and methoxy- and ethoxyresorufins were metabolized by fplant microsomes. Dealkylation of pentoxy- and benzyloxyresorufins was not detected. All dealkylating activities were enhanced by aging plant tissues in the presence of xenobiotics, in some cases up to 20-fold relative to the activities detected in control tissues. Increases in total P450 in the same tissues never exceeded 3-fold. The isozymes induced by prototype P450 inducers clearly differed from those in mammalian liver. That multiple P450s with overlapping substrate specificities were involved in the metabolism of both alkoxycoumarins and alkoxyresorufins was demonstrated by (1) the differential induction of the activities in response to exposure to xenobiotics, (2) the differential inhibition of the activities by clotrimazole, paclobutrazole and tetcyclacis in aminopyrine and benzo(a)pyrene-treated tissues, and(3) the selective inhibition observed with antibodies raised against purified ethoxycoumarin deethylase fractions. Our results suggest that the measurement of the dealkylation of such fluorescent substrates in plants might be useful to monitor environmental pollution.     

Semen alloantigens and lymphocytotoxic antibodies in AIDS and ICL

More than 90% of people with AIDS develop circulating immune complexes (CICs) and lymphocytotoxic antibodies (LCTAs). Animals infected with HIV, however, never display CICs or LCTAs, and remain healthy. Similarly, HIV-infected people who do not develop CICs or LCTAs also do not progress to AIDS. The appearance of CICs and LCTAs is, however, highly prognostic for AIDS and death. Since HIV infection does not,per se, lead to the development of CICs and LCTAs, other causes are likely. One such cause, for which both epidemiologic and experimental evidence exists, is semen. Semen components include sperm, seminal fluid, lymphocytes, and sometimes infectious agents, including HIV, mycoplasmas, and herpes and hepatitis viruses, all of which independently cause immune suppression. Extensive evidence demonstrates sperm (and various viruses) contains many proteins mimicking the CD4 protein of T-helper cells, while HIV, mycoplasmas, and seminal fluid mimic class II MHC proteins of other lymphocytes. We identify a large number of protein sequences that display such mimicry using computer homology searching, and demonstrate experimentally that sperm antibodies specifically precipitate antibodies against class II MHC mimics such as mycoplasmas, which in turn precipitate antibodies to lymphocyte antigens. These data prove that immunologic exposure to sperm and lymphocytes (as may occur in receptive anal intercourse, needle sharing, or blood transfusions) is theoretically capable of initiating lymphocytotoxic autoimmunity. Such autoimmunity may play a significant role in the pathogenesis of AIDS, and will need to be addressed clinically in high risk individuals regardless of HIV status and regardless of the success of anti-HIV prophylaxis and treatment.     

Purification and immunological characterization of superoxide dismutase of the onion maggot,Delia antiqua

Cytosolic superoxide dismutase (SOD) of the onion maggot, Delia antiqua, was purified to apparent homogeneity by ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and gel filtration chromatographies. Native molecular mass was estimated as 32,000 daltons. SDS-PAGE revealed only one subunit of 16,000 daltons, indicating that SOD is a homodimer. Isoelectric focusing revealed 3 charge isomers of pls 5.3, 5.5, and 5.7. The specific activity of purified SOD was 4,250 U/mg protein. A monoclonal antibody (MAb, aSOD2B7) raised against Delia SOD recognized only SOD of the same genus, but another MAb (aSOD1H11) recognized SOD of Drosophila melanogaster as well. © 1995 Wiley-Liss, Inc.