Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of cluster picking, and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class 11 determinants.Abbreviations used in this paper HLA human major histocompatibility - EBV Epstein-Barr virus - B-LCL Blymphoblastoid cell line - NA not absorbed - PBS phosphate-buffered saline - SPA Sepharose protein A - NRS normal rabbit serum     

Structural and functional variability among DQ β alleles of DR2 subtypes

Homozygous lymphoblastoid cell lines representing various Dw subtypes of DR2 were examined for polymorphism at the DQ locus by molecular and cellular techniques. The subtypes studied included Dw2, Dw12, and a group heterogenous by cellular typing that we shall refer to as non-Dw2/non-Dw12. Restriction fragment length polymorphism analysis of cell lines representing these subtypes revealed DQ -specific patterns consistent with cellular typing. Two-dimensional gel electrophoresis of DQ molecules from representative cell lines revealed a structural polymorphism of DQ among the three subtypes. The DQ chain migrated to a position that was unique to each subtype and was consistent among various representative cell lines of each subtype. Nucleotide sequence analysis of cDNA clones of DQ from Dw2, Dw12, and non-Dw2/non-Dw12 lines confirmed that the variability resided at the genetic level. Variability was found in the form of numerous scattered nucleotide substitutions throughout the first domain of these alleles. The DQ gene of the non-Dw2/non-Dw12 cell line AZH was further found to be almost identical with the DQ gene of a DR1 line (Bell et al. 1985b), implicating a common evolutionary origin of these alleles. The only difference between these two sequences was due to an apparent gene conversion event at amino acid 57. T-cell cloning experiments resulted in the derivation of Epstein-Barr virus-specific, DQw1-restricted clones that proliferated against only those cell lines that exhibited the DQ gene common to AZH and the DR1 cell line. Thus, the polymorphism among DQ alleles within DR2 results in subtype-specific restriction.     

Potential for transduction of plasmids in a natural freshwater environment: effect of plasmid donor concentration and a natural microbial community on transduction in Pseudomonas aeruginosa

Transduction of Pseudomonas aeruginosa plasmid Rms149 by the generalized transducing bacteriophage phi DS1 was shown to occur during a 9-day incubation of environmental test chambers in a freshwater reservoir. Plasmid DNA was transferred from a nonlysogenic plasmid donor to a phi DS1 lysogen of P. aeruginosa that served both as the source of the transducing phage and as the recipient of the plasmid DNA. When the concentration of donors introduced into the chambers was varied while the recipient concentration in each chamber was at a level equivalent to natural concentrations of P. aeruginosa, the concentration of plasmid-containing donor cells introduced was shown to affect the frequency of transduction significantly. Transduction was observed both in the absence and in the presence of the natural microbial community. The presence of the natural community resulted in a rapid decrease in the numbers of the introduced donors and recipients and a decrease in the number of transductants recovered. These results demonstrate the potential for naturally occurring transduction in aquatic environments and indicate that donor load may be an important parameter in assessing this potential.     

Development of a fermentation procedure to produce a tempe-related food using common beans as substrate

Summary A basic procedure was developed to produce a tempe-like product using the mouldRhizopus oligosporus and black common beans (Phaseolus vulgaris) as substrate. The initial pH of the substrate was 5.8, and fermentation was conducted at 37°C with a relative humidity of 70% for 72 hrs. Levels of soluble solids and soluble protein increased dramatically as a result of fermentation. Some changes were as well observed in fatty acid contents of fermented samples. It was concluded that the common bean used was an acceptable substrate for preparing this product.     

Purification of the parasporal crystals ofBacillus thuringiensis subsp.israelensis and development of a novel bioassay technique

Summary A spore-free parasporal crystal suspension was prepared fromBacillus thuringiensis subsp.israelensis by an aqueous biphasic separation technique and the waste fractions quantified with regard to spore numbers and insecticidal potencies. The technique proved efficient in selectively removing spores from the spore-crystal mixture. The final crystal suspension was used to develop a novel bioassay system which allowed rapid determination of crystal effectiveness in 1/8 to 1/6 of the time required in the conventional mortality versus concentration bioassay.     

Studies on the simultaneous production of single cell protein and polygalacturonase fromKluyveromyces fragilis

Summary Kluyveromyces fragilis produces polygalacturonase (PG) on a lactose medium. Although the enzyme is normally repressed at high aeration levels, significant amounts of PG can be produced under such conditions when pectin is added as inducer. The productivity and yield of cell mass were not significantly affected by the presence of inducer, suggesting potential applications to current single cell protein processes from whey.     

Effect of temperature and long-term operation on passively immobilizedZymomonas mobilis for continuous ethanol production

Summary A fibrous support was used forZ. mobilis immobilization. The system showed a broad optimum temperature range (25–35°C) for highest ethanol productivity, ethanol yield and glucose conversion during continuous fermentation of a 100 g/L glucose medium. Ethanol production and glucose conversion kept steady during two months of continuous operation at D=1h^–1.     

Immobilization and nonspecific adsorption of proteins to pyrogeneous highly dispersed silicon dioxide

Summary A support based on pyrogeneous silicon dioxide of particle size 0.01 to 0.1/um, modified by 3-(amino)propyltriethoxysilane and activated by glutaraldehyde was employed for the immobilization of concanavalin A, immunoglobulins, basic pancreatic trypsin inhibitor, and chymotrypsin. Its binding capacity is comparable with that of porous supports while the biological activity of the proteins immobilized is retained. Nonspecific adsorption of these proteins to the support is low compared to its binding capacity.     

Effect of high concentration of nutrients onBacillus thuringiensis cultures

Summary Bacterial insecticide production using a strain ofBacillus thuringiensis var. kurstaki was studied in batch culture considering the influence of increasing concentration of components of a glucose — yeast extract — mineral salts medium.It was found that spore counts were increased from 1.08×10¹² spores. 1^–1 to 7.36×10¹² spores. 1^–1 and toxin level from 1.05 mg.ml^–1 to 6.85 mg.ml^–1, when the concentration of glucose was increased from 8 to 56 (g 1^–1), with the corresponding increase in the rest of medium components. Higher concentration of nutrients inhibit either spore count or toxin production.Preliminary experiments of fed-batch cultures which allows the use of high amounts of nutrients were also carried out. In this case spore counts of 1.2×10¹³ spores.1^–1 were achieved.     

Functionally important lysine residues in inorganic pyrophosphatase from E. coli. I. Interaction of inorganic pyrophosphatase with pyridoxal-5'-phosphate

Interaction of inorganic pyrophosphatase from E. coli with pyridoxal-5'-phosphate includes binding of the reagent at the active site through the phosphate group and then a reversible modification of one lysine residue in each of the enzyme's subunit. In the equilibrium state the protein's molecules contain both inactive modified and native subunits. A stable secondary amine is formed upon the sodium borohydride reduction of the modified protein.