Production of alloantibodies against bovine B-lymphocyte antigens.

A series of alloimmunizations were carried out between BoLA class I antigen typed bulls, with the aim of generating class II specific reagents. Of the antisera produced, seven demonstrated exclusively B cell reactivity. Another 19 sera reacted with both T and B cells from some animals and with B cells only in other cases. Suitable buffy coat absorptions removed T cell reactivity from some sera and shortened broader reactivities in certain B cell specific sera. Typing of separated T and B cells from related and unrelated animals permitted clustering of the sera into four groups. These groups behave as allelic specificities. The class II nature of the recognized structures was strongly indicated by two further pieces of evidence. The presence or absence of particular B cell antigens correlated with reactivity of cells in one-way mixed lymphocyte cultures. In addition, a number of the B cell specific sera were characterized by immunoprecipitation of radiolabelled lymphocytes. The precipitated products corresponded in molecular weight to alpha and beta chains of MHC class II dimers, as has been found in this and other species.

Biochemical evidence that equine leucocyte antigens W13, W22 and W23 are present on horse major histocompatibility complex class II molecules.

A number of horse alloantisera were characterized biochemically as being directed against MHC class I or class II antigens by immunoprecipitation of the corresponding antigens from lysates of biosynthetically radioactively labelled lymphocytes and determination of their molecular weights by SDS-PAGE and fluorography. Sera recognizing A2 and A3 specificities precipitated antigens of 44,000 Daltons molecular weight (class I heavy chain), whereas sera with specificities W13, W22 and W23 precipitated antigens corresponding to class II dimers (30,000 and 32,000 Daltons). Comparison with antigens precipitated from horse lymphocyte lysates using (cross-reacting) antibodies to human class I and class II MHC molecules confirmed the results obtained.

Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha- and beta-chain cDNA probes.

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.

Rapid data acquisition from a microtiter plate fluorescence reader and applications in kinetic measurements.

Programs written in Applesoft BASIC for the rapid acquisition and evaluation of data from a commercially available microtiter plate fluorescence reader are presented. Using the data acquisition program, the relative fluorescence readings from all 96 wells of the microtiter plate (one read cycle of the fluorescence reader) can be stored in each of up to 90 consecutively numbered files on a single-sided diskette. A simple timer circuit is described which, when used in conjunction with the above program, initiates the fluorescence reading process at preset time intervals, thus making automatic acquisition of data possible. A further program plots the data from consecutive files on the computer monitor and prints a hard copy if required. The feasibility of applying the above system and software to kinetic measurements in enzyme systems is demonstrated using methylumbelliferyl phosphate and an alkaline phosphatase/immunoglobulin conjugate. In addition, its use in following the formation of extracellular hydrogen peroxide by stimulated polymorphonuclear leukocytes using horseradish peroxidase-coupled oxidation of the fluorescent compound 7-hydroxy-6-methoxy-coumarin (scopoletin) is described.

Localization of galactosyl- and sialyltransferase by immunofluorescence: evidence for different sites.

Polyclonal rabbit antisera against soluble human milk galactosyltransferase and bovine colostrum sialyltransferase were used to localize by indirect immunofluorescence the respective intracellular enzymes in primary cultures from bovine fetal kidneys and established cell lines of human and bovine fibroblasts. Staining for galactosyltransferase was juxtanuclear and crescent shaped in epitheloid cells; a similar staining, occasionally perinuclear and sparsely distributed in the cytoplasm, was found in fibroblasts. In contrast, staining for sialyltransferase in epitheloid kidney cells derived from the same primary culture was observed predominantly in cytoplasmic vesicles that were spread over the whole cytoplasm. Sialyltransferase-positive vesicles had a similar distribution in fibroblasts and often appeared concentrated around an unstained Golgi area. Thus, in both cell types galactosyl- and sialyltransferase were localized in different subcellular compartments. Since both galactosyl- and sialyltransferase participate in formation of the terminal glycan NeuAc(alpha 2----6)Gal(beta 1----4)GlcNAc(Neu, neuraminic acid) present in many N-glycosidic complex types of glycans, different subcellular compartments for these enzymes support a model of functional compartmentalization of the Golgi apparatus that is compatible with an assembly-line model for glycan chain elongation and termination.

Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site.

Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-alpha-D-galactopyranosylpeptide galactose beta(1 lead to 3) transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements. Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.

Identification of the product formed by human erythrocyte galactosyltransferase.

Sepharose 4B-immobilized desialylated ovine submaxillary mucin was used as an acceptor for galactose transfer from UDP-galactose, catalyzed by a Triton X-100-solubilized galactosyltransferase from human erythrocyte ghosts. The product could be cleaved from the insoluble acceptor substrate by alkaline borohydride treatment and identified on Bio-Gel P-2 as a disaccharide. The nature of the glycosidic bond of the isolated material was elucidated by periodate oxidation/NaB[3H]4 reduction/acid hydrolysis and subsequent identification of the aminopolyol formed as L-threosaminitol. Specific cleavage of the enzymatic product by beta-galactosidase indicated a beta-configuration for incorporated galactose. These data permit classification of the enzyme as UDP-galactose: alpha-D-N-acetylgalactosaminyl-protein beta (1 leads to 3) transferase. Furthermore, in the presence of Triton X-100, the enzyme from normal erythrocytes catalyzed transfer of galactose to the glycan moieties of asialo-agalacto-glycophorin in Tn-erythrocytes from a patient with permanent mixed-field polyagglutinability.

The application of a new synthetic substrate to the determination of enteropeptidase in rat small intestine and human intestinal biopsies.

The application of a new synthetic substrate to the direct determination of enteropeptidase is described. The substrate Gly-(L-Asp)4-L-Lys-2-naphthylamide contains the amino acid sequence of the activation peptides of trypsinogen linked via an amide bond to the fluorophore 2-naphthylamine. The sequence of amino acids is responsible for the specificity and substrate recognition of the enteropeptidase-catalyzed activation of trypsinogen. Interference in the assay by trypsin is prevented by the addition of soybean trypsin inhibitor to the substrate solution. The fluorimetric determination of the liberated 2-naphthylamine allows the direct observation of the reaction kinetics. For the hyrolysis of the synthetic substrate by purified enteropeptidase the pH optimum was 8.2 and the Km 0.17 mmol/l. The new substrate was used to determine the distribution of enteropeptidase along the rat small intestine and also to measure enteropeptidase activity in human intestinal biopsies.

Determination of chromium sesquioxide in faeces by a spectrophotometric method.

Chromium sesquioxide Cr2O3, present as a non-absorbable marker in faeces, may be determined spectrophotometrically as chromate ion in aqueous solution after ashing and alkaline fusion. Recovery of this substance is excellent. The method described is simpler, more suited to the clinical laboratory and less hazardous than previously reported methods.