Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 antigens.     

Syrticola mediterraneus n. sp., a harpacticoid copepod from the Bay of Calvi,Corsica

Syrticola mediterraneus n. sp. belonging to the family Cylindropsyllidae Sars (sensu Lang, 1948) is described from the Bay of Calvi (Corsica). The species is closely related to (?) Notopontia galapagoensis Mielke, 1982 and Syrticola flandricus Willems & Claeys, 1982. The former species is now transferred to the genus Syrticola as Syrticola galapagoensis n. comb.     

A feline class II α gene with striking similarity to the HLA-DPA pseudogene

A genomic library was constructed from DNA of a domestic cat and screened with a human HLA-DR probe at low stringency. Several positive clones were isolated, and the DNA sequence of one of these clones was determined. Comparison with class II gene sequences from other species suggested that the feline gene is a DPA homologue (FLA-DPA) showing 84% similarity with HLA-DP1 in the exon encoding the second domain. The FLA-DPA gene that was isolated is a pseudogene, as two frame-shift mutations are present: one in the exon encoding the second domain, causing premature termination of translation, and one in the exon encoding the transmembrane region. The latter mutation and the further deletion of two codons in the transmembrane exon show a remarkable resemblance to the same exon of the human pseudogene, HLA-DPA2. Hence, both pseudogenes evolved from the same ancestral gene. The inactivation of this DPA gene could therefore have occurred prior to the major mammalian divergence.     

Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (baker's yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.     

Direct assay for phosphotransacetylase and acetyl-coenzyme A carboxylase by high-performance liquid chromatography

A simple and specific assay to measure the activity of two coenzyme A derivative-processing enzymes, i.e., phosphotransacetylase (EC 2.3.1.8) and acetyl-coenzyme A carboxylase (EC 6.4.1.2), is described. The assay is based on the HPLC analysis of the short-chain coenzyme A derivatives formed by the enzymatic reaction, viz., acetyl-CoA and malonyl-CoA. For this purpose, ion-pair reversed-phase HPLC conditions are optimized. Furthermore, the influence of several variables on the enzyme reaction is studied in order to get maximum activity. Due to its short analysis time, good selectivity, and chromatogram information, HPLC proves to be an excellent method for the assay of these enzymes.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

DNA sequence of the coding region of the HLA-B44 gene

An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.     

Mechanism of complement-induced stimulation of prostacyclin production by isolated rabbit peritoneum

The interaction between the complement system and prostaglandin synthesis has not thoroughly been explored, although both mediators are known to be involved in inflammatory reactions and endotoxic shock. When rabbit peritoneum, a rich source of prostacyclin forming activity was incubated in serum in which the complement system was activated (CVF, LPS, zymosan), the tissue produced significantly more PGI2, when compared with appropriate controls, indicating that by activation of the complement, factors were generated that stimulated PGI2 biosynthesis. Further results indicated that tryptic cleavage products of complement factor C3 and C5 also led to the appearance of PGI2 releasing principles with a molecular weight of about 7000-11000. The stimulation of PGI2 biosynthesis was explained by enhanced release of AA, and not due to increased activity of cyclo-oxygenase or PGI2 synthetase. Our results suggest that complement-derived products may promote the supply of prostaglandins at the site of inflammation.     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Fully automated measurements by light microscopy of tissue sections using a cellular array computer

Software was developed for the acquisition, segmentation and analysis of microscopic OD-images on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board. The delineation algorithms for peroxisomes, lysosomes, and nuclei in liver, kidney, and adrenal gland sections start by thresholding the difference between the original image and a low pass filtered version. The resulting binary mask is then processed by morphological operations in order to produce an object overlay. The efficiency of the programs is evaluated by comparing delineated objects at different OD-levels, created by varying the stain or by multiplying the original pixel values with constant factors. Manual delineation on some images is also used as a reference. More complex algorithms are used for the delineation of muscle fibres in ATP-ase-stained sections and immunocytochemically labelled cells in monolayer preparations. Muscle images from parallel sections with different stainings are matched with a coordinate transform, enabling the transfer of the object mask from a single delineated image to the unprocessed images and thus obtain all necessary information for fibre classification. After segmentation, the OD-images and their object overlays are fed into a data extraction program, measuring for each delineated object user-selected features. Data are sent to a VAX for statistical interpretation.