Human immunoglobulin variable region genes: V κ polymorphisms suggest variation in V-gene repertoires

The present study characterizes four potentially informative polymorphic bands of 5.2, 2.3, 1.9, and 1.2 kb, detected by Southern blot hybridization of Eco RI digests of human DNA using HK101/80 (an immunoglobulin V I probe). These restriction fragment length polymorphisms (RFLP) show Mendelian segregation and they are linked to each other and to Km(1), the allotypic marker on the kappa constant region. There is strong linkage disequilibrium between the 2.3 and 1.2 kb polymorphisms. A 0.7 kb Pst I polymorphic band and a 2.9 kb Sac I polymorphic band were also identified; the latter may reflect a region of tandem repeats in the V region. No bands representing the alternative forms of any of the polymorphic restriction sites were identified. This implies either that genes are missing from the V repertoire or that such bands are hidden because of comigration of fragments due to conservation of restriction sites. Evidence for comigration of fragments was obtained from independent V clones and suggests that dark bands on Southern blots of Pst I digests must often represent several superimposed genes which have conserved restriction sites. The demonstration of RFLP within the V region provides circumstantial evidence for polymorphic variation in the repertoire of V structural genes. The RFLP reported here should be useful as genetic markers in future studies on the immune response and disease susceptibility in man.     

Carbohydrate moieties of rat MHC class I antigens

The rat major histocompatibility complex class I antigens RT1.A^u and RT1.E^u from the u haplotype and RT1.Aⁿ from the n haplotype were labeled with ¹⁴C-asparagine or with ³H-fucose, mannose, galactose, and N-acetylglucosamine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed complete removal of radioactivity from the sugar-labeled antigen heavy chains by digestion with glycopeptidase F, an enzyme that removes N-linked glycans completely. High performance liquid chromatography analysis of the tryptic digests of the mixed sugar-labeled and asparagine-labeled antigens demonstrated that all the sugar-labeled peptides were coincident with asparagine-labeled peptides. The Aⁿ antigen showed three glycopeptides, each of which had different amounts of sugar radioactivity. The antigens A^u and E^u showed two glycopeptides with different amounts of radioactivity but at identical positions in the two antigens. Antigen E^u had an additional glycopeptide with a lower amount of radioactivity. The positions of the glycopeptides from the A^u and E^u antigens were different from those of the Aⁿ antigen. The peptide profiles of the ¹⁴C-asparagine-labeled A^u and E^u antigens demonstrated distinct differences between the molecules. The results of this study show that: (a) all the glycans on rat class I antigens are N-linked, as they are on H-2 and HLA class I antigens; (b) there are compositional differences among the glycans in each of the three antigens; (c) the glycosylation pattern of the rat class I antigens is similar to that of the mouse class I antigens, which contain two or three glycans, in contrast to that of the human class I antigens, which contain only one glycan; and (d) the antigens A^u and E^u from the same haplotype are more closely related to each other than they are to the Aⁿ antigen.     

Gene transfection of the HuLy-m2 (Leu-9) antigen into mouse L cells

Human DNA was transfected into mouse L cells and tk⁺ HuLy-m2⁺ (= CD7⁺) transfectants isolated after growth in hypoxanthine, aminopterin, thymidine medium and repeated cloning. After several cycles of transfection, > 90% of HuLy-m2⁺ L cells could be detected, by rosetting and by cytofluorography, which showed the transfectants to have a density of CD7 two to five times that found on peripheral blood lymphocytes. Despite this, the 37 kd CD7⁺ dimer could only be identified with difficulty using cell-surface radioiodination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques. An antiserum was produced (C3H anti-HuLy-m2⁺ L cells) which, after absorption, was shown to react with HuLy-m2 antigens present on human thymocytes and lymphocytes and on CD7⁺ transfected L cells.Abbreviations BSA bovine serum albumin - DME Dulbecco's modified Eagle's medium - EDTA ethylenediamine-tetraacetate - HAT hypoxanthine, aminopterin, thymidine - HSV herpes simplex virus - PBL peripheral blood lymphocyte - PBS phosphate-buffered saline - RFC rosette-forming cell - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - tk thymidine kinase     

The mouse Fc receptor for IgG (Ly-17) : molecular cloning and specificity

A cDNA clone encoding the mouse Ly-17⁺ Fc receptor for IgG, isolated from a myelomonocytic cell line, was sequenced and expression of mRNA and the functional FcR investigated. The receptor is a 301 amino acid transmembrane glycoprotein with two homologous extracellular domains that are also homologous to members of the Ig superfamily. The receptor has four sites of N-linked glycosylation and a long 94 amino acid cytoplasmic tail. Northern analysis, immune complex binding, and serological studies demonstrate that the receptor encoded by the cDNA clone binds mouse IgG_1/2b and rabbit IgG complexes.     

Stable isotope dilution gas chromatography/mass spectrometry of prostaglandins and leukotrienes

The group of arachidonic acid metabolites comprising the prostaglandins, thromboxanes, and leukotrienes (eicosanoids) are extremely potent, biologically active compounds. Their properties include proaggregatory anti-aggregatory activity for platelets, chemotactic activity for neutrophils, vasoactive activity, and contractile activity to smooth muscle. In order to determine the role of these substances in pathophysiological conditions, it is essential to have highly sensitive methods available for their analysis. It is generally accepted that combined gas chromatography/mass spectrometry is the most specific technique available for the quantitative analysis of eicosanoids. However, methods based on electron impact ionization and positive ion chemical ionization are relatively insensitive, and many investigators have preferred the use of less specific but more sensitive methods based on radioimmunoassay. We have explored the use of negative ion chemical ionization mass spectrometry to improve sensitivity coupled with capillary column chromatography to maximize specificity. Conversion of the terminal carboxyl group (present in all eicosanoids) to the pentafluorobenzyl ester derivative confers excellent electron capturing properties to the molecule. The derivative undergoes highly efficient thermal electron capture in the gas phase, and any fragmentation that occurs subsequently is directed almost entirely away from the analyte molecule. The stabilized carboxylate anion that results carries at least 30% of the total ion current. Using selected ion monitoring techniques it is possible to detect eicosanoids in the range 1–8 pg on column. This methodology has been applied to the development of stable isotope dilution assays for plasma 6-oxo-prostaglandin (PG) F_1α (1) and for the simultaneous analysis of six biologically important PGs in biological fluids (2). In addition, stable isotope dilution techniques have been developed for the analysis of serum thromboxane B₂ and serum leukotriene B₄ (3). The application of this technology to understanding the role of arachidonic acid metabolism in humans will be discussed.     

Some factors affecting the composition of tropical ichneumonid faunas

An increasing accumulation of data shows that tropical ichneumonid faunas are no more species-rich than extra-tropical ones, despite the fact that most of their host groups show increased tropical species-richness. This lack of increase in ichneumonid species-richness can be attributed to the absence of groups whose hosts are not present (e.g. Ctenopelmatinae) and poor tropical representation by many groups of diurnal koinobionts (e.g. Campopleginae). Low host density has been postulated as a barrier to tropical koinobiont species-richness, but it is here suggested that this is not the only limiting factor as groups of nocturnal koinobionts, such as the Ophioninae, show increased tropical species-richness. It is postulated that koinobionts have the capability of being able to locate sparse hosts, but as they host-search in flight, a prolonged daytime host-searching period in the tropics would expose them to a high level of predation pressure. By being active at night koinobionts can avoid diurnally active predators. It is also postulated that sparse hosts may be located more easily at night and more habitats may be climatically suitable for ichneumonid activity when they are not subject to direct sunlight. Idiobionts, such as the Mesostenini and Pimplini, are more species-rich and morphologically diverse in the tropics than they are in extra-tropical regions. It is suggested that this results from the fact that tropical idiobionts can be active during the whole of the diapausing period, when their hosts are available, whereas activity by temperate idiobionts is prevented by inclement weather. Although many idiobionts are probably less exposed to predators than koinobionts, many have evolved obvious protective devices.     

Mg2+-ATPase activity in wheat root plasma membrane vesicles: Time-dependence and effect of sucrose and detergents

Purified plasma membrane vesicles were isolated in the presence of 250 mM sucrose from 7-day-old roots of Triticum aestivum L. cv. Drabant by aqueous polymer two-phase partitioning. When added to a low-salt medium containing 9-aminoacridine (9-AA), the vesicles caused a much larger total decrease in 9-AA fluorescence when sucrose was absent than when sucrose was present. A slow component of the decrease was also larger in the absence of sucrose. Triton X-100 reduced the decrease in 9-AA fluorescence upon vesicle addition and abolished completely the slow component of the decrease. There was no correlation between the time-dependence of 9-AA fluorescence and that of the Mg²⁺-ATPase described below. The time course of Mg²⁺-ATPase activity was followed by sampling at short intervals (down to 10 s) and analyzing for P, released. In the absence of detergent, the rates of P, release were linear from zero minutes, whether 250 mM sucrose was present or not, but the rate was 10^?50% higher in the absence of sucrose than in its presence. Sucrose (250 mM) added during a minus-sucrose assay lowered Mg²⁺-ATPase activity within 2 min to the level observed with 250 mM sucrose present from the start. The effect of 25-1 100 mM sucrose was tested and there was little or no effect below KM) mM. Above 100 mM sucrose the rate of P, release decreased drastically; at 1 100 mM sucrose the rate was ca 20% the rate at 25 mM sucrose. The inhibitory effect of sucrose was not alleviated by increased concentrations of Mg²⁺ and/or ATP. nor was it affected by the presence or absence of Triton X-100. We conclude that sucrose somehow inhibits the Mg²⁺-ATPase directly or affects the conformation of the plasma membrane in such a way as to inhibit the enzyme. The presence of detergents increased Mg²⁺-ATPase activity in the order Triton X-100 (4–5-fold) > Zwittergent 3–14 = Na-cholate = octylglucoside > digitonin (2-fold). In all cases optimal activity was observed at detergent concentrations at or below the critical micellar concentration. The detergent concentration curves could be simulated by the sum of a stimulatory and an inhibitory reaction. At the optimal concentration, digitonin gave a linear time-course of P, release, whereas all the other detergents showed a distinct lag of 1–3 min before maximal rates were attained. The problems of using detergents in polarity assays are discussed.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Workshop on algal polysaccharides

Summary This workshop was an impromptu event, but the fact that a number of interesting problems were identified by the participants from the floor may indicate that it is worth repeating. If it is to be repeated, however, it is important that notice be given, and that the scope of the workshop be defined in advance, so that participants can be better informed and bring supporting data. The best approach would be to identify a convenor and define the scope of the workshop prior to the first circular for the next Seaweed Symposium; invitations could then be issued by the convenor for specific topics and data, and a very brief program could be issued with the third circular. Such a procedure may very well allow the identification of new areas for research.