Pulsed-field gel analysis of human immunoglobulin heavy-chain constant region gene deletions reveals the extent of unmapped regions within the locus

The human immunoglobulin heavy-chain constant region gene locus is organized in three main gene groups, the physical distances of which are unknown. Different types of gene deletions, originated by unequal crossingover, have been found to encompass one or more genes in the locus. We have analyzed some of these deletions by means of pulsed-field gel electrophoresis, which allows resolution of large DNA fragments. By identifying a fragment containing two of the main gene groups and by observing the size reduction of this fragment in subjects with deletions, we were able to estimate the distance between the two groups and better locate the pseudogene in-between.     

2,3,7,8, tetrachlorodibenzo-p-dioxin induces oxygen activation associated with cell respiration

We have investigated the influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on bioenergetic functions of isolated heart-mitochondria. Electron transfer and energy conservation activities were found to be decreased in the presence of very low amounts of the polychlorinated biphenyl compound (1.5 nmol/mg mitochondrial protein). The effect was greatest when substrates for complex I were used. In this case coupling of oxidative phosphorylation to respiration was drastically diminished, essentially at the expense of state 3 respiration, and P/O values were found around 2 instead of 3. Succinate-related energy conservation remained practically unaffected in the presence of TCDD, suggesting an interference of the toxic compound at coupling site I. SOD plus catalase were found to protect energy-linked respiration from the effect of dioxin indicating the involvement of superoxide radicals and H2O2 in the development of the observed phenomena. The present contribution provides experimental evidence on the formation of these oxygen species in the presence of TCDD. Furthermore, the site of action of TCDD is demonstrated and discussed in relation to the oxygen radical formation observed.     

Lack of sensitivity to ouabain in natural killer activity

Natural killer (NK) activity against K562 target cells is not an ouabain-sensitive process. Inhibition of 40% of cytotoxicity was achieved only with an ouabain concentration much higher than that required to inhibit cell activation in other systems such as leukocyte chemotaxis and B lymphocyte plaque formation. Pretreatment of effector cells with biological agents such as phorbol-ester 12-O-tetradecanoylphorbol-13-acetate or interferon increased the cytotoxicity. This activation was not counteracted by ouabain. The effect of ouabain on NK activity was compared with a well-known ouabain-sensitive process, for example, phytohemagglutinin-induced peripheral blood lymphocyte proliferation. Ouabain completely blocked [3H]thymidine incorporation, independent of the stage of the culture when the drug was added, with exception of the last 6 h. This inhibition could be partially reversed by addition of KCl. Ouabain was equally effective when whole blood cultures were used. These results suggest that NK activity is ouabain resistant, unlike other systems of cell activation that lead or do not lead to proliferation.     

The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.     

Salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection

Susceptibility to Salmonella typhimurium infection was compared in H (high Ab responder) and L (low Ab responder) mice obtained by several selective breeding experiments (Selections I, II, III, IV and IV A). H mice were always much more susceptible to infection than their L mice counterparts within a continuous LD 50 variation range. In three of the selections (I, II and IV A) the low responsiveness character is known to result mainly from rapid Ag degradation in L mice macrophages. It was hypothesized that resistance to multiplication of intracellular pathogens could be related to an increased catabolic activity towards Ag. This was actually demonstrated, in F2 segregant hybrids of selection IV A, by the significant inverse correlation between capacity for Ab production and resistance to infection.     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.     

Glutamate agonists from marine algae

The occurrence of three glutamate agonists — glutamic acid, D-homocysteic acid and kainic acid — in a spontaneous mutant of Palmaria palmata is reported. Glutamic acid and D-homocysteic acid, but not kainic acid, were found in the wild-type plant. The closely related glutamate agonist, domoic acid, was found in the red alga Chondria baileyana and in the diatom Nitzschia pungens forma multiseries. In the diatom, domoic acid can build up to high levels in excess of 3% (dry wt.), making N. pungens a potential commercial source of this neuroactive amino acid. Information is also presented on the distribution, chemistry and biological activity of neuroactive amino acids from algae, and a possible biogenic relationship among kainoid metabolites is discussed. author for correspondence     

Polarized distribution of binding sites for concanavalin A and wheat-germ agglutinin in the zona pellucida of goodeid oocytes (teleostei)

Summary Zonae pellucidae of the viviparous goodeid teleosts Girardinichthys viviparus, Xenoophorus captivus, and Xenotoca eiseni were investigated ultrastructurally, and binding sites for ConA and WGA were localized on cross-sections using a colloidal gold technique. In late stages of development, the oocytes are surrounded by a three-zonated acellular matrix multiply perforated by pore canals allowing long microvilli of the oocyte to penetrate interstices of the follicle epithelium. Together, the surface of the microvilli and zona pellucida is coated by a thin layer of homogeneous slightly electron-dense material. In early oogenesis, the thin acellular layer is entirely packed with binding sites for WGA, whereas those for ConA occur only sparsely. Three-zonated zonae pellucidae amply contain both WGA and ConA receptors. The asymmetric labelling pattern obtained with both lectin protein gold preparations indicates a polarized organization of the different glycoconjugates. WGA receptors are concentrated within the outer region of the zona pellucida. Labelling with ConA-HRP-Au complexes produced heavy deposits of marker beads within the inner two thirds of the zona pellucida and weak labelling of the superficial coat. After prolonged digestion with neuraminidase, WGA binding sites were no longer detectable.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Summary Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicleassociated nucleation.