Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Allelic profile at 37 biochemical loci of two inbred strains of the house mouse derived from wild Mus musculus musculus

Two newly established inbred strains derived from Mus musculus musculus, designated PWD/Ph (F29) and PWK/Ph (F33), were examined for their alleles at 37 biochemical loci located on 12 different chromosomes. The allelic pattern showed characteristic differences from those observed in common inbred strains. The genetic distance D between PWK/Ph and PWD/Ph was 0.027, whereas the corresponding values for the genetic distances between PWK/Ph and C57BL/6J, DBA/2J, BALB/cJ and SWR/J were 0.777, 0.721, 0.721 and 0.838 respectively. New allozymes are described as being controlled by the loci Es-23, Pre-2 and Tam-1. The genetic relationship to M.m.molossinus is indicated by identical alleles at six other loci (Es-2, Es-9, Es-10, Es-11, Es-18 and Es-22).     

Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.     

Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.     

Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist

The rise in cytosolic free Ca2+, shape change, superoxide formation, and granule exocytosis induced in human neutrophils by N-formyl-Met-Leu-Phe (fMLP) and by a newly discovered activating peptide, neutrophil-activating factor, termed NAF, were compared. NAF was effective in the concentration range of 0.1-10 nM and was 10- to 100-fold more potent than fMLP. In qualitative terms, the single responses to either stimulus were remarkably similar: they showed virtually identical onset and initial kinetics, and were all inhibited by pretreatment of the neutrophils with Bordetella pertussis toxin. In addition, the respiratory burst elicited by either stimulus was inhibited by 17-hydroxywortmannin and staurosporine. Two conclusions are drawn from these results: 1) neutrophil activation by NAF (as by fMLP) is dependent on a GTP-binding protein and on protein kinase C; 2) a similar, or even identical, mechanism of signal transduction must be assumed on stimulation of human neutrophils with NAF, fMLP, and other chemotactic agonists. Human monocytes, lymphocytes, and platelets did not show cytosolic free Ca2+ changes when exposed to NAF, which suggests that NAF is selective for the neutrophils.     

Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator.

Lac repressor, lambda cro protein and their operator complexes are structurally, biochemically and genetically well analysed. Both proteins contain a helix-turn-helix (HTH) motif which they use to bind specifically to their operators. The DNA sequences 5'-GTGA-3' and 5'-TCAC-3' recognized in palindromic lac operator are the same as in lambda operator but their order is inverted form head to head to tail to tail. Different modes of aggregation of the monomers of the two proteins determine the different arrangements of the HTH motifs. Here we show that the HTH motif of lambda cro protein can replace the HTH motif of Lac repressor without changing its specificity. Such hybrid Lac repressor is unstable. It binds in vitro more weakly than Lac repressor but with the same specificity to ideal lac operator. It does not bind to consensus lambda operator.     

Ultrastructural identification and distribution of the adhesion molecules ICAM-1 and LFA-1 in the vascular and extravascular compartments of the human palatine tonsil

Summary Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.