Developmental regulation of presence of binding sites for neoglycoproteins and endogenous lectins in various embryonic stages of human lung,liver and heart

Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs.     

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum

Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na₃VO₃ as first described by Dux and Martonosi. These crystals exhibit repeat rows 11 nm apart which contain discrete units with 7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freezedrying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.Abbreviations Tris (tris[hydroxymethyl])aminomethane - TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), 2-(2-hydroxy-1-bis[hydroxymethyl]ethyl)aminoethanesulfonic acid - SR sarcoplasmic reticulum - CPP calcium pump protein Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Structural alterations of tapetal cells in the retina of cats induced by prolonged treatment with chloroquine

Summary Cats were treated with high doses of chloroquine for one year during which the ocular fundus was periodically examined. After completion of the treatment, the tapetal cells were investigated by light and electron microscopy. Prolonged treatment with the retinotoxic drug chloroquine reduced the light reflection of the fundus, and examination by light and electron microscopy revealed a destruction of the rod-like structures in the cytoplasm of the tapetal cells.     

SE- and SW-migrating Blackcap (Sylvia atricapilla) populations in Central Europe: Orientation of birds in the contact zone

In the Blackcap (Aves: Sylvia atricapilla), a widespread passerine noctural migrant, a “migratory divide” between SE- and SW-migrating populations exists in Central Europe at about 14° E and south of 52° N. The autumn migratory directions are known to have a genetic basis and are expressed in orientation cages in captivity. Migratory directions of birds in the contact zone between the two populations were studied by analysing ringing data and by testing three groups of hand-raised individuals in orientation cages. Available ringing data are insufficient to establish migratory directions in the contact zone north of the Alps. Hand-raised birds from south-west Germany and the most eastern part of Austria oriented SW and SE, respectively, confirming directions known from ringing recoveries. A sample of birds from the contact zone near Linz (Austria) oriented SW to NW (mean = 268°) and was significantly different from both adjoining populations. This contrasts with results of a cross-breeding experiment with mixed pairs of SW- and SE-migrants bred in captivity: The F1-offspring chose southerly directions, intermediate between both parental populations (Helbig, 1991). It is suggested, therefore, that a distinct subpopulation with a large fraction of birds wintering in the British Isles has established itself in the contact zone. Differences in directional choices between groups of siblings from this area indicate that intrapopulation genetic variability is present. This may have led to a rapid spread of the novel W-NW migratory direction, because north of the Alps strong selection seems to be acting against mixing of SE- with SW-migrating populations.     

Luminol-dependent chemiluminescence in bovine eosinophils and neutrophils: Differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (> 90%), maximally stimulated with 1 nmol/l phorbol 12-myristate-13-acetate (PMA) showed ten times more extracellular luminol-dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (> 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27-fold difference) and platelet-activating factor (PAF) at 2 μmol/l (9-fold difference), but not by calcium ionophore A23187 (15 μmol/l). Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet-activating factor induced a dose-dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL. Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.     

Levels of tropinone-reductase activities influence the spectrum of tropane esters found in transformed root cultures of Datura stramonium L.

The nortropane sulphur analogues 8-thiabicyclo[3.2.1] octan-3-one, 8-thiabicyclo[3.2.1]octan-3a-ol and 8-thiabicyclo[3.2.1]octan-3-ol have been found to have differential effects in vitro on the activities of tropinone reductase I and tropinone reductase II from Datura stramonium L. It has been demonstrated that only tropinone reductase I is able to metabolise 8-thiabicyclo[3.2.1]octan-3-one and that only this enzyme is inhibited by 8-thiabicyclo[3.2.1]octan-3-ol and 8-thiabicyclo[3.2.1]octan-3-ol. A K _m of 0.035 mM was determined for 8-thiabicyclo[3.2.1]octan-3-one and I₅₀ values of 0.081 mM and 0.021 mM for 8-thiabicyclo[3.2.1]octan-3-ol and 8-thiabicyclo[3.2.1]octan-3-ol, respectively. The influence that these differential interactions might have on metabolism was investigated in transformed root cultures of D. stramonium. It was found that when these cultures were grown in the presence of either 8-thiabicyclo[3.2.1]octan-3-one or 8-thiabicyclo[3.2.1]octan-3-ol the spectrum of alkaloids that accumulated was altered from that found in control roots in the manner predicted from the observed effects of these inhibitors on the isolated reductases. The effect could be mimicked by feeding pseudotropine, the product of tropinone reductase II. It is concluded that the relative levels of activity of the two tropinone reductases might play an important role in regulating the balance of tropan-3-ols to tropan-3-ols seen in the spectrum of tropane-alkaloid-producing plants.Abbreviations GC/MS gas chromatography/mass spectrometry; - I₅₀ concentration of inhibitor required to reduce the rate of reaction to half the maximal value; - -TBOL 8-thiabicyclo[3.2.1]octan-3-ol; - -TBOL 8-thiabicyclo[3.2.1]octan-3-ol; - TBON 8-thiabicyclo[3.2.1]octan-3-one; - TR tropinone reductase We are most grateful to J. Eagles (I.F.R., Norwich) for GC/MS analysis, to colleagues at I.P.B.P. and I.F.R. for helpful discussions, to the technical staff (Chemistry, Glasgow) and to W. Millar (Chemistry, Glasgow) for assistance with the reduction of TBON. This work was, in part, supported by a grant to B Dräger from the Deutsche Forschungsgemeinschaft (Dr227/I-I). The research reported here was supported by an Academic Research Collaboration Cooperative Award (project No. 215) from the British Council and the Deutscher Akademischer Austauschdienst to R.J. Robins and B. Dräger.