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.     

The effect of copper and nitrogen on the amino acid composition of oat straw

Summary The effect of fertilization with nitrogen and copper on the amino acid composition of oat straw has been studied.The plants (Avena sativa cv Yielder) were grown in peat with a very low copper content and supplied with two levels of nitrogen (NH₄ or NO₃) and three levels of copper sulphate.The higher level of nitrogen stimulated growth only when copper was added, whereas, without copper, it had an adverse effect on growth and prevented grain formation altogether. The higher level of nitrogen increased the nitrogen content of the straw at all levels of copper, but particularly in plants receiving no copper.Total amino acids in the straw hydrolysate of copper sufficient oats accounted for about 50% of the total N and was about 20% higher in copper-deficient tissues. The addition of copper caused a decrease in the amounts of all amino acids. The relative proportions of most of the amino acids to glycine remained fairly constant. Threonine, serine, alanine, iso-leucine, histidine and arginine showed small significant differences with copper treatment, whereas valine, tyrosine, phenylalanine, proline, lysine and cysteic acid (derived from cysteine and cystine) showed no differences. The proportion of aspartic acid relative to glycine in the straw hydrolysate was greatly increased in copper deficient plants supplied with the higher level of nitrogen, particularly as ammonium. The proportion of glutamic acid was also increased by the higher level of nitrogen, but showed no effect of added copper. Most of the difference in aspartic acid could be accounted for as free asparagine. The possible reasons for higher proportions of asparagine are discussed in relation to the metabolism of the oat plant.     

Phosphorylation of a 100 000 dalton component and its relationship to calcium transport in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcomplasmic reticulum from rabbit fast skeletal muscle contains intrinsic protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) and a substrate. The protein kinase activity was Mg2+ dependent and could also phosphorylate exogenous protein substrates. Autophosphorylation of sarcoplasmic reticulum vesicles was not stimulated by cyclic AMP, neither was it inhibited by the heat-stable protein kinase inhibitor protein. The phosphorylated membranes had the characteristics of a protein with a phosphoester bond. An average of 73 pmol Pi/mg protein were incorporated in 10 min at 30 degrees C. Addition of exogenous cyclic AMP-dependent protein kinase increased the endogenous level of phosphorylation by 25-100%. Sarcoplasmic reticulum membrane phosphorylation, mediated by either endogenous cyclic AMP-independent or exogenous cyclic AMP-dependent protein kinase, occurred on a 100 000 dalton protein and both enzyme activities resulted in enhanced calcium uptake and Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3), in a manner similar to cardiac microsomal preparations. Regulation of Ca2+ transport in skeletal sarcoplasmic reticulum may be mediated by phosphorylation of a 100 000 dalton component of these membranes.     

Selective effects of anti-Ia serum and complement treatment upon polyclonal B lymphocyte responses to dextran sulfate and lipopolysaccharide.

Subpopulations of B lymphocytes have been shown to vary in their expression of Ia alloantigens and polyclonal responsiveness to thymic independent antigens. We have demonstrated that the polyclonal B cell antibody response to dextran sulfate is less sensitive to removal of Ia-positive cells than is the response to LPS. This is a consistent finding whether alloantibody and complement (C) pretreatment is directed toward cells bearing Ia antigens coded for by the entire I region or by the I-A or I-E subregions. Heterogeneity appears to exist within the dextran sulfate-sensitive population in that using high antibody; cell ratios during antibody and C-mediated cell selection results in an inhibition of the proliferative but not the antibody response. This result may indicate a differential expression of Ia antigens on dextran sulfate-sensitive B cells that respond by proliferation versus those cells that produce antibody. Alternatively, proliferative responses to dextran sulfate may be more dependent upon Ia-positive accessory cells than is the polyclonal antibody response.     

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.     

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.     

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.