Purification and partial characterization of lysosomal neuraminidase from human placenta

Lysosomal neuraminidase and beta-galactosidase are present in a complex together with a 32-kDa protective protein. This complex has been purified and the different components have been dissociated using potassium isothiocyanate (KSCN) treatment. beta-Galactosidase remains catalytically active, but neuraminidase loses its activity upon dissociation. The inactive dissociated neuraminidase was purified by removing the remaining non-dissociated beta-galactosidase/protective protein complex using beta-galactosidase-specific affinity chromatography. The dissociated neuraminidase material shows two major polypeptides on SDS-PAGE with an apparent molecular mass of 76 kDa and 66 kDa. Subsequently the 32-kDa protective protein was dissociated from the beta-galactosidase/protective protein complex, and purified. Antibodies raised against the dissociated inactive neuraminidase preparation specifically immunoprecipitate the active neuraminidase present in the complex with beta-galactosidase and protective protein. By immunoblotting evidence is provided that the 76-kDa protein is a subunit of neuraminidase which, in association with the 32-kDa protective protein, is essential for neuraminidase activity.     

B-cell epitopes of scleroderma-specific autoantigens

Conclusions It can be concluded that the precise localization of the epitopes on autoantigens associated with scleroderma has not been determined yet, and further subcloning experiments will be required to map the epitopes more precisely. However, the fact that the antigenicities of the C-terminal ends of topo I as well as of CENP-B are highly affected by the length of the fusion segments suggest that most, if not all, antigenic determinants on these parts of the autoantigens are conformational epitopes. Studies based upon molecular modelling of antibodies reacting with antigens suggest that over 90% of B-cell epitopes are conformational [50]. This implies that the most successful approach to allocate B-cell epitopes on autoantigens in the near future may be the use of techniques for mapping conformational epitopes. Such techniques are currently being developed [reviewed in 51]. Until now, the limited data available indicate that the B-cell epitopes on the scleroderma-associated autoantigens are distributed over the entire proteins. The C-terminal parts of the antigens seem to be good candidates for harboring the major autoimmune epitopes, but more experimental data will be necessary to confirm this suggestion.     

Identification of a reversible inhibitor of plasminogen activators in blood plasma

Inhibition of tissue-type plasminogen activator (t-PA) by pooled plasma could be ascribed for only 60% to the endothelial cell type PA inhibitor. The residual inhibition is ascribed to a so-far undescribed plasma component present at 0.2 nmol/l. This component shows reversible binding to t-PA with an apparent K_i of 10 pmol/l (does not hinder t-PA binding to fibrin); also reacts with urokinase, but not with DIP-t-PA; is stable at 37°C and does not occur in media of endothelial cells, hepatocytes and fibroblasts. This PA binding component in plasma adds to the regulation of plasminogen activator activities.

Fibrinolysis Tissue-type plasminogen activator Urokinase Blood plasma Endothelial cell type plasminogen activator inhibitor Protease inhibitor     

Glucose transport in lysosomal membrane vesicles. Kinetic demonstration of a carrier for neutral hexoses

Lysosomal membrane vesicles isolated from rat liver were exploited to analyze the mechanism of glucose transport across the lysosomal membrane. Uptake kinetics of [14C]D-glucose showed a concentration-dependent saturable process, typical of carrier-mediated facilitated transport, with a Kt of about 75 mM. Uptake was unaffected by Na+ and K+ ions, membrane potentials, and proton gradients but showed an acidic pH optimum. Lowering the pH from 7.4 to 5.5 had no effect on the affinity of the carrier for the substrate but increased the maximum rate of transport about 3-fold. As inferred from the linearity of Scatchard plots, a single transport mechanism could account for the uptake of glucose under all conditions tested. As indicated by the transstimulation properties of the carrier, other neutral monohexoses, including D-galactose, D-mannose, D- and L-fucose were transported by this carrier. The transport rates and affinities of these sugars, measured by the use of their radiolabeled counterparts, were in the same range as those for D-glucose. Pentoses, sialic acid, and other acidic monosaccharides including their lactones, aminosugars, N-acetyl-hexosamines, and most L-stereoisomers, particularly those not present in mammalian tissues, were not transported by this carrier. Glucose uptake and transstimulation were inhibited by cytochalasin B and phloretin. The biochemical properties of this transporter differentiate it from other well-characterized lysosomal sugar carriers, including those for sialic acid and N-acetylhexosamines. The acidic pH optimum of this glucose transporter is a unique feature not shared with any other known glucose carrier and is consistent with its lysosomal origin.     

Assignment of the gene(s) involved in the expression of the proliferation-related Ki-67 antigen to human chromosome 10

Summary The antigen recognized by the monoclonal antibody Ki-67 is a proliferation-related nucleolus-associated constituent used as a marker for cycling cells in tumor diagnosis. Antibody Ki-67 reacts with human proliferating cells, but not with hamster and mouse cells. Expression of the Ki-67 antigen was studied in a panel of human-rodent somatic cell hybrids. The results indicate that a gene involved in the expression of the antigen is located on chromosome 10.     

Complementation between urokinase-producing and receptor-producing cells in extracellular matrix degradation.

The respective roles of urokinase plasminogen activator (u-PA) and the u-PA receptor in extracellular matrix degradation was investigated. Human pro-u-PA and the human u-PA receptor were expressed independently by two different mouse LB6 cell lines. The matrix degradation capacity of these cell lines individually or in coculture was studied. Although pro-u-PA-producing cells alone degrade the matrix in the presence of plasminogen, u-PA-receptor producing cells do not. Cocultivation of a small fraction of pro-u-PA-producing cells with the receptor-producing cells increases the rate of matrix degradation at least threefold. By immunoprecipitation it was shown that cocultivation of the two cell lines increases the conversion of the inactive pro-u-PA to the active two chain u-PA. The enhancement of matrix degradation and of pro-u-PA activation requires actual binding of pro-u-PA to its receptor because it is inhibited by u-PA-receptor antagonists. The u-PA receptor must be cell associated, as binding of pro-u-PA to a receptor solubilized from the cell surface with phosphatidyl-inositol specific phospholipase C did not enhance the activation of pro-u-PA in the presence of plasminogen. The finding that activity of u-PA is enhanced when it is bound to its receptor, even when the receptor is produced by a different cell, might have important implications for the mechanisms of u-PA-induced extracellular proteolysis in vivo.     

Discovery of 2-arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-cyclo[3.2.1]octane in the 4-position as potent inhibitors of mTOR with selectivity over PI3K

2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC₅₀ <1 nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC₅₀ <50 nM).     

Human MUC1 mucin: a multifaceted glycoprotein

Human MUC1 mucin, a membrane-bound glycoprotein, is a major component of the ductal cell surface of normal glandular cells. MUC1 is overexpressed and aberrantly glycosylated in carcinoma cells. The role MUC1 plays in cancer progression represents two sides of one coin: on the one hand, loss of polarity and overexpression of MUC1 in cancer cells interferes with cell adhesion and shields the tumor cell from immune recognition by the cellular arm of the immune system, thus favoring metastases; on the other hand, MUC1, in essence a self-antigen, is displaced and altered in malignancy and induces immune responses. Tumor-associated MUC1 has short carbohydrate sidechains and exposed epitopes on its peptide core; it gains access to the circulation and comes into contact with the immune system provoking humoral and cellular immune responses. Natural antibodies to MUC1 present in the circulation of cancer patients may be beneficial to the patient by restricting tumor growth and dissemination: early stage breast cancer patients with a humoral response to MUC1 have a better disease-specific survival. Several MUC1 peptide vaccines, differing in vectors, carrier proteins and adjuvants, have been tested in phase I clinical trials. They are capable of inducing predominantly humoral responses to the antigen, but evidence that these immune responses may be effective against the tumor in humans is still scarce.     

Inclusion of ecologically based trait variation in plant functional types reduces the projected land carbon sink in an earth system model

Earth system models demonstrate large uncertainty in projected changes in terrestrial carbon budgets. The lack of inclusion of adaptive responses of vegetation communities to the environment has been suggested to hamper the ability of modeled vegetation to adequately respond to environmental change. In this study, variation in functional responses of vegetation has been added to an earth system model (ESM) based on ecological principles. The restriction of viable mean trait values of vegetation communities by the environment, called ‘habitat filtering’, is an important ecological assembly rule and allows for determination of global scale trait–environment relationships. These relationships were applied to model trait variation for different plant functional types (PFTs). For three leaf traits (specific leaf area, maximum carboxylation rate at 25 °C, and maximum electron transport rate at 25 °C), relationships with multiple environmental drivers, such as precipitation, temperature, radiation, and CO₂, were determined for the PFTs within the Max Planck Institute ESM. With these relationships, spatiotemporal variation in these formerly fixed traits in PFTs was modeled in global change projections (IPCC RCP8.5 scenario). Inclusion of this environment‐driven trait variation resulted in a strong reduction of the global carbon sink by at least 33% (2.1 Pg C yr^?1) from the 2nd quarter of the 21st century onward compared to the default model with fixed traits. In addition, the mid‐ and high latitudes became a stronger carbon sink and the tropics a stronger carbon source, caused by trait‐induced differences in productivity and relative respirational costs. These results point toward a reduction of the global carbon sink when including a more realistic representation of functional vegetation responses, implying more carbon will stay airborne, which could fuel further climate change.     

Sequence-variant repeats of MUC1 show higher conformational flexibility, are less densely O-glycosylated and induce differential B lymphocyte responses

The human epithelial cancer mucin MUC1 is able to break tolerance and to induce humoral immune responses in healthy subjects and in cancer patients. We recently showed that clusters of sequence-variant repeats are interspersed in the repeat domain of MUC1 at high frequency, which should contribute to the structural and immunological features of the mucin. Here we elucidated the potential effects exerted by sequence-variant repeats on their O-glycosylation. Evidence from in vitro glycosylation with polypeptide N-acetylgalactosaminyltransferases GalNAc-T1 and GalNAc-T2 in concert with mass spectrometric analyses of in vivo glycosylated MUC1 probes from transiently transfected HEK293 cells indicated reduced glycosylation densities of repeats with three concerted replacements: AHGVTSAPESRPAPGSTAPA. The Pro to Ala replacement in STAPA exerts not only proximal effects on the ppGalNAc-T2 preferred site at -3 and -4, but also more distant effects on the ppGalNAc-T1 preferred site at -15 (TSAPESRPAPGSTAPA). We also examined the conformational changes of MUC1 glycopeptides induced by the concerted DT to ES replacements and revealed a higher conformational flexibility of ES/P peptides compared to DT/P peptides. Differences in conformational flexibilities and in O-glycosylation densities could underlie the observed differential humoral responses in humans. We were able to show that the natural immunoglobulin G (IgG) responses to the repeat domain of MUC1 in sera from nonmalignant control subjects are preferentially directed to variant repeat clusters. In contrast, the IgG response in patients with adenocarcinoma shifted to higher frequencies of preferential DTR peptide binding.