Lectin-like characteristics of recombinant human interleukin-1beta recognizing glycans of the glycosylphosphatidylinositol anchor

We found that 35S-labeled recombinant human interleukin-1beta (rhIL-1beta) binds phosphatidylinositol-specific phospholipase C-treated human placental alkaline phosphatase, phosphatidylinositol-specific phospholipase C-treated trypanosome surface variant glycoproteins, and urinary uromodulin immobilized on plates or immobilized on CNBr-activated Sepharose 4B. The interaction between rhIL-1beta and these glycoproteins was lectin-like, since it was inhibited in the presence of specific saccharides, i.e. mannose 6-phosphate or synthetic Ac-NH.CH2.CH2. PO4--->6Manalpha1-->(+/-2Manalpha1-->+/-6Manalpha1-->) propyl at about 1 microM. On the other hand, a wide variety of compounds including biantennary sugar chains derived from these glycoproteins as well as ethanolamine phosphate, inositol phosphate, mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol 6-phosphate did not show any inhibitory effect at concentrations up to 1 mM. These results indicate that rhIL-1beta interacts with these glycoproteins via the mannose 6-phosphate diester of glycans on the glycosylphosphatidylinositol (GPI) anchor. Furthermore, when monolayers of polarized Madin-Darby canine kidney cells on polycarbonate filter membranes were incubated with 35S-rhIL-1beta in either the apical or basolateral chamber, 35S-interleukin-1beta was found to bind specifically to the apical membranes with a Ka value of 4.6 x 10(7) M-1, and the specific interaction was inhibited by 1 microM mannose 6-phosphate. Since the mannose 6-phosphate diester moiety exists only in the GPI glycans on plasma membranes, it was evident that interleukin-1beta can directly interact with the mannose 6-phosphate diester component of the intact glycan of GPI anchors on plasma membranes.     

Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18 degrees C with the membrane-impermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37 degrees C originated exclusively from the membranes containing the MesNa-accessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37 degrees C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18 degrees or 37 degrees C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18 degrees C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.     

The transjugular stent implantation for the treatment of malignant portal and hepatic vein obstruction in cancer patients

BACKGROUND: The increase in portal vascular resistance is a significant complication of metastatic disease to the liver or locally advanced cancer, e.g., biliary cancer. PATIENTS AND METHODS: This paper describes the successful palliative treatment of two cancer patients with portal hypertension presenting with the symptoms of tense ascites, mesenteric congestion, and severe variceal bleeding. By creating a stenttract between a hepatic vein and a main branch of the portal vein and/or by placing an extendable stent into the portal vein, the transjugular intrahepatic portosystemic stent-shunt (TIPS) technique was used to decompress the portovascular system. RESULTS: The TIPS-technique offers a new, safe and effective palliation for malignant portal hypertension. In both patients, the symptoms of the portal hypertension disappeared after the procedure. This was accompanied by a significant improvement of the patients performance status allowing an early ambulation. CONCLUSION: Our findings demonstrate the feasibility and effectiveness of the TIPS procedure as a minimal invasive treatment for portal vein decompression in selected tumor patients.     

The contribution of epidemiology to psychosomatic medicine

The G1 glycoprotein of California encephalitis (CE) virus plays a critical role in the infection of mosquito and mammalian cells. We found that CE virus enters baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cells by the endocytic pathway. Ammonium chloride, a lysosomotropic amine that prevents release of virus from endosomes, inhibited infection of both cell types when added within 10 min after viral adsorption. In addition, infected cells formed polykaryons when the extracellular pH was lowered to 6.3; optimal fusion occurred at pH 5.8 and 6.0 (C6/36 and BHK-21 cells, respectively). Two neutralizing G1 MAba, 6D5.5 and 7D4.5, inhibited low pH-induced syncytia formation without affecting viral attachment, suggesting a role for G1 in viral entry. Since viral fusion proteins have been demonstrated to undergo conformational changes at low pH, acid-induced changes in G1 and G2 were assessed. While both G1 and G2 demonstrated low pH-induced alterations in detergent binding, only G1 displayed an altered protease cleavage pattern at the fusion pH. These results indicate that the G1 protein of CE virus undergoes conformational changes necessary for low pH-mediated entry into both mosquito and mammalian cells.     

Kinetic evidence for low chemical processivity in ncd and Eg5

We have examined the formation of hydroxyphenols, nitrophenols, and the minor products 4-nitrosophenol, benzoquinone, 2,2'-biphenol, and 4,4'-biphenol from the reaction of peroxynitrite with phenol in the presence and absence of added carbonate. In the absence of added carbonate, the product yields of nitrophenols and hydroxyphenols have different pH profiles. The rates of nitration and hydroxylation also have different pH profiles and match the trends observed for the product yields. At a given pH, the sum of the rate constants for nitration and hydroxylation is nearly identical to the rate constant for the spontaneous decomposition of peroxynitrite. The reaction of peroxynitrite with phenol is zero-order in phenol, both in the presence and absence of added carbonate. In the presence of added carbonate, hydroxylation is inhibited, whereas the rate of formation and yield of nitrophenols increase. The combined maximum yield of o- and p-nitrophenols is 20 mol% (based on the initial concentration of peroxynitrite) and is about fourfold higher than the maximal yield obtained in the absence of added carbonate. The o/p ratio of nitrophenols is the same in the presence and absence of added carbonate. These results demonstrate that hydroxylation and nitration occur via two different intermediates. We suggest that the activated intermediate formed in the isomerization of peroxynitrous acid to nitrate, ONOOH*, is the hydroxylating species. We propose that intermediate 1, O=N-OO-CO2-, or secondary products derived from it, is (are) responsible for the nitration of phenol. The possible mechanisms responsible for nitration are discussed.     

Inference, inquiry, evidence censorship, and explanation inconnectionist expert systems

The combination of the techniques of expert systems and neural networks has the potential of producing more powerful systems, for example, expert systems able to learn from experience. In this paper, we address the combinatorial neural model (CNM), a kind of fuzzy neural network able to accommodate in a simple framework the highly desirable property of incremental learning, as well as the usual capabilities of expert systems. We show how an interval-based representation for membership grades makes CNM capable of reasoning with several types of uncertainty: vagueness, ignorance, and relevance commonly found in practical applications. In addition, we show how basic functions of expert systems such as inference, inquiry, censorship of input information, and explanation may be implemented. We also report experimental results of the application of CNM to the problem of deforestation monitoring of the Amazon region using satellite images     

Goodpasture syndrome involving overlap with Wegener's granulomatosis and anti-glomerular basement membrane disease

A 68-year-old Caucasian woman presented to the hospital with nodular pulmonary infiltrates and acute renal failure. Wegener's granulomatosis was initially considered to be most likely because of the presence of increased serum levels of c-anti-neutrophil cytoplasmic antibodies (c-ANCA). A consultation through the Internet after a renal biopsy demonstrated crescentic, necrotizing glomerulonephritis and linear deposits of immunoglobulin G (IgG) and complement C3, typical of anti-glomerular basement membrane (GBM) disease. Hemodialysis was instituted; however, the patient suddenly developed a massive cerebral hemorrhage and died before full therapy could take effect. Postmortem analysis of the patient's sera revealed high titers of IgG against the alpha 3 NC1 domain of type IV collagen. Serologic evidence of both p-ANCA and anti-GBM antibodies are becoming more frequently recognized in the setting of rapidly progressive glomerulonephritis. The patient reported here had the unusual combination of c-ANCA antibodies with anti-GBM disease, and this association raises complex questions regarding the pathogenesis of this type of renal injury.     

Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines

Certain bacterial immunostimulatory (i.s.) DNA sequences containing unmethylated CpG motifs stimulate antigen-presenting cells (APC) to express a full complement of costimulatory molecules and to produce cytokines including interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha. While IL-12 is key to their T helper cell (Th)1-promoting adjuvant activity, secretion of toxic levels of TNF-alpha is harmful in that it promotes toxic shock. Given the beneficial as well as harmful consequences of i.s. DNA, we investigated the possibility of identifying DNA sequences, i.e. CpG oligodeoxynucleotides (ODN) which differentially activate IL-12 versus TNF-alpha cytokine production in APC. Here, we describe an i.s. DNA sequence with these characteristics. While its potential to induce IL-12 is preserved, its ability to trigger TNF-alpha release is strongly curtailed both in vitro and in vivo. I.s. DNA could be segregated into lethal and non-lethal in a mouse toxic shock model. The non-toxic i.s. DNA was useful as an adjuvant, thus allowing cytotoxic T cell responses to the soluble protein ovalbumin and conferring a resistant Th 1 phenotype to BALB/c mice lethally infected with Leishmania major. This i.s. CpG motif may thus be prototypic for a useful immunostimulating DNA sequence that lacks harmful side effects.