Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the ß-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis, EB 188. Part 1. Zoospore development and physiology.

Experiments were performed to determine the effect of Aspergillus oryzae (AO) fermentation extract on zoospore development in the rumen fungus Neocallimastix frontalis EB 188. Powdered product, or liquid extract prepared from such powder, was added at the recommended value for supplementation in dairy cattle. Stationary and stirred cultures were periodically sampled and assayed for extracellular and intracellular protein and enzymes, gas production, zoospore production and maturation, and carbon source utilization. Soluble extract increased fungal physiology when grown in stirred vessels or stationary cultures. Treated cultures produced higher levels of enzymes (nearly double). Mobile zoospores matured into germination entities more rapidly in treated cultures, and when powdered product was used, nearly 3 times more motile zoospores were produced at 56 h of fungal growth. Levels of the intracellular enzyme malate dehydrogenase increased by 6-fold in the presence of powdered product. Product wheat bran carrier used as soluble extract or powder had very little effect on fungal cultures. Medium cellulose was completely hydrolyzed in all cultures but this occurred earlier in those containing AO treatment.

Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis, EB 188. Part 2. Carbon source utilization and effects on zoospore production.

The effect of a commercial Aspergillus oryzae fermentation extract on the utilization of carbon source and zoospore production by the rumen fungus Neocallimastix frontalis EB 188 was determined. In addition, the composition of a soluble extract prepared from the commercial product was analyzed. This extract was added to N. frontalis EB 188 cultures grown on a variety of substrates and periodically assayed for protein, enzymes, zoospore production, and carbon source utilization. The powdered product contained 93% dry matter, more than 3,000 A. oryzaespores per gram, and did not contain strong buffers or high concentrations of salt. Measurable concentrations of DNA, protein, carbohydrate and several enzymes including cellulase and amylase were also found. Soluble extract increased fungal physiology and treated cultures produced significantly higher levels of supernatant protein and enzymes including amylase, cellulase and beta-glucosidase. The fungal response depended on culture carbon source. However, culture zoospore production was increased regardless of substrate provided. Culture utilization of glucose was more rapid in treated cultures, yet high levels of the extract greatly inhibited glucose utilization.

The 3' untranslated region of messages in the rumen protozoan Entodinium caudatum.

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.

The role of peroxisome proliferator-activated receptor gamma in bladder cancer in relation to angiogenesis and progression.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) immunohistochemical expression was analyzed in 75 human bladder tumor specimens, where the expression of some angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PDECGF), and tumor progression markers, such as epidermal growth factor receptor (EGFr), p16, mutated p53, and normal pRB, were also analyzed. The results were then compared to the clinical and pathological characteristics of the disease. PPAR gamma was expressed more significantly in papillary tumors than in solid cancers, and its presence was associated with statistical significance to low incidence of tumor recurrence or progression. This significant association was observed also when PPAR gamma was expressed in the presence of PDECGF, which resulted, when considered alone, to an angiogenic factor typical of solid cancers and appeared related to poor prognosis. In the presence of bFGF, on the contrary, PPAR gamma expression no longer resulted to a significant association with low incidence of tumor recurrence or progression, suggesting a possible worsening role of this angiogenic factor, typical of papillary cancers, in its interaction with PPAR gamma.

Protozoal sequences may reveal additional isoforms of the 14-3-3 protein family.

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.

Fermentation extract effects on the morphology and metabolism of the rumen fungus Neocallimastix frontalis EB188.

The effects of Aspergillus oryzae fermentation extract, Amaferm, on the rumen fungus Neocallimastix frontalis EB188 were studied. The secretion of cellulase was increased by 67% and rhyzoid development was increased 3.8-fold in the presence of extract. Strength of fungal response increased in a dose-dependent manner and demonstrated a positive correlation between cell surface area and enzyme secretion. Above certain concentrations of extract, however, the development of the fungus and enzyme secretions remained at control values or slightly diminished. Supernatant fluid appearance of the intracellular enzyme, malate dehydrogenase, paralleled the secretion of cellulase both in the presence and absence of extract. Ether solubilization of extract demonstrated that the active component(s) possessed a moderately polar value between 2.7 and 2.8. Thin layer chromatography separated extract into inert, inhibitory and intensely stimulating fractions. These results support the idea that by accelerating fungal growth and metabolism, Amaferm increases the rate (or extent) of fibre degradation caused by rumen fungi and that this, in turn, may contribute to enhanced animal performance.

Phylogenetic position and codon usage of two centrin genes from the rumen ciliate protozoan, Entodinium caudatum.

A lambda phage cDNA expression library was constructed from washed suspensions of the rumen ciliate protozoan, Entodinium caudatum, which had been maintained in an isolated, monofaunated sheep. The library was screened using an anti-E. caudatum antiserum raised in rabbits against sonically disrupted protozoa, DNA sequences for two centrins or caltractins, a subfamily of the EF-hand Ca(2+)-modulated proteins which are closely related, highly conserved cytoskeletal proteins, were identified and characterised. Their phylogenetic position was established relative to other centrin gene sequences. The two proteins showed homology to Paramecium tetraurelia centrins: 50 and 52% identities and 71 and 75% similarities in the protein sequence, over 99 and 110 amino acids fragments. Codon usage and indices revealed the E. caudatum follows universal codon usage, but with a restricted number of codons, and has a low G&C content.

Reorganization of contractile systems and protein tyrosine phosphorylation in platelet aggregation.

Platelet aggregation is accompanied with reorganization of contractile systems. This event involves tyrosine phosphorylation of various proteins. When tyrosine phosphorylation is induced by an intracellular trigger as vanadate, a phosphorylated cytosolic 60 Kd protein acts as a nucleating center on which new actin filaments grow. When aggregation is induced by an extracellular signal as ristocetin, actin filaments of the membrane-associated skeleton and the 60 Kd protein bound to this structure migrate toward the cytosol and only in this soluble state the 60 Kd protein undergoes tyrosine phosphorylation and consequently triggers reorganization of contractile systems.

Enzymatic method for measuring subnanomolar concentrations of oxygen in gaseous or aqueous solutions.

A method that combines glucose oxidase with [U-14C]glucose to measure subnanomolar to decamillimolar concentrations of oxygen is presented. The method is inexpensive and easy to use. It is independent of sample pH and enzyme inhibitors, including detergents and metal ions. This method can be directly applied to gaseous or aqueous samples and multiple assays can be run simultaneously.

Production of reactive oxygen-derived species by redox reactions between Fe(II)cytochrome c and oxygen. A kinetic study.

In aerated water solutions Fe(II)Cytochrome c is slowly oxidized to Fe(III)Cytochrome c by molecular oxygen, from which superoxide anion radicals are produced (kobs = 2.7 x 10(-4) min-1 at 37 degrees C and pH = 7.3). The biological importance of this reaction has been evidenced by kinetic investigations in the presence of scavengers. In the presence of Superoxide dismutase the oxidation rate is strongly enhanced (kobs = 3 x 10(-3) min-1 at 37 degrees C). Catalase and mannitol reduce the rate constant values by 50% and 25% respectively either in the presence or in the absence of Superoxide dismutase: differences between rate constants correspond to the differences in stoichiometric redox ratios indicating that hydrogen peroxide and hydroxyl radicals are formed subsequently to the production of superoxide anion radicals.

Phospholipids metabolism in platelets stimulated with collagen.

Collagen addition to platelets suspended in Calcium-free medium induces slow shape change followed by fast aggregates formation. Time courses of membrane phospholipids metabolism and arachidonic acid oxidative metabolism indicate that phospholipase C is the immediate target of the stimulus, and subsequently phospholipase A-2 is activated by synergistic action of released calcium and protein kinase C.

Two distinct mechanisms of inhibition of platelets aggregation by acetylsalicylic acid.

The von Willebrand Factor-Ristocetin adduct activates Platelets aggregation and secretion. Acetylsalicylic acid inhibits Platelets activation by two distinct mechanisms indicating that the adduct activates Platelets by triggering at least two distinct intraplatelet metabolic pathways. The first starts from the activation of Phospholipase A-2 that produces Arachidonic acid, which, in turn, undergoes the metabolic pathway leading to Thromboxane A-2; this pathway can be blocked by the intraplatelet Acetylsalicylic acid by irreversible inactivation of Cyclooxygenase but it is insensitive to the extra-platelet Acetylsalicylic acid. The second pathway is triggered by intact von Willebrand Factor, but not by the Acetylsalicylic acid-treated one; it is insensitive to intraplatelet Acetylsalicylic acid and therefore unrelated to the Arachidonic acid metabolism.

[Effect of ASA on the interaction of von Willebrand factor with the platelet membrane]. / Effetto di ASA sull'interazione del fattore di von Willebrand con la membrana piastrinica.

Ristocetin induces a conformational change on von Willebrand Factor (vWF) similar to that due to the interaction with the subendothelium, by which the former can interact with the Glycoprotein-1 B (GPIB) of the platelet membrane and trigger aggregation and granule content secretion. Platelet Rich Plasma (PRP) treated with Acetyl Salicylic Acid (ASA) loses completely the aggregability induced by addition of Ristocetin whereas ASA-treated and successively Washed Platelets (AWP) supplemented with normal plasma (PPP) give an aggregation and a secretory response to Ristocetin similar to that given by PRP; similarly normal Washed Platelets (WP) supplemented with ASA-treated plasma (APPP) give identical aggregation, and secretion by Ristocetin addition. Ours results indicate that the Ristocetin-vWF complex can trigger two distinct intraplatelet metabolic pathways. A first well known way starts from the activation of Phospholipase A-2 (PL-A2), by which arachidonic acid is produced, that, in turn, undergoes the metabolic pathway leading to Thromboxane A-2; this pathway can be blocked by the intraplatelet ASA by irreversible inactivation of Cyclooxygenase, but it is insensitive to the extra-platelet ASA. A second, independent metabolic pathway, can be triggered by intact vWF, but not by the ASA treated one. It is insensitive to intraplatelet ASA and therefore unrelated to the arachidonic acid metabolism. This pathway could start from the activation of Phospholipase C (PL-C).

Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188.

Cellulases from the ruminal fungus Neocallimastix frontalis EB188 were separated by using hydroxylapatite column chromatography. Seven carboxymethylcellulases, six avicelases, and four beta-glucosidases accounted for the majority of the activities. The separation of enzymes was confirmed by using polyacrylamide gel electrophoresis. Electrophoretic migration, analysis of hydrolysis products, and substrate specificity measurements suggested that several different cellulases were secreted in N. frontalis EB188. The possible relationship of cellulase diversity to protein glycosylation is discussed.

Kinetic study of a cellobiase purified from Neocallimastix frontalis EB188.

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.

Glucose metabolic pathways in the anaerobic rumen fungus Neocallimastix frontalis EB188.

Primary pathways for glucose metabolism were established in the anaerobic rumen fungus Neocallimastix frontalis EB188. This highly capable cellulolytic organism demonstrated a strict anaerobic integration of metabolic pathways. Glycolysis in N. frontalis EB188 was coupled to malate dehydrogenase, 'malic' enzyme and specified hydrogenosome reactions. Pyruvate, as in most life forms, was a pivotal compound. The major fermentation products of N. frontalis EB188 were acetate, ethanol and lactate, with the concomitant generation of H2. On the basis of its unique characteristics and streamlined fermentation pathways, it was concluded that N. frontalis EB188 should be an important contributor to programs generating energy and selected chemicals from currently intractable biomass.

Supernatant protein and cellulase activities of the anaerobic ruminal fungus Neocallimastix frontalis EB188.

Protein and cellulose activities were measured in culture supernatants of the anaerobic ruminal fungus Neocallimastix frontalis EB188 established in glucose medium and switched to either glucose, cellobiose, or cellulose media. Polyacrylamide gel electrophoresis was used to show differences caused by changing medium carbon source. Culture supernatants contained proteins with molecular weights ranging from greater than 116,000 to about 19,000. Low levels of cellulose activity were evident in glucose-grown cultures. Increased amounts of slowly migrating cellulase activities appeared in the supernatants of glucose-grown cultures switched to cellulose. Cellulase activities which reacted differentially during colorimetric and in situ assays were produced. Isoelectric points of cellulase activities varied from 3.7 to 8.3, and activities possessed optimal pHs of between 5.9 and 6.5.

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants.

Messenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector gt11 with an efficiency of cloning of approximately 10(4) recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the -galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

Changes in gene position are accompanied by a change in time of replication.

The globin and immunoglobulin multigene families have been used to study the effect of chromosomal organization on the time of gene replication. Some of the genes are late-replicating, providing the first identification of late-replicating sequences that are not highly repetitive. One is a member of the mouse alpha-globin gene family, which consists of genes mapping to three different chromosomes. The other genes in this family replicate early during S. Our studies demonstrate that immunoglobulin gene rearrangements and rearrangements between these genes and the c-myc oncogene are accompanied by dramatic differences in their temporal order of replication. We conclude that a gene's position in the chromosome, rather than its sequence, determines the time of replication. We suggest that the differences in association with gene rearrangement result from changes in the proximity of the affected gene to sites that control the temporal order of replication during S.