Search PubMed⌕ Search

Biomedical subjects

A Corti

Publications and source records attributed to A Corti.

At least 73 records · Page 4Linked to original sources
PubMed1995

Production of soluble tumor necrosis factor receptor type I in Escherichia coli: optimization of the refolding yields by a microtiter dilution assay.

In this study optimization of the soluble tumor necrosis factor receptor type I (sTNF-RI) refolding by the use of a micro-renaturation assay in 96-well microplates is described. Microplate wells were filled with buffers varying in pH and urea and substrate concentration. Denatured and reduced sTNF-RI was then rapidly diluted and allowed to refold for a variable time at different temperatures. The extent of renaturation was measured by a sandwich enzyme-linked immunosorbent assay (ELISA), based on the use of two monoclonal antibodies obtained against urinary sTNF-RI. Among about 100 different combinations tested, a maximum refolding yield of 21.5% has been obtained in 100 mM Tris, pH 8-8.5, 1 mM EDTA, 0.1% bovine serum albumin, 2 M urea, at a denatured protein concentration of 10 micrograms/ml and at 26 degrees C. Folded sTNF-RI was purified by batchwise immunoaffinity chromatography and its activity evaluated by immunological and biological assays. A good correlation was observed between the data obtained with different assays (biological assay, ligand-directed ELISA, and double-determinant sandwich ELISA) indicating that the refolded receptor has gained biological and immunological reactivity comparable to those of the soluble TNF-receptor type I expressed in eukaryotic cells.

Animals↗
PubMed1995

Mechanism of suramin-induced deoligomerization of tumor necrosis factor alpha.

Deoligomerization of human tumor necrosis factor alpha (TNF), spiked with 125I-labeled form, was studied quantitatively using size-exclusion chromatography and off-line monitoring with a gamma-counter. A detailed investigation of the oligomeric state of TNF was carried out as a function of its own concentration (0.3-7500 nM referred to the subunit, M(r) 17,000) in the absence or in the presence of various amounts (10, 100, 1000 microM) of suramin, an inhibitor of TNF biological activity in vitro, which promotes TNF deoligomerization. The dependence of trimeric form content on total TNF concentration was modeled with a sequential dissociation process (trimer-->dimer-->monomer) assuming an identical dissociation constant for each step, Kd1 = 0.2 nM. This model was used as the simplest for data fitting although, generally, no chromatographic resolution of dimeric species could be obtained. Best fitting of all data could be achieved with a model including a conformational change of TNF trimer into a state more prone to deoligomerization (Kd2 = 400 nM), which was favored by suramin binding. A kinetic study of TNF dissociation by the same method produced values for the deoligomerization rate of trimer: on the average, koff approximately 4 x 10(-5) S-1 (t1/2 approximately 5 h) between 4 and 20 degrees C with little dependence on suramin concentration; at 37 degrees C, a sizable increase is observed in the presence of 1 mM suramin (koff = 2.3 x 10(-4) S-1, t1/2 = 0.8 h). Data of suramin inhibition on TNF receptor binding, as obtained after incubation times much shorter than the above half-life of trimer, indicate that suramin binding to TNF trimer is the early mechanism of receptor binding inhibition.

Humans↗
PubMed1995

Biodegradation of nonionic surfactants. I. Biotransformation of 4-(1-nonyl)phenol by a Candida maltosa isolate.

Results are reported concerning biodegradation of 4-(1-nonyl)phenol by cultures of a Candida maltosa strain isolated from aerobic sludge samples collected at a depuration plant treating wastewaters from a textile industry. The yeast was able to utilize 4-(1-nonyl)phenol as a sole carbon and energy source. Preliminary attempts to draw the actual metabolic pathway evidenced microbial attack on the alkyl chain with the production of 4-acetylphenol. To the best of our knowledge this is the first report describing a microorganism capable of attacking nonylphenol in axenic culture and at the same time allowing for the identification of its degradation products.

Journal Article↗
PubMed1995

Identification of two forms (31-33 and 48 kD) of the urinary soluble p55 tumor necrosis factor receptor that are differentially N- and O-glycosylated.

The structure and the activity of urinary soluble TNF receptor type 1 (sTNF-R1), isolated from the urine of normal individuals, has been characterized and compared with that of recombinant sTNF-R1 expressed in CHO cells and with that of a nonglycosylated form expressed in Escherichia coli. Urinary sTNF-R1 was resolved in a major band of 31-33 kD and in a 48 kD band (less than 5% of total) by reducing SDS-PAGE; CHO sTNF-R1 was resolved in two bands of 29 and 31 kD. All bands were recognized by various anti-sTNF-R1 antibodies as well as by TNF-alpha in western and ligand blotting assays. No cross-reaction was observed with anti-TNF-R2 antibodies. N- and O-glycosylation studies indicated that (1) the 29-31 kD recombinant form as well as the 31-33 kD urinary form are N-glycosylated; (2) the differences between the 29-31 and 31-33 kD recombinant and natural products are mainly related to differences in the N-linked sugar content; and (3) the 48 kD sTNF-R1 isolated from urine also contains O-linked sugars. The urinary sTNF-R1 antigen mixture was able to inhibit TNF-alpha cytotoxicity with a potency comparable to that of nonglycosylated E. coli sTNF-R1. At variance, urinary sTNF-R1 was able to inhibit TNF-beta sevenfold more efficiently than E. coli sTNF-R1. In conclusion, two subtypes of sTNF-R1 have been isolated from urine: a main N-glycosylated form of 31-33 kD and a N- and O-glycosylated form of 48 kD that appears to be a minor constituent of the urinary sTNF-R1 antigen.

Animals↗
PubMed1994

Tumor necrosis factor (TNF) alpha quantification by ELISA and bioassay: effects of TNF alpha-soluble TNF receptor (p55) complex dissociation during assay incubations.

It has been previously reported that different quantitative results can be obtained when TNF alpha is measured in biological fluids by bioassay and immunoassay. This is thought to be related to the presence of antigenic forms of TNF alpha that cannot be detected by bioassay, such as complexes with soluble receptors (sTNF-R) or TNF alpha monomers. In this work we have observed discrepancies between antigenic and bioactive TNF alpha even when we used a sandwich-ELISA, unable to detect TNF alpha monomers, based on antibodies that bind epitopes overlapping with the soluble-receptor binding site of TNF alpha. Moreover, we found that antigenic TNF alpha levels in the presence of p55-sTNF-R (sTNF-R1) measured by different immunoassays were variable, depending on the immunoreagents and incubation time. To investigate whether TNF alpha-soluble receptor complex dissociation occurring during assay incubations contributes to the variability of results, we studied the kinetics of TNF alpha-soluble receptor interactions and examined the effect of complex dissociation using different analytical systems. TNF alpha association (k(on)) and dissociation (koff) rate constants with sTNF-R1, measured by real-time biospecific interaction analysis, were 5.01 x 10(5) s-1 M-1 and 2.8 x 10(-4) s-1, corresponding to an equilibrium constant (Kd) of 0.59 nM and to a half life for these complexes of 38 min. Complex dissociation and differential changes in the TNF alpha-sTNF-R1 bound:free ratio, in different analytical systems, markedly affects TNF alpha quantification.

Binding, Competitive↗
PubMed1994

Gene relaxation and aging: changes in the abundance of rat ventral prostate SGP-2 (clusterin) and ornithine decarboxylase mRNAs.

Sulfated glycoprotein 2 (SGP-2) mRNA progressively increased in the ventral prostate of the aging rat, reaching, at 24 months, 4-fold higher than at 3 months. Ornithine decarboxylase (ODC) mRNA peaked at 6 months (4-fold increase), and at 12 and 24 months was maintained at higher levels than at 3 months. ODC enzymatic activity was enhanced at 6 months to a much smaller extent than its own mRNA, the values at 12 and 24 months dropping to below those at 3 months. Putrescine (Put), spermidine (Spd) and spermine (Sp) concentrations also peaked at 6 months (100% increase for Put, 50% for Sp and Spd). At 24 months, Put and Spd were diminished, and Sp was unchanged with respect to the 3-month values. Under the same conditions, glyceraldehyde-3-phosphate dehydrogenase mRNA did not undergo significant alterations.

Aging↗
PubMed1994

Idiotope determining regions of a mouse monoclonal antibody and its humanized versions. Identification of framework residues that affect idiotype expression.

The contribution of framework regions (FRs) of antibody-variable domains to idiotype expression was studied by examining the interaction of various "humanized" versions of a mouse anti-TNF alpha monoclonal antibody (mAb78) with polyclonal and two monoclonal antibodies (mAb1G3 and mAb9F1), generated against the mAb78 idiotype. Humanized mAb78, bearing human constant domains and mouse complementarity-determining regions (CDRs) inserted with human FRs, was found to be five to sevenfold less reactive than mAb78 with polyclonal anti-idiotype antibodies and 200 to 300-fold less active in neutralizing TNF alpha. The substitution of heavy-chain FRs residues of the humanized antibody with original mouse residues 28 to 30, 48 to 49, 67 to 68, 70 to 71, 78, 80 and 82 progressively restored the immunoreactivity with polyclonal immunoglobulin Gs to the level of a version having mouse heavy chain and human light chain FRs, and increased 10 to 20-fold the TNF alpha neutralizing activity. This suggests that at least some of these residues are critical for TNF alpha binding as well as for the expression of idiotopes that are strongly immunogenic in syngeneic animals. All antibody versions with either human or mouse FRs were able to bind to various extents mAb1G3, a gamma-type anti-Id antibody that inhibits mAb78/TNF alpha interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an alpha-type anti-Id antibody unable to block the access of TNF alpha to mAb78 paratopes. Substitution of heavy chain FR residues 28 to 30 markedly decreased the binding of mAb1G3 (100 to 1000-fold). This suggests that these antibodies recognize CDR and FR idiotopes, respectively, that can be drastically modified by changes in the FRs. In conclusion, the results suggest that CDRs as well as FRs markedly contribute to antibody Id expression. Although strongly immunogenic idiotopes are probably located within the CDRs, the results also suggest that some FR residues are critically involved in shaping antibody Id diversity by affecting the structure of CDR-related idiotopes.

Amino Acid Sequence↗
PubMed1994

Changes in circulating levels of atrial natriuretic factor (ANF) during orthotopic liver transplantation in humans.

Atrial natriuretic factor (ANF) is a 28 amino acid peptide secreted by the atrial cardiocytes. Clearance is via the lung (50%) and the liver (25%). The main stimulus to ANF secretion is atrial distension but vasoconstrictors, sympathetic stimulation, catecolamines and tachycardia are able to enhance its circulating blood levels. ANF blood concentrations were measured during orthotopic liver transplantation in six postnecrotic cirrhotic patients. Significant increases in ANF blood levels occurred at the end of the anhepatic phase (P < or = 0.02 vs baseline) associated with low cardiac filling pressures (P < or = 0.02 vs baseline) and increased systemic vascular resistances (P < or = 0.02 vs preanhepatic phase). Aldosterone blood levels showed a similar behaviour, increasing significantly (P > or = 0.001 vs baseline) at the end of the anhepatic phase. ANF fell after reperfusion of the graft and returned towards baseline values at the end of the procedure. Since most of the total body clearance of ANF is performed by the lungs, its sharp increase at the end of the anhepatic phase could be considered a counterregulatory response to vasoconstricting stimulation and to fluid-sparing mechanisms in the presence of relative hypovolaemia. Its decrease after reperfusion could be related to volume normalization and partly to the enhanced clearance performed by the newly grafted liver.

Adult↗
PubMed1994

Identification of an epitope of tumor necrosis factor (TNF)-receptor type 1 (p55) recognized by a TNF-alpha-antagonist monoclonal antibody.

The relationships between epitope topography and agonistic/antagonistic effects of anti-TNF receptor type 1 (TNF-R1) antibodies on TNF-alpha cytotoxic activity have been studied. To this purpose various monoclonal antibodies (mAbs) against the soluble form of TNF-R1 (sTNF-R1) have been generated and characterized. Epitope topography studies identified at least four distinct epitopes located outside (4E10) or within (or close to) the TNF-alpha binding site of urinary sTNF-R1 (7H3, 4C1, 9B11). mAbs 7H3 and 4C1 were able to neutralize the inhibition of human TNF-alpha cytotoxicity on L-M cells by sTNF-R1, while 4E10 was unable. Moreover, 7H3 and 4C1 were able to antagonize the TNF-alpha cytotoxicity on human U937 cells, while they were uneffective on mouse L-M cells, suggesting that these antibodies recognize, in a species-specific mode, also the membrane form of the human receptor. No agonistic effects were observed when these antibodies were used in the absence of TNF-alpha. Epitope topography studies carried out using overlapping decapeptides covering most of the sTNF-R1 sequence showed that residues 143-148 of the fourth cysteine-rich domain of the receptor (FFLREN) contain antigenic determinants recognized by the antagonist antibody 7H3. These results suggest that at least part of residues 143-148 of sTNF-R1 are surface exposed on the soluble as well as on the membrane forms of TNF-R1 and are accessible to TNF-alpha antagonists.

Amino Acid Sequence↗
PubMed1993

Suramin induces deoligomerization of human tumor necrosis factor alpha.

Suramin inhibits the biological activity of human tumor necrosis factor alpha (TNF) through a direct action on the ligand rather than on its receptors (Grazioli, L., Alzani, R., Ciomei, M., Mariani, M., Restivo, A., Cozzi, E., and Marcucci, F. (1992) Int. J. Immunopharmacol. 14, 637-642). In order to clarify the mechanism whereby suramin leads to inhibition of TNF, we investigated the possibility that suramin might modify the quaternary structure of TNF which is biologically active as a trimer. For this purpose we used a new assay (double streptavidin sandwich assay) designed for the rapid detection of oligomer-monomer conversion of proteins. Taking advantage of this assay we observed, upon incubation with suramin, dissociation of TNF. Suramin-induced dissociation of TNF was confirmed by gel filtration chromatography. Under conditions of partial dissociation, two molecular species were separated. One of higher molecular weight, corresponding to trimeric TNF, was biologically active, whereas the other, corresponding to monomeric TNF, was inactive. These results are at variance with others recently reported, where suramin has been shown to induce microaggregation of several polypeptides (Middaugh, C. R., Mach, H., Burke, C. J., Volkin, D. B., Dabora, J. M., Tsai, P. K., Bruner, M. W., Ryan, J. A., and Marfia, K. E. (1992) Biochemistry 31, 9016-9024). This suggests that suramin inhibits the bioactivity of different protein molecules through opposite effects on their quaternary structure. The present results are, to our knowledge, the first demonstration of a drug inhibiting a target molecule through dissociation of its quaternary structure.

Animals↗
PubMed1993

Evidences that syngeneic alpha-type anti-idiotypic antibodies may non-competitively inhibit idiotype/oligomeric antigen interactions by affecting idiotype avidity.

The effects of syngeneic anti-Id antibodies on the multivalent interaction between human TNF-alpha, a homotrimeric Ag, and an anti-TNF mAb (mAb(1)78) have been studied. Eight anti-mAb(1)78 Ig secreting hybridoma, able to inhibit TNF binding in a competitive or non-competitive mode, have been generated. Two representative clones (mAb(2)1G3 and mAb(2)9F1) were selected for studying the inhibition mechanism of TNF-mAb(1)78 interaction. Idiotype-paratope topography studies indicated that mAb(2)1G3 (IgG2a) and mAb(2)9F1 (IgG1) bind two sterically distinct idiotopes on mAb(1)78 (IgG1) V regions. In particular, mAb(2)1G3 was found to bind an idiotope located within (or spatially close to) the Ag combining site suggesting that competitive inhibition of TNF binding to mAb(1)78 by mAb(2)1G3 occurs through paratope blockade. On the other hand, mAb(2)9F1 recognizes an idiotope located outside the paratope, being able to bind mAb(1)78 even in the presence of saturating amounts of TNF. mAb(1)78-TNF molar ratio in complexes, at stoichiometric equivalence, was unchanged in the presence of a large excess of mAb(2)9F1, suggesting that the functional bivalency of mAb(1)78 was not impaired by this anti-Id antibody. However, bivalent mAb(2)9F1 was able to partially inhibit the binding of bivalent mAb(1)78 to oligomeric TNF in liquid-phase as well as in solid-phase assays, whereas no inhibition was observed with monovalent mAb(2)9F1-F(ab) or mAb(1)78-F(ab). This suggests that inhibition is based on a decrease of the avidity of bivalent mAb(1)78 and not on allosteric effects on antigen binding sites. The effect of mAb(2)9F1 on mAb(1)78 arm flexibility and paratope orientation is discussed. In conclusion, the results indicate that anti-Id antibodies may inhibit Ag-antibody multivalent interactions by paratope blockade or by affecting the antibody avidity.

Animals↗
PubMed1993

Kinetic analysis of TNF-alpha oligomer-monomer transition by surface plasmon resonance and immunochemical methods.

In this work we have studied the kinetic parameters of oligomeric tumour necrosis factor alpha (TNF-alpha) dissociation using biospecific interaction analysis (BIA), based on surface plasmon resonance (SPR) of TNF-alpha immobilized on a sensor chip, and by an ELISA technique able to detect TNF-alpha oligomers in solution. Validation studies, carried out with sensor chips bearing TNF-alpha oligomers or bovine albumin monomers, verified that: (a) TNF-alpha can be immobilized in the oligomeric form onto sensor chips; (b) the covalent linkage between TNF-alpha and sensor chips is stable under the experimental conditions; (c) TNF-alpha monomers are present on the sensor chips after dissociation; (d) immobilization and dissociation rate constant (kdiss) measurements are reproducible. The kdiss of recombinant TNF-alpha, measured under non denaturing conditions at pH 7.4 by BIA and ELISA were in good agreement, being 0.92 x 10(-5)/s and 1.1 x 10(-5)/s respectively (corresponding to a half life of about 20.9 h and 17.5 h, respectively). The dissociation rate was found to be significantly affected by the presence of detergents and by the pH of the solution, suggesting that TNF-alpha, at low concentrations, exists in solution with different molecular forms depending on the time of storage and buffer composition. Real-time BIA is rapid and does not require particular antibodies or reagents. Thus, the stability of the quaternary structure of natural or recombinant TNF-alpha from human or animal species as well as that of other oligomeric cytokines can probably be studied using this method.

Animals↗
PubMed1993

Mode of interaction between tumor necrosis factor alpha and a monoclonal antibody expressing a recurrent idiotype.

Tumor Necrosis Factor alpha (TNF alpha) is an inflammatory cytokine which exists mainly as a 51kD complex built up of 3 identical, noncovalently-linked polypeptide subunits. We have raised monoclonal antibodies (mAb) against human TNF alpha (huTNF alpha). One of these mAb (mAb78, mouse IgG1k) was studied in detail. mAb78 expresses a recurrent idiotype typical of the BALB/c anti-huTNF alpha antibody response. HuTNF alpha bound to mAb78 with an affinity constant (Kobs) of 3.2 x 10(10)M-1. The number of huTNF alpha-binding sites per mAb78 molecule was approximately 0.7. At concentrations higher than the Kobs mAb78 neutralized huTNF alpha at a approximately 1.3:1 molar ratio. mAb78 precipitated huTNF alpha in a double immunodiffusion assay in agar. Gel-filtration experiments of mAb78-huTNF alpha mixtures, that had been set up in large antigen excess, detected complexes of 570 kD as the smallest ones formed under these conditions. We propose that these results are accommodated best by a model according to which cyclic complexes built up of 3 mAb78 and 2 huTNF alpha molecules are the smallest units formed upon interaction of the reagents. In view of this model we discuss how huTNF alpha and mAb78 can undergo a precipitin reaction.

Animals↗
PubMed1992

Antithrombin III supplementation during orthotopic liver transplantation in cirrhotic patients: a randomized trial.

Severe intraoperative bleeding is one of the main problems during liver transplantation. Acquired hemostatic defects, namely primary or secondary hyperfibrinolysis, are considered significant pathogenetic events. Antithrombin III (ATIII), the main physiological serine protease inhibitor, has a critical role in the regulation of hemostasis. 29 patients with post necrotic cirrhosis undergoing liver transplantation were randomized to receive or not ATIII replacement therapy before the induction of anaesthesia and thereafter throughout surgery. Activation of both coagulation and fibrinolysis (increase of thrombin-antithrombin complexes, fibrin and fibrinogen degradation products) were demonstrated in both groups. Blood loss and transfusion requirements were not affected by ATIII administration.

Adult↗