Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation.

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. Here we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in cell proliferation, suggesting involvement of the Ras-MAP kinase pathway. Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein Shc to beta4. Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min. These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Bound Tris confounds the identification of binding site residues in a paraquat single chain antibody.

We produced an anti-paraquat single chain antibody (scFv) to investigate its potential use in immunotherapy for paraquat poisoning. However, this scFv was expressed in an insoluble form and only displayed moderate binding affinity. An earlier examination of the pH dependence of antigen binding by the parent paraquat-specific mAb (7D7-3) suggested that the electrostatic effects of a tyrosine residue were important. The aims of the current study were to obtain expression of a soluble scFv (D10) and to increase its binding affinity. The former was achieved by expression in a phagemid vector. Site-directed mutagenesis of tyrosine residues in CDR H3 did not result in improved affinity for paraquat, suggesting that the original pH dependence required re-examination. Nuclear magnetic resonance studies of 7D7-3 Fab revealed that the original observation of the pH-dependent paraquat binding with a mid-point of approximately pH 8.9 was due to tightly bound Tris. It appears that as Tris is titrated to a neutral species the energetically unfavourable juxtaposition of its positive charge with that of paraquat is reduced. These findings have broad implications in the interpretation of the pH or salt dependence of any antibody-antigen interaction which should be made cautiously and with regard to the possible interference of buffer components introduced during the preparation of the antibody.

Studies of protein interactions by biosensor technology: an alternative approach to the analysis of sensorgrams deviating from pseudo-first-order kinetic behavior.

A procedure for evaluating the thermodynamic equilibrium constant by kinetic analysis of sensorgrams which deviate from the pseudo-first-order kinetic behavior predicted for 1:1 interactions between ligate and affinity sites on the sensor surface is described. This analysis employs quantitative expressions that are used in conventional kinetic characterization of protein interactions by biosensor technology, but with the equilibrium sensorgram response fixed at a predetermined magnitude. Simulated sensorgrams for situations in which the aberrant kinetic behavior reflects (i) heterogeneity of affinity sites and (ii) isomerization of the complex between ligate and affinity sites are used to explore the feasibility of the approach. Its application is then illustrated with BIAcore studies of the interaction between the Fab fragment of an anti-paraquat monoclonal antibody and immobilized antigen in the form of a paraquat analog attached covalently to the sensor surface. Studies with an extremely high degree of antigen substitution on the sensor surface yielded sensorgrams that deviated markedly from pseudo-first-order kinetic behavior. However, they yielded the same binding constant (3 x 10(6) M-1) as the value deduced by conventional analysis of sensorgrams that conformed with pseudo-first-order kinetics because of a much lower concentration of immobilized antigen on the sensor surface. Such identity of binding constants eliminates heterogeneity of immobilized paraquat sites as the likely source of the aberrant kinetic behavior.

Inhibition of chicken liver carboxylesterase by activated carbonyls and carbonyl hydrates.

Identical Kcat values (approximately 40 s-1) are obtained for the chicken liver carboxylesterase catalyzed hydrolysis of phenyl, p-nitrophenyl and o-nitrophenyl benzoates providing support for the involvement of an acyl-enzyme pathway, with the rate-limiting deacylation of a common benzoyl-enzyme intermediate. Chicken liver carboxylesterase catalyzed fragmentation of (E)-benzilmonoxime O-2,4-dinitrophenyl ether shows a pH dependence on a group active in the free base form with a pK'a approximately 5.0. The Ki-pH profile for benzil inhibition shows a dependence on a similar group with a pK'a = 5.4. The reactions between chicken liver carboxylesterase and the hydrated aldehyde, chloral hydrate, have shown this compound to be at once a substrate and potent inhibitor of the enzyme. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is first dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent aldehyde produces a hemiacetal adduct. The Ki value for chloral hydrate inhibition calculated from the kinetic analysis (90 nM) compares favourably with the value measured from the steady-state kinetics (87 nM).

New catalytic roles for serine esterases: a 19F-NMR study of the interaction of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone with chicken liver carboxylesterase.

The reactions of 3,3,3-trifluoro-2,2-dihydroxy-1-phenyl-1-propanone (TDPP) with chicken liver carboxylesterase have shown that this ketone hydrate is not only a potent inhibitor of the enzyme, but also a substrate for a number of enzyme-catalyzed reactions. The kinetics of inhibition are consistent with a mechanism in which the bound hydrate is initially dehydrated in a rate-limiting step catalyzed by the enzyme. Nucleophilic attack by the active-site serine on the parent ketone then produces a hemiketal adduct. However, the slow reactivation (by dialysis) of TDPP-inhibited enzyme indicates that the interaction with this inhibitor is more complex. At equilibrium, a dissociation constant of 2.4 pM was obtained for this interaction. 19F-NMR studies of the enzyme-TDPP complex show that after pre-equilibration, the major adduct is not the hemiketal adduct. It is proposed that this final adduct is a cross-linked adduct formed between TDPP, the active-site serine and the active-site histidine. 19F-NMR studies reveal that chicken liver carboxylesterase catalyses the cleavage of TDPP to yield either fluoride ion or trifluoroacetate, and also the benzilic acid rearrangement of TDPP to alpha-trifluoromethylmandelate. These products have also been identified in model studies of the reaction between TDPP and imidazole.

Conversion of irinotecan (CPT-11) to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), by human liver carboxylesterase.

We have investigated the conversion of the novel anti-topoisomerase I agent CPT-11 (irinotecan; 7-ethyl-10[4-(1-piperidino)-1-piperidno]carbonyloxycamptothecin ) to its active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin), by human liver carboxylesterase (HLC). Production of SN-38 was relatively inefficient and was enzyme deacylation rate-limited with a steady-state phase occurring after 15-20 min of incubation. This later phase followed Michaelis-Menten kinetics with an apparent Km of 52.9 +/- 5.9 microM and a specific activity of 200 +/- 10 mumol/sec/mol. However, the total enzyme concentration estimated from the intercept concentrations of SN-38 was much lower than that estimated directly from the titration of active sites with paraoxon (0.65 vs. 2.0 microM, respectively). Because deacylation rate-limiting kinetics result in the accumulation of inactive acyl-enzyme complex, we postulated that incubation of CPT-11 with HLC would result in an inhibition of the HLC-catalysed hydrolysis of p-nitrophenylacetate (p-NPA), an excellent substrate for this enzyme. Indeed, this was found to be the case although complete inhibition could not be attained. Analysis of possible kinetic schemes revealed that the most likely explanation for the disparity in estimated enzyme concentrations and the incomplete inhibition of p-NPA hydrolysis is that CPT-11 also interacts at a modulator site on the enzyme, which profoundly reduces substrate hydrolysis. Furthermore, loperamide, a drug often used for the treatment of CPT-11-associated diarrhea, was found to inhibit both CPT-11 and p-NPA HLC-catalysed hydrolysis, most likely by a similar interaction. These observations have direct implications for the clinical use of CPT-11.

Production of a paraquat-specific murine single chain Fv fragment.

Producing an effective antidote against poisoning by the herbicide paraquat (PQ) has proven to be an elusive goal. One approach that holds some promise is immunotherapy with antibody fragments. In this study we detail the production of a single chain Fv fragment (scFv) specific for paraquat by linking cloned heavy (Vh) and light chain (VL) variable region genes via the peptide spacer (Gly4-Ser)3. These genes were obtained from hybridoma cells secreting a PQ-specific murine monoclonal antibody. The scFv (28 kDa) was expressed at 4% of the total cell protein by the Escherichia coli vector, pPOW, but was associated with the membranes. After solubilization and reduction, the scFv was renatured by rapid dilution. Western blotting confirmed that the refolded scFv had similar structural properties to the parental mAb. The isoelectric point of the scFv (7.0) is equal to the value calculated from the deduced amino acid sequence. Surface plasmon resonance was used to demonstrate specific PQ binding by the refolded scFv(Ka=1.24x10(6)M(-1)) which is similar to that determined for the parent Fab fragment (4.6x10(6)(-1)).

Prevention of paraquat toxicity in suspensions of alveolar type II cells by paraquat-specific antibodies.

1. The herbicide, paraquat, is accumulated by the energy-dependent polyamine uptake pathway of alveolar type II cells. There it undergoes redox cycling that results in an amplified production of toxic reactive oxygen species and depletion of NADPH and other reducing equivalents. These processes account for the lung being the major target organ for paraquat toxicity. 2. We postulated that paraquat-specific antibodies would inhibit the uptake of the herbicide by type II cells and prevent its toxicity. Accordingly, we examined the effects of paraquat-specific monoclonal antibodies and Fab fragments on the uptake, efflux and cytotoxicity of 50 microM paraquat in suspensions of alveolar type II cells isolated from the rat. 3. The uptake of paraquat was linear over 40 min. Over this time, the uptake rate was inhibited significantly (% inhibition, 73-89) by IgG (25 or 50 microM) or Fab fragments (50 or 100 microM). 4. The apparent efflux rate of paraquat, studied over 16 h, was increased significantly from 0.12 h-1 for the control cells in medium to 0.17 h-1 by paraquat-specific Fab fragments but was unaffected by the specific IgG. 5. Cytotoxicity was determined by measuring the release of 51Cr from the cells. The cytotoxicity of 50 microM paraquat was decreased significantly (percent decrease, 56-80%) in the presence of specific antibodies. 6. These studies in vitro suggest some potential for immunotherapy in selected cases of paraquat poisoning.

Polyclonal amanitin-specific antibodies: production and cytoprotective properties in vitro.

The amanitins found in several mushroom species are responsible for many deaths every year. Based on its successful application to cardiac glycoside overdose, immunotherapy could be applicable to amanitin toxicity. Therefore, we produced polyclonal amanitin antibodies by immunizing rabbits with a novel conjugate of alpha-amanitin. Purified antibodies had an average association constant for alpha-amanitin of 1.3 x 10(9) M-1. A partially protective effect of the antibodies against amanitin toxicity in vitro in Chang cells was evident at a molar ratio of antibody binding sites to alpha-amanitin of 4:1. Together with reported studies in vivo, these investigations indicate the potential of immunotherapy for amanitin poisoning.

Quantitation of paraquat in biological samples by radioimmunoassay using a monoclonal antibody.

We have developed a sensitive and specific radioimmunoassay for the quantitative determination of paraquat in plasma, urine, and bronchoalveolar lavage fluid using a monoclonal antibody. The synthesis of the iodinated paraquat tracer is novel and less complicated than a previously reported method. Regardless of the type of biological sample analyzed, the sensitivity of the assay was 0.46 ng/ml in a 200-microliters aliquot. The results correlated well with those of another assay performed in a separate laboratory. Paraquat concentrations were determined in plasma and in urine of a dog over several days after the intravenous administration of 7.48 mg paraquat cation/kg and in bronchoalveolar lavage fluid obtained 34 hr after the 2-hr infusion was discontinued. Concentrations of paraquat measured ranged from 14.1 to 0.03 micrograms/ml in plasma and 2034 to 0.36 micrograms/ml in urine. Concentrations of paraquat in plasma obtained at various times after the ingestion of paraquat by three patients ranged from 51.0 to 0.010 micrograms/ml.

Competition between paraquat and putrescine for uptake by suspensions of rat alveolar type II cells.

Paraquat and the structurally similar polyamines, such as putrescine and spermidine, are accumulated actively and selectively by the alveolar type II cells via the polyamine uptake system. We report the uptake kinetics of paraquat and putrescine and their mutual inhibition in freshly isolated rat type II cell suspensions. The uptake of paraquat by type II cells exhibited saturation kinetics and could be inhibited in a concentration-dependent manner by putrescine. By applying enzyme kinetic analysis to our experimental data it was demonstrated that the uptake of paraquat or putrescine is inhibited in a partially competitive manner by the respective inhibitor. Thus, we postulate that the polyamine uptake pathway in type II cells for paraquat and putrescine has two separate sites, one for each substrate, and that binding of one leads to a conformational change in the other.

Cloning and analysis of a cDNA encoding a human liver carboxylesterase.

A human liver carboxylesterase (CE)-encoding cDNA has been cloned using synthetic oligodeoxyribonucleotides (oligos) based on the known amino acid (aa) sequences of rabbit and rat liver CEs. The oligos hybridize specifically to DNA encoding liver CEs. The longest cDNA obtained from screening several cDNA libraries encodes about 80% of the protein and translates into an aa sequence which has a high degree of similarity with the sequences of liver CEs from other species. On hybridization to mRNA isolated from human liver, the cDNA gave a single band of about 2.0 kb consistent with its encoding a protein of less than 68 kDa. DNA obtained from a number of human livers and probed with the CE cDNA gave identical hybridization patterns. These patterns were moderately complex by comparison with published data.

Adaptation of the Müller method to allow quantitative characterization of the affinity and cross-reactivity of antibodies by competitive radioimmunoassay.

A quantitative expression is derived for the evaluation of antigen-antibody affinity constants from radioimmunoassays for the completely general situation in which antigen and antibody are both multivalent. The theoretical analysis is then extended to encompass quantitative characterization of the competitive inhibition observed in screening tests for cross-reactivity of antibody with structural analogs of the eliciting antigen. These procedures are illustrated with a radioimmunological study of the cross-reactivity of a desipramine-elicited monoclonal antibody with other tricyclic antidepressants. An unexpected finding to emerge from this immunochemical study is the demonstration that a single affinity constant suffices to describe the interaction of desipramine with a polyclonal antibody elicited by this univalent antigen.

Survival after unexpected high serum methotrexate concentrations in a patient with osteogenic sarcoma.

An 18-year-old female patient receiving adjuvant chemotherapy for osteogenic sarcoma developed a pruritic erythematous rash during infusion of the eighth dose of methotrexate (8 g/m2) in the series. In other respects, the infusion proceeded normally but the 24-hour serum concentration of methotrexate was unexpectedly and extremely high, 574 mumols/L. Dosing error was excluded, as was the hypothesis that the high concentrations were due to the presence of methotrexate-specific antibodies. Acute oliguria and renal failure were the primary manifestations of the drug-induced toxicity and the high concentrations can be attributed to decreased renal elimination of the drug over the first 24 hours. Treatment consisted of folinic acid rescue, forced diuresis, sequential charcoal haemoperfusion and haemodialysis, and repeated oral doses of activated charcoal. After examination of the contribution of the extracorporeal procedures and the charcoal to the elimination of the drug, the relative lack of morbidity was attributed primarily to the folinic acid rescue and the intensive supportive care.

The importance of electrostatic interactions in the binding of paraquat to its elicited monoclonal antibody.

In this study the pH-dependent interactions interactions between a paraquat-specific murine monoclonal antibody and two antigens: paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride), and a p-nitrophenol analogue (1-(N-methyl-4,4'-bipyridinium)-1-(2-hydroxy-5-nitrophenyl)methane dichloride; PQNP) were determined by ELISA. In each case the KAS-pH profile reflected the titration of a single amino acid residue. pK'a values derived from these plots were 8.90 (paraquat) and 8.13 (PQNP). Increasing pH led to a significant increase in the association constant for each antibody complex. A spectrophotometric titration of PQNP in the presence and absence of excess antibody indicated the presence of another charged amino acid residue at the binding site, which could be assigned as a carboxylic acid. From these studies, a model for paraquat-antibody binding has been proposed.

Purification and characterization of two human liver carboxylesterases.

1. Two carboxylesterases (EC 3.1.1.1) purified from human livers were distinguished by pI (isoelectric point), nondenaturing polyacrylamide gel electrophoresis, molecular weight, catalytic activity, N-terminus and immunological cross-reactivity. 2. The low pI carboxylesterase has not been reported previously. 3. Numerous bands seen when each enzyme was focused on analytical IEF gels could not be separated. 4. When sections of the band pattern was refocused, the original complete band pattern was generated. 5. Both the mid and low pI carboxylesterases had catalytic activity for xenobiotics as well as medium and long chain fatty acid esters.

Evaluation of equilibrium constants for antigen-antibody interactions by solid-phase immunoassay: the binding of paraquat to its elicited mouse monoclonal antibody.

A procedure is devised for simple graphical evaluation of the association constant for antibody-antigen interactions from data obtained by conventional solid-phase immunoassay for antigen. Application of the method to situations involving a univalent antigen is illustrated by means of ELISA data for the interactions of paraquat with its elicited murine monoclonal antibody, and the Fab fragment derived therefrom. Although the completely general situation with both antibody and antigen multivalent is not amenable to study by the present procedure, the quantitative expression is readily modified to accommodate antigen multivalency provided that the univalent Fab fragment may be substituted for the multivalent antibody (IgG or IgM) as partitioning species in the solid-phase immunoassay.