Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule.

Recombinant monoclonal antibodies undergo extensive posttranslational modifications. In this article, we characterize major modifications, separated by cation exchange chromatography, on an immunoglobulin G1 (IgG1) monoclonal antibody (mAb). We found that N-terminal cyclization of glutamine residues to pyroglutamate on the light and heavy chains are the major isoforms resolved during cation exchange chromatography. However, using CEX, we also separated and identified isoforms with unpaired cysteine residues in the V(H) domain of the molecule (Cys22-Cys96). Omalizumab, a therapeutic anti-IgE antibody, has unpaired cysteine residues in the V(H) domain between Cys22 and Cys96, and the Fab fragment, containing the unpaired cysteine residues, is reported to have reduced potency. Dynamic interchain disulfide rearrangement, with slow kinetics, was recently reported to take place in serum for an IgG2 molecule and resulted in predictable mature isoforms. Analytical evaluation of our mAb, after recovery from serum, revealed that the unpaired intrachain cysteine residues (Cys22-Cys96) reformed their disulfide bond. The significance of this study is that correct pairing occurred rapidly, and we speculate that thiol molecules such as cysteine, homocysteine, and glutathione in serum provide an environment, outside the endoplasmic reticulum, for correct linkage.

A catalytic mechanism for caspase-1 and for bimodal inhibition of caspase-1 by activated aspartic ketones.

We have evaluated 619 aspartic ketones with 9 different types of prime-side groups (acyloxymethyl, aryloxymethyl, arylthiomethyl, alkylthiomethyl, acylamino-oxymethyl, sulfonylaminomethyl, alpha-ketoamide, alpha-(1-phenyl-3-trifluoromethyl-pyrazol-5-yl)oxymethyl (PTP), and aliphatic ketones) as inhibitors of caspase-1. The inhibitory behaviors could be classified as reversible, inactivating, or bimodal (i.e. reversible inhibition followed by slow inactivation) based on the kinetically observed formation of reversible thiohemiketal complexes and conversion to an irreversible thioether adduct, and the mechanism of any given ketone was only poorly predictable on the basis of leaving group structure and chemistry. Among 201 bimodal inhibitors, the rate of conversion of the reversible thiohemiketal complex to the inactive thioether (k(i)) was strictly first-order, consistent with direct conversion of the thiohemiketal to the thioether with no intermediate collapse to free ketone and thiolate. We have examined 22 crystallographic structures of caspase-1 complexed as a thiohemiketal with the inhibitors from 8 different ketone classes, and found the Cys285S-C-C(alpha)-leaving group dihedral angle to be near either to 60 degrees or to 180 degrees. Only the 180 degrees conformation was permissive for SN2 displacement of the leaving group and, furthermore, positioned His237Ndelta to stabilize developing charge on the leaving group. Among these structures and 19 additional complexes, all showed a strong interaction between His237Ndelta and the ketone or thiohemiketal oxygen. We therefore propose a proteolytic mechanism for caspase-1 involving polarization of the scissile carbonyl by the His237 imidazolium group. During thiohemiketal/thioether conversion (but probably not during peptide hydrolysis), the leaving group is stabilized by the His237 imidazolium.

N-acetylcysteine protects from glutathione depletion in rats exposed to hyperoxia.

BACKGROUND: N-acetylcysteine (NAC) may protect against oxidative injury by providing cysteine for glutathione (GSH) biosynthesis or by direct reactions with electrophiles. We have recently shown that hyperoxic exposure of rats prior to liver perfusion is associated with significant decreases in hepatic GSH and significant changes in biliary amino acid concentrations. We hypothesized that NAC administration during hyperoxic exposure would prevent depletion of hepatic GSH by providing cysteine for GSH biosynthesis. METHODS: NAC was administered during two conditions known to induce GSH depletion: hyperoxic exposure and biochemical inhibition of GSH synthesis using buthionine sulfoximine (BSO). After 48 hours, GSH concentrations in bile, liver and perfusate and biliary amino acid concentrations were determined using isolated perfused liver preparations. RESULTS: Administration of NAC to rats maintained in normoxic or hyperoxic conditions, prior to liver perfusion, resulted in dose-dependent increases in GSH concentrations in bile, liver and perfusate, increases in bile flow rates and changes in biliary amino acid concentrations. When BSO was given concurrently with NAC in normal or hyperoxic conditions, these effects were not observed, and oxidant stress was evident. CONCLUSIONS: NAC prevents oxidant stress during hyperoxic exposure, most likely by supplying cysteine as a precursor for GSH synthesis.

Hyperoxia and glutathione depletion in the isolated perfused rat liver.

BACKGROUND: Hepatic stores of glutathione may be depleted by hyperoxic exposure or poor nutritional status. We studied the effects of hyperoxia or hepatic glutathione depletion on bile flow rates, and on biliary concentrations of glutathione and amino acids. METHODS: Glutathione depletion was induced in vivo by 1) hyperoxic exposure (O2) for 48 hours, 2) inhibition of glutathione synthesis by treatment with buthionine sulfoximine (BSO), 3) a combination of BSO + O2, or 4) inhibition of cysteine synthesis by propargyglycine (PPG). Livers were then isolated and perfused. RESULTS: Glutathione concentrations in bile, liver, and perfusate were significantly decreased by all treatments. Bile flow was significantly decreased in groups treated with BSO or O2 + BSO, and perfusate LDH was increased by O2 + BSO or PPG. Significant changes in biliary amino acid concentrations included decreased sulfur-containing amino acids and increased branched-chain amino acids in groups treated with BSO, PPG, or O2; and increased essential amino acids in groups treated with O2 or PPG. CONCLUSION: Oxygen exposure or inhibition of glutathione synthesis results in significant decreases in hepatic, perfusate and biliary glutathione concentrations, and increases in biliary amino acids. A decrease in bile flow rate was associated only with the most severe glutathione depletion.

The acute effects of TNF-alpha on the isolated perfused rat liver.

BACKGROUND: TNF-alpha mediates the hepatic response to sepsis by mechanisms which are not well understood. TNF-alpha is known to stimulate the hepatocellular uptake of specific amino acids in vivo; however, little is known about the direct effects of TNF-alpha on hepatic amino acid or glutathione homeostasis, which is a potential factor in the acute hepatic response to sepsis. METHODS: Using the isolated perfused rat liver, we characterized the effects of TNF-alpha on the secretion of amino acids and glutathione into bile and perfusate. Livers taken from adult male Sprague-Dawley rats were perfused with TNF-alpha at a dose of 1 or 2 micrograms. Bile and perfusate were collected for the quantitation of amino acid and glutathione concentrations. RESULTS: Administration of 2 micrograms TNF-alpha resulted in significant increases in biliary and perfusate concentrations of branched chain, gluconeogenic, and total amino acids. TNF-alpha was also associated with dose-related increases in oxygen uptake, and greater biliary concentrations of glutathione. CONCLUSION: TNF-alpha has direct effects upon hepatic amino acid metabolism, which represent some of the early events involved in the mechanism of response to sepsis.

The effects of light exposure on the in vitro hepatic response to an amino acid-vitamin solution.

BACKGROUND: Administration of parenteral nutrition (PN) that has been irradiated with light is associated with hepatic dysfunction in rats in vivo. Using the isolated perfused rat liver, we report the in vitro hepatic response to a light-exposed amino acid-vitamin (AAV) solution, compared with a light-protected solution. METHODS: The amino acid-vitamin solution (3 g Aminosyn and 2.5 mL MVI-12 added to buffer) was placed under a lamp in a beaker that was covered completely with foil (light-protected) or with a transparent wrap (light-exposed) for 24 hours before liver perfusion. Livers from adult male rats were isolated and perfused with buffer for 30 minutes, with the AAV solution for 60 minutes, and again with buffer for 30 minutes. RESULTS: Infusion with the AAV solution resulted in decreases in bile flow rates. Compared with light-protected groups, light-exposure was associated with significantly lower bile flow rates, significant increases in biliary concentrations of oxidized glutathione (GSSG), and significantly decreased biliary concentrations of free amino acids, including the glutathione precursors glutamate and glycine. CONCLUSIONS: Perfusion of the isolated rat liver with an AAV solution that has been irradiated with light for 24 hours results in a decrease in bile flow rates and an increase in biliary GSSG concentrations, suggesting oxidant stress. Consideration should be given to protecting solutions from light in the clinical setting.

LU103793 (NSC D-669356): a synthetic peptide that interacts with microtubules and inhibits mitosis.

LU103793 (NSC D-669356) is a new synthetic derivative of Dolastatin 15, an antiproliferative compound which was isolated from the mollusk Dolabella auricularia. Like Dolastatin 15, LU103793 is highly cytotoxic in vitro (IC50 = 0.1 nM). To investigate the mechanism of action of LU103793, we used a combination of biochemical and cellular methods. Turbidity assays with bovine brain microtubules demonstrated that LU103793 inhibits microtubule polymerization in a concentration-dependent manner (IC50 = 7 microM). Treatment with this compound also induced depolymerization of preassembled microtubules. Cell cycle analysis of tumor cell lines treated with LU103793 indicated a block in the G2-M phase. At the cellular level, it induced depolymerization of microtubules in interphase cells and development of abnormal spindles and chromosome distribution in mitotic cells. Although these effects are very similar to the cellular alterations caused by vinblastine, LU103793 does not inhibit vinblastine binding to unpolymerized tubulin in vitro. Our results suggest that LU103793 exerts its cytotoxic activity primarily through disruption of microtubule organization.

Biliary amino acid and glutathione secretion in response to amino acid infusion in the isolated rat liver.

The intravenous infusion of amino acid solutions has been associated with cholestatic liver injury in hospitalized patients and in laboratory animals. In the isolated rat liver, we recently showed that the acute decrease in bile flow, previously reported by other investigators, is dose related, reversible, and associated with dose-related increases in total biliary amino acid concentrations. In the present study, we characterized the effects of graded infusions of amino acid solutions, with and without taurocholate, on biliary secretion of individual amino acids and glutathione, an important regulator of bile flow. Livers from young adult male rats were perfused with an amino acid solution for 1 hour and allowed to recover for 30 minutes. Infusion of the amino acid solution was associated with dose-related increases in biliary concentrations of most amino acids included in the amino acid solution. Infusion of amino acid solutions resulted in a decreased bile/perfusate ratio of most amino acids, which were secreted into bile in amounts approximating their calculated uptake from the infusate. The inclusion of taurocholate in the infusate was associated with lower biliary concentrations of each individual amino acid and significant decreases in biliary total, reduced, and oxidized glutathione. Further investigation of the relationship between these changes in biliary amino acids and glutathione concentrations and the cholestasis associated with infusion of amino acid solutions may provide insights into the mechanism by which amino acids induce such cholestasis.

Amino acid infusions induce reversible, dose-related decreases in bile flow in the isolated rat liver.

Parenteral infusion of amino acid solutions is known to produce cholestasis in experimental animal models and in the isolated perfused rat liver. To characterize the dose responsiveness and reversibility of amino acid-induced cholestasis, isolated rat livers were perfused with solutions containing 1.5, 3.0, or 6.0 g of amino acids for 1 hour and allowed to recover for 30 minutes. Perfusion of livers resulted in a rapid, dose-related decrease in bile flow (p < .0001 at doses of 3.0 and 6.0 g). When the amino acid solution was discontinued, bile flow recovered to near control rates. Infusion of taurocholate reduced the magnitude of the decrease in bile flow associated with amino acid infusion but did not prevent it. Infusion of amino acid solutions was associated with the following changes in bile: (1) dose-related increases in total free amino acid concentrations; (2) increased osmolarity; (3) increased glucose concentrations; (4) increased potassium concentrations; (5) decreased chloride concentrations; (6) increased oxygen uptake in livers not perfused with added taurocholate; and (7) increased total bile acid concentrations in livers perfused with added taurocholate. Additional investigations are needed to determine whether these associations are attributable to individual amino acids or the total metabolic load of the amino acids.

Genetic control of serum antibody responses of inbred mice to type 1 and type 2 fimbriae from Actinomyces viscosus T14V.

Antibodies reactive with type 1 and type 2 fimbriae from Actinomyces viscosus T14V specifically inhibit the adherence of A. viscosus T14V to salivary pellicle-coated tooth surfaces and other bacteria, and these antibodies are thought to modulate colonization by this microorganism. These studies were done to determine whether previously noted differences in the antibody responses of inbred mice to type 1 and type 2 fimbriae might be under genetic control. The serum immunoglobulin G (IgG) and IgM antibody responses of inbred, F1 hybrid, and H-2 congenic mice, immunized with A. viscosus T14V cells, were analyzed by enzyme-linked immunosorbent assays for antibodies reactive with A. viscosus T14V whole-cell type 1 and type 2 fimbriae. The results confirmed earlier findings and indicated striking variations in the amounts of IgG anti-type 1 (23-fold) and anti-type 2 (48-fold) fimbria antibodies elicited. The responses of the 17 inbred strains tested showed a relatively continuous distribution from high to low, as well as marked differences in the responses of H-2 and Igh-C identical strain pairs. An analysis of the responses of F1 hybrid and H-2 congenic mice indicated dominance of the low-responder gene(s) and control by H-2-linked genes. Antisera from two high-responder strains inhibited in vitro bacterial adherence to a much greater degree than antisera from a low-responding strain. These data suggest polygenic control of the magnitude of the IgG anti-type 1 and anti-type 2 fimbria antibody responses by H-2-linked genes as well as background genes not associated with H-2 or Igh-C loci.

Dominant low responsiveness in the IgG response of mice to the complex protein antigen type 1 fimbriae from Actinomyces viscosus T14V.

Type 1 fimbriae from Actinomyces viscosus T14V, composed of a complex protein of Mr 65,000, mediate the adherence of A. viscosus T14V to the host, whereas type 1 fimbriae-specific antibodies inhibit adherence. Genetic control of the serum IgG response to type 1 fimbriae was evaluated in a series of inbred, hybrid, recombinant inbred, and back-cross mice. Mice were given i.p. injections of 10(8) A. viscosus T14V cells in saline on days 0 and 14, and IgG anti-type 1 fimbriae in sera obtained on day 26 were measured by ELISA. Segregation analysis of the responses of (BALB/cJ x A/J)F1 x A/J backcross mice suggested polygenic control. Linkage analysis in (BALB/cJ x A/J)F1 x A/J backcross and SWXL recombinant inbred mice suggested control by genes linked with H-2 and with Ly-17 and Akp-1. In several F1 hybrid strains derived from H-2-disparate high and low responder parental strains, low responsiveness was dominant. The F1 derived from the H-2-identical high and low responder strains CBA/J and C3H/HeJ was a low responder, suggesting that dominant low responsiveness was mediated by non-H-2-linked genes. A three-gene model is proposed for regulation of the type 1 fimbriae response, including an MHC-linked gene, a gene linked with Ly-17 and Akp-1 on the telomeric portion of chromosome 1, and a background gene whose linkage is unknown.

Analysis of the serum antibody responses to type 1 and type 2 fimbriae in mice immunized with Actinomyces viscosus T14V.

The antibody responses of inbred mice immunized with Actinomyces viscosus T14V cells were analyzed using enzyme-linked immunoabsorbent assays (ELISAs) for measuring serum antibodies reactive with A. viscosus T14V cells and type 1 and type 2 fimbriae. In A/J mice immunized intraperitoneally on days 0 and 14, the serum antibody responses approached peak levels during d 19-35, and a dose of 10(8) cells/injection elicited optimal responses. Analysis of the responses of three genetically diverse strains of inbred mice indicated striking variations in the amounts of anti-type 1 (6.5-fold) and anti-type 2 (14.3-fold) antibodies elicited. The observed variations in the magnitude of the anti-fimbrial antibody responses are theoretically of sufficient magnitude to account for significant differences between mouse strains in their ability to inhibit adherence of A. viscosus T14V to saliva-coated hydroxyapatite and other bacteria. These studies provide a model with which the effects of variations in anti-fimbrial antibody responses on bacterial adherence may be analyzed.