Selective suppression of in vitro T-dependent humoral immunity by synthetic food additive antioxidants.

Effect of antioxidants on humoral immune responses, such as butylated hydroxytoluene (BHT), n-propyl gallate (PG) and dimethyl sulfoxide (DMSO) is suppression in vitro antibody production. These antioxidants all inhibited T-dependent B cell response, not T-independent and polyclonal B cell response. These data suggest that antioxidants suppress humoral immunity by suppression of regulation of T cells or action of macrophages on B cells, not by direct suppression of B cells. The other possible explanation for antioxidant action is the lack of T-B cell contact required for the triggering of the B cell response with T-dependent antigens.

Human IgA monoclonal antibodies specific for a major ragweed pollen antigen.

Human hybridoma cell lines secreting IgG specific for the major allergen in the pollen of short ragweed, Amb a I, were established from patients who had been receiving antigen injections for immunotherapy. Recombinant Ig genes were then constructed by cloning the heavy and light chain variable region genes of the human hybridoma cell line and joining them to the human alpha or kappa constant region genes in mammalian expression vectors. Amb a I-specific IgA was expressed in two mouse myeloma cell lines, NS0 and Sp2/0. In both systems, transfected alpha and kappa chains were assembled into IgA monomers or into dimers covalently linked by the endogenous murine J chains. We propose that recombinant IgA monoclonal antibodies specific for airborne allergens may be applied to the mucosal surface of the nasal linings or of the lower airway of sensitized individuals to inhibit the entry of allergenic molecules across the mucosal epithelium and, therefore, to prevent the development of allergic responses.

Role of Asp274 in lac repressor: diminished sugar binding and altered conformational effects in mutants.

The role of Asp274 in inducer binding of lac repressor has been explored by spectroscopic measurements, fluorescence quenching, in vitro induction assays, and chemical modification of mutants with conservative substitutions at this site. Although no fluorescence emission shift or characteristic UV difference spectrum was observed at high inducer concentration, fluorescence quenching, effects on operator binding, and chemical modification results indicate indirectly that the mutants Asp274-->Asn and Asp274-->Glu bind sugar, albeit with very low affinity (> 0.1 M). Consistent with very weak inducer binding indicated by protection from fluorescence quenching by iodide, operator binding activity of these two mutant proteins is altered at very high IPTG concentration, although in opposite directions. The distinct effects of inducer on operator binding in these two mutant proteins as well as substantial differences in the effect of sugar ligand on chemical modification of Cys107 and Cys140 by 2-(bromoacetamido)-4-nitrophenol suggest that the conformation of the protein before and after association with sugar may differ in these mutant proteins. Fluorescence quenching assays of lac mutant proteins at Asp274 indicate the proximity of Trp220 to the side chain at position 274, consistent with the location of this residue in the structural model of lac repressor and in the crystallographic structure of the homologous purine repressor. From these results, we conclude that Asp274 is in the inducer binding site, that the character of this residue is crucial to inducer binding, and that interaction of sugar with the side chain at this position may be associated with the conformational change necessary for generating high affinity ligand binding.

Lysine 84 is at the subunit interface of lac repressor protein.

Mutations have been generated at the Lys84 site of the lac repressor to explore its predicted role in inducer binding and/or subunit interaction. Four single mutations, Lys84-->Ala, Lys84-->Leu, Lys84-->Arg, and Lys84-->Glu, have been generated by site-specific mutagenesis. In addition, the mutation Tyr282-->Asp, which results in a monomeric repressor, has been coupled with these four single mutants to generate the four corresponding double mutants. Unchanged inducer binding affinities in all Lys84 mutants except Lys84-->Arg suggest that Lys84 does not contribute energy to inducer binding and is not found in the inducer-binding site as previously proposed (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). Interestingly, the double mutants with hydrophobic side chains at the Lys84 site are tetramers, while those with charged side chains remain monomers. This result agrees with the recent model of the lac repressor (Nichols, J. C., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612), in which Lys84 is mapped by sequence alignment to the same face of the subunit as Tyr282. More detailed inducer binding, operator binding, and immunoblotting studies show that all the mutations at Lys84 have quaternary structures that deviate from wild-type protein, providing supportive evidence for the model placing this residue on the surface of the monomer subunit. Substitution of Lys84 by Ala or Leu results in 100-200-fold decreased association and dissociation rate constants for inducer binding and biphasic character. This decrease can be rescued at least partially in the respective double mutants at elevated pH, at which wild-type repressor shows a 10-fold decrease in affinity and cooperativity in inducer binding. In all substitutions with Ala or Leu, immunoblotting patterns with monoclonal antibody, an assay sensitive to alterations in quaternary structure, are distinct from wild-type repressor. Although substitution with Arg at position 84 yields a protein with 10-fold lower inducer binding affinity, the mutant shows decreased pH dependence of inducer binding. Substitution at this site with Glu results in cooperativity at neutral pH with no change in inducer binding at elevated pH. In addition, operator binding affinity of this mutant is affected by elevated pH, a phenomenon not observed in wild-type repressor. These changes in inducer and operator binding properties appear to be related to the altered quaternary structure of these mutants at Lys84.

Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: aspartate 274 is essential for inducer binding in lac repressor.

To explore the roles of three aspartate residues, Asp88, Asp130, and Asp274, found in the proposed inducer binding site of lac repressor [Sams, C. F., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1984) Nature 310, 429-430], each site was substituted with alanine, glutamate, lysine, or asparagine by site-specific mutagenesis. The mutations at the Asp88 site resulted in a 5-13-fold decrease in inducer binding affinity, largely due to an increase in the inducer dissociation rate constants for these mutants. In addition, the mutant proteins Asp88-->Ala and Asp88-->Lys exhibited altered allosteric behavior for inducer binding. These data conflict with the original hypothesis placing Asp88 in the inducer binding site, but are in agreement with a recent model that places this amino acid close to the subunit interface involved in cooperativity associated with inducer binding [Nichols, J. C., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. Substitution at Asp130 did not alter the inducer binding affinity nor other binding activities. Thus, this amino acid is not crucial in the binding to beta-substituted monosaccharides or in protein function. In stark contrast, all mutant proteins with substitutions at the Asp274 site exhibited no detectable inducer binding. With the exception of Asp274-->Lys, the structures of these mutant proteins appear to be similar to wild-type. The data demonstrate that Asp274 plays a crucial role in inducer binding of this transcriptional regulator.

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma.

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane-isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-microm particle size, C18-bonded silica column and water-sodium acetate-heptanesulfonate-acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4+/-3.1% for TMB and 89.4+/-4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10-5000 ng/ml for TMB and 25-25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.

A 13 kb resolution cosmid map of the 14 Mb fission yeast genome by nonrandom sequence-tagged site mapping.

We present the application of a nonrandom sequence-tagged site (STS) content detection method in mapping an entire genome, that of fission yeast. The novelty of our strategy is in the use of STS probes made from both ends of cosmid clones, selected on the basis of "sample without replacement" (only library clones that show no previous positive hybridization are selected and made into probes). We developed powerful techniques, based on consistency analysis, for error detection and contig assembly. In addition, we probed our library with genetically mapped markers and Notl or Sfil linking clones, thereby anchoring contigs onto chromosomes. Our map contains more than 1000 sites, including genes (most were previously unmapped), occurrences of known repetitive elements, and Notl-Sfil restriction sites.

Video-assisted thoracic surgery as a primary therapy for primary spontaneous pneumothorax. Decision making by the guideline of high-resolution computed tomography.

BACKGROUND: Because blebs are confirmed in most of the patients undergoing thoracotomy, identification of blebs by high-resolution computed tomography (HRCT) can be proposed as a surgical indication in primary spontaneous pneumothorax (PSP). If an apical bleb is identified, we treat the patient by video-assisted thoracic surgery (VATS). METHODS: From May 1995 to September 1997, 61 patients (21.9 +/- 4.6 years) were seen for initial episodes of PSP. Only seven showed bullae on simple chest radiography. However, by HRCT, 48 had sizable blebs (>5 mm), and 45 were treated surgically by VATS. RESULTS: The mean duration of chest tube use after surgery was 3.2 +/- 1.9 days, and the mean hospital stay was 4.5 +/- 1.9 days. Only one recurrence developed 5 weeks after VATS. CONCLUSIONS: Our protocol is effective in controlling an initial episode of PSP. It shortens the observation time before definitive surgical treatment, shortens the hospital stay, and decreases the likelihood of recurrence.