Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position.

The effects of pH in the range 6.0-8.0 on the thermodynamics of the reversible thermal unfolding of Escherichia coli thioredoxin in the oxidized state have been determined over a range of concentrations using differential scanning calorimetry.

Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues.

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase.

Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques.

We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1-n-propane, 1-n-butane, and 1-n-pentane thiols.

The hydrophobic core of Escherichia coli thioredoxin shows a high tolerance to nonconservative single amino acid substitutions.

A set of single amino acid substitutions has been constructed at positions Leu42 and Leu78 in the hydrophobic core of Escherichia coli thioredoxin. This protein is required for the in vivo assembly of filamentous bacteriophages such as M13. Almost all the mutants retain this activity regardless of the change in size, hydrophobic nature, or charge of the substitution.

Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli.

We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature E1 alpha and E1 beta subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, the mature E1 alpha subunit was fused with the affinity ligand E. coli maltose-binding protein (MBP) through an endoprotease Factor Xa-specific linker peptide.

Comparison of two self administered psychiatric questionnaires (GHQ-12 and SRQ-20) in primary care in Chile.

The General Health Questionnaire (GHQ-12) and the Self Report Questionnaire (SRQ-20) were simultaneously validated against the criterion of the Revised Clinical Interview Schedule (CIS-R) in a primary care clinic in Santiago, Chile. A Relative Operating Characteristic (ROC) analysis was used to determine the optimal threshold point for case definition and to compare the performance of these two questionnaires. The validation coefficients for the GHQ-12 and the SRQ-20 were, respectively: sensitivity 76% and 74%; specificity 73% and 77%; overall misclassification rate: 26% and 25%.

IV sedation in pediatric dentistry: an alternative to general anesthesia.

This prospective study was conducted to determine the sedative effects of IV ketamine and fentanyl on vital signs and behavior. Twenty-seven children, classified as ASA I, with a mean age of 34 months, were studied. The dosages of IV ketamine and fentanyl given were 0.

Chaperonins groEL and groES promote assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase in Escherichia coli.

We have investigated the possible role of chaperonins groEL and groES in the folding and assembly of heterotetramers (alpha 2 beta 2) of mammalian mitochondrial branched-chain alpha-keto acid decarboxylase (E1) in Escherichia coli. The mature E1 alpha subunit fused to maltose-binding protein (MBP) was coexpressed with mature E1 beta on the same vector in ES- and EL- mutant strains. Only small or trace amounts of active E1 component were obtained.

Use of designer recombinant mitochondrial antigens in the diagnosis of primary biliary cirrhosis.

The appearance of autoantibodies against mitochondria in patients with primary biliary cirrhosis has been known for more than 25 yr. In the past, based on the biochemical complexity of the mitochondrion and the use of crude extracts for immunodiagnosis, a degree of nonspecificity in assaying for antibodies to mitochondria has been present. This problem has been largely circumvented by the cloning of the mitochondrial antigens and the identification of the E2 subunits of the pyruvate dehydrogenase complex and the branched chain 2-oxo-acid dehydrogenase complex as the major and immunodominant autoantigens of primary biliary cirrhosis.

Cloning and expression in Escherichia coli of mature E1 beta subunit of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex. Mapping of the E1 beta-binding region on E2.

A cDNA encoding the mature E1 beta subunit of the bovine branched-chain alpha-keto acid dehydrogenase complex was isolated from a lambda ZAP expression library. The bovine E1 beta cDNA is 1,393 base pairs in length. It encodes the entire mature E1 beta subunit consisting of 342 amino acid residues and a partial mitochondrial targeting presequence of 26 residues.

"America responds to AIDS," but did college students? Differences between March, 1987, and September, 1988.

To assess thoughts, perceptions, attitudes, and behavior related to AIDS, data were collected from 2 similar samples of college students. The sets of data were collected 18 months apart. Results indicate increased awareness and knowledge and changed attitudes and behaviors in some cases, but the absolute magnitudes of the differences are small for most between-group comparisons.