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Biomedical subjects

R Wetzel

Publications and source records attributed to R Wetzel.

At least 73 records · Page 4Linked to original sources
PubMed1988

[Small intestine obturation and perforation in a calf caused by a foreign body. Case report].

Obturation and perforation of the jejunum by an ingested fir branch was diagnosed in a 3 months old male German Fleckvieh calf. Following resection of the altered segment of intestine an end-to-end anastomosis was performed. The abdominal cavity was flushed with a 0.4% solution of polyvidon-iodine in normal saline, and an antibiotic preparation was instilled. Systemic antibiotic therapy was maintained for 6 days. Recovery was uneventful, and the animal was discharged 9 days after admission.

Animals↗
PubMed1987

[Arthrodesis of the wrist joint in patients with polyarthritis].

In rheumatoid arthritis, extreme damage to the wrist joint with rupture of the wrist extensor tendons can only be treated effectively by arthrodesis. The authors describe the technique of wrist fusion by an AO-plate. Twenty-two arthrodeses have been performed between July 1974 and June 1984, representing 7.4% of all surgical procedures on rheumatoid wrists (n = 294). Radial deviation and dorsiflexion of the hand must be avoided. Follow-up was possible in nine patients (eleven wrist-fusions). All these patients were satisfied with the result.

Adult↗
PubMed1986

Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme.

We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation [Perry, L.J., & Wetzel, R. (1984) Science (Washington, D.C.) 226, 555-557]. Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds. By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys. Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97. While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents. This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers. Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation.

Base Sequence↗
PubMed1985

Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme.

The phage T4 gene coding for lysozyme has been cloned into a plasmid under control of the (trp/lac) hybrid tac promoter and expressed in Escherichia coli with no significant toxic effect to actively growing cells. E. coli D1210 (lacIq) transformed with this plasmid produced active T4 lysozyme at levels up to 2% of the cellular protein after induction with isopropyl-beta-D-thiogalactoside. A strain producing active lysozyme was shown to be under a selective disadvantage when co-cultured with a similar strain producing inactive lysozyme. Purified strains, however, are reasonably stable in culture and under normal storage conditions.

DNA, Recombinant↗
PubMed1984

Disulfide bond engineered into T4 lysozyme: stabilization of the protein toward thermal inactivation.

By recombinant DNA techniques, a disulfide bond was introduced at a specific site in T4 lysozyme, a disulfide-free enzyme. This derivative retained full enzymatic activity and was more stable toward thermal inactivation than the wild-type protein. The derivative, T4 lysozyme (Ile3----Cys), was prepared by substituting a Cys codon for an Ile codon at position 3 in the cloned lysozyme gene by means of oligonucleotide-dependent, site-directed mutagenesis. The new gene was expressed in Escherichia coli under control of the (trp-lac) hybrid tac promoter, and the protein was purified. Mild oxidation generated a disulfide bond between the new Cys3 and Cys97, one of the two unpaired cysteines of the native molecule. Oxidized T4 lysozyme (Ile3----Cys) exhibited specific activity identical to that of the wild-type enzyme when measured at 20 degrees C in a cell-clearing assay. The cross-linked protein was more stable than the wild type during incubation at elevated temperatures as determined by recovered enzymatic activity at 20 degrees C.

Chemical Phenomena↗
PubMed1984

Roles of the 29-138 disulfide bond of subtype A of human alpha interferon in its antiviral activity and conformational stability.

Human alpha (leukocyte) interferons contain two disulfide bonds between Cys-1 and Cys-98 and between Cys-29 and Cys-138. Reduction of interferon under native conditions leads to irreversible loss of antiviral activity; reduction in denaturant, followed by oxidation in native conditions, leads to restoration of activity. This behavior, unusual for disulfide-containing proteins, was studied by using a thiosulfonate derivative of subtype A of human alpha interferon (IFN-alpha A). The disulfide-free thiosulfonate formed at 25 degrees C has essentially no antiviral activity, while maintaining a conformation related to that of native IFN-alpha A. This species can regain activity after regeneration of its 29-138 disulfide, by thiol-disulfide interchange in native buffer. Incubation of the disulfide-free thiosulfonate under nonreducing conditions at 37 degrees C generates a monomeric species that has lost its native conformation as well as its ability to regain antiviral activity after thiol-disulfide interchange. These results explain the difficulty in obtaining, under native conditions, a reduced species that regains activity upon oxidation; complete reduction of IFN-alpha A in 100 mM 2-mercaptoethanol requires 37 degrees C, a temperature that promotes conformational decay of the disulfide-free form.

Animals↗
PubMed1984

Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli.

Plasmids have been constructed that direct the synthesis in Escherichia coli of heavy chains and/or light chains of an anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was constructed for expression of a truncated form of heavy chain (Fd' fragment) in E. coli. Functional CEA-binding activity was obtained by in vitro reconstitution in E. coli extracts of heavy chain or Fd' fragment mixed with extracts containing light chain.

Amino Acid Sequence↗
PubMed1982

Structural features of human leukocyte interferon A as determined by circular dichroism spectroscopy.

The solution structure of human leukocyte (alpha) interferon-A (LeIF-A) purified from E. coli extracts was investigated by circular dichroism spectroscopy. At pH 8.2, the native molecule exhibits 40-70% alpha-helix and no clearly detectable beta-structure. The tertiary structure includes a closely-packed interior containing at least one tryptophan side-chain. Titration to pH 1.5 produces a partial loss of structural features which are rapidly regained on return to neutral pH.

Circular Dichroism↗
PubMed1981

Assignment of the disulphide bonds of leukocyte interferon.

Nucleotide sequencing of cloned cDNA can lead to rapid and precise preduction of the primary amino acid sequence of gene products, but it cannot establish such post-translational modifications as the existence and arrangement of disulphide bonds. For example, the complete sequences of at least one fibroblast and seven leukocyte interferon genes are already known, but knowledge derived from analysis of the proteins is confined to information on the N-termini and some tryptic fragments. The presence of at least one disulphide bond in leukocyte interferon is suggested by that molecule's sensitivity to reducing agents. In addition, comparison of all leukocyte interferon gene sequences so far reported indicates four highly conserved cysteines. One of these genes has been engineered for efficient direct expression in Escherichia coli and we have purified the gene product, leukocyte interferon A (IFN-alpha A, Fig. 1) from bacterial extracts to a single species of molecular weight (MW) 19,400. I report here the determination of the disulphide bonds of the purified protein by analysis of tryptic fragments. The results indicate that Cys 1 is bonded to Cys 98, and Cys 29 is bonded to Cys 138.

Cloning, Molecular↗
PubMed1981

Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin.

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.

Amino Acid Sequence↗
PubMed1981

Evidence for rhythmic attention.

Three studies compared effects of different rhythmic contexts on order judgments of targets embedded in auditory patterns designed to manifest auditory stream segregation. Of interest was the effect of rhythmic structure upon the frequency-based captor effect, wherein tones in one stream "capture" those of similar frequencies from another. Three rhythmic structures were studied. Experiment 1 compared a rhythm conducive to pairwise-target stream formation with one conducive to a four-tone stream, and one based on an isochronous rhythm in a within-subjects design. Patterns embedding the target tone pair contained a preliminary string of captor tones at frequencies either close or far from frequencies of distracting tones that flanked the target pair. No effect of captor distance was found. Experiments 2 and 3 varied rhythm between subjects using 9- and 11-tone patterns, respectively. In general, the magnitude of the captor effect was found to vary with temporal predictability of flanking and/or target tones. The pairwise rhythm was most likely to improve order judgments by facilitating frequency-based capture. Results are discussed in terms of a rhythmic attentional hypothesis.

Attention↗
PubMed1981

Properties of a human alpha-interferon purified from E. coli extracts.

A human alpha interferon, designated HuIFN-alpha A, produced in E. coli by direct expression of cloned cDNA [Goeddel et al., Nature 287, 411--416 (1980)] has been purified from bacterial extracts and characterized. The protein has a molecular weight (19,400 by SDS/PAGE) and amino acid composition consistent with the DNA sequence. The pI was determined to be 6.1. The molecule has a specific activity of 1.5 x 10(8) NIH reference units/mg of protein. The sequence of the first 35 amino acids is identical to that expected from the nucleotide sequence. About 50% of the molecules begin with the expected cysteine, and 50% begin with the initiator methionine which E. coli apparently did not remove efficiently. Analysis of a trypsin digest of the native molecule showed that all four of the molecule's cysteines are involved in disulfide bonds: Cys1 is bonded to Cys98, and Cys29 is bonded to Cys 138.

Amino Acids↗
PubMed1980

Production of biologically active N alpha-desacetylthymosin alpha 1 in Escherichia coli through expression of a chemically synthesized gene.

Thymosin alpha 1, an immune restorative polypeptide hormone, was synthesized in Escherichia coli by using recombinant DNA cloning techniques. Based on the known amino acid sequence, a gene coding for the thymosin alpha 1 polypeptide chain was designed and enzymatically assembled from chemically synthesized oligodeoxyribonucleotide fragments. The gene was ligated into plasmid pBR322 and placed under lac operon control, and N alpha-desacetylthymosin alpha 1 was expressed as part of a beta-galactosidase chimeric protein. Cyanogen bromide cleavage of this protein gave a mixture of polypeptides, among which thymosin alpha 1 activity was detected by radioimmunoassay (RIA). The E. coli product is identical with native thymosin alpha 1 isolated from calf thymus in the amino acid sequence but lacks the N-terminal acetyl group. Results of a guinea pig migration inhibition factor (MIF) assay, a terminal deoxyribonucleotidyl transferase (TdT) assay, and radioimmunoassay indicate that the N alpha-desacetylthymosin alpha 1 produced by deoxyribonucleic acid (DNA) cloning techniques has biological activity equivalent to that of the native hormone.

Amino Acid Sequence↗
PubMed1980

Temperature behaviour of human serum albumin.

Structural alterations of albumin, their dependence on concentration and the role of free --SH groups at thermal denaturation, as well as the reversibility of thermally induced structural changes, were studied. Application of various physical methods provides information on a series of structural parameters in a major concentration range. Apart from changes of the helix content, heat treatment gives rise to beta structures which are amplified on cooling and which are correlated with the aggregation of albumin. With rising temperature and concentration the proportion of beta structures and aggregates increases. At degrees of denaturation of up to 20% complete renaturation is possibly in every case. The structure content is concentration-dependent even at room temperature. It may be that intermolecular interactions induce additional alpha-helix structures which are less stable, however, than the ones stabilized by intramolecular interactions. Unfolding of the pocket containing the free --SH group of cysteine-34 enables disulphide bridges to be formed leading to stable aggregates and irreversible structural alterations. Through binding of N-ethymaleimide to free --SH groups, which blocks the formation of disulphide bridges, it is possible to prevent aggregation and irreversible conformational changes. At temperatures below 65--70 degrees C, oligomers are formed mainly via intermolecular beta structures.

Circular Dichroism↗