Biomedical subjects
R Wetzel
Publications and source records attributed to R Wetzel.
[Supinator tunnel syndrome--a differential diagnosis of so-called tennis elbow].
The increasing activity in sport lead especially in untrained persons to overstress of the elbow joint. Often one tends to diagnose a epicondlylits humeri radialis i.E. tennis elbow. In 1873, Runge (16) first described what we now call epicondylitis humeri radialis, i.e. what Morris (10) 1882 called "Tennis elbow." Its etiology is still under discussion, and the diagnosis of tennis elbow necessitates exact and detailed differential diagnostic efforts. In the work presented, we describe one major differential diagnosis, the posterior interosseous nerve syndrome or supinator entrapment syndrome. With sketches and anatomical preparations we describe the course of the radial nerve around the elbow joint and of its ramus profundus within the supinator muscle pouch. Listing representative causes of this syndrome, we also comment on clinical aspects and our surgical method of choice. We have interviewed and reexamined 6 of our patients suffering from a so called therapy-resistant epicondylitis humeri radialis. Due to their clinical picture all six had been operated for posterior interosseous nerve syndrome/supinator muscle entrapment syndrome. We present our results including neurophysiological evaluations. In relapsing, therapy resistant and diagnostically untypical humeral radial epicondylitis, a posterior interosseous nerve syndrome should be considered. After clinical and electromyographic examination a revision of the supinator channel is justified in selected cases.
Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.
A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)
Pustular osteoarthropathy and its differential diagnosis.
A combination of costo-sterno-clavicular hyperostosis and palmo-plantar pustulosis, sometimes with hyperostotic spondylosis and spondylarthritis, is called pustular osteoarthropathy. In the Western hemisphere 40 cases have been reported and in the Far East the condition occurs more commonly. Five cases are described. Diagnosis is difficult because the skin lesions can occur before the costo-sterno-clavicular hyperostoses. The clinical condition is discussed together with the differential diagnosis. Pustular osteoarthropathy seems to be an enthesopathy and the palmoplantar pustulosis is interpreted as a form of psoriasis.
Protein engineering.
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Mutations in human interferon gamma affecting inclusion body formation identified by a general immunochemical screen.
High level expression of the gene for human interferon-gamma (HuIFN-gamma) in E. coli JM101 cultured at 37 degrees C results in the distribution of over 90 percent of the total accumulated gene product into inclusion bodies (IBs). We have identified mutations throughout the molecule that alter the distribution between the soluble and inclusion body fractions without greatly affecting total expression level. Some mutants retain high biological activity but are localized almost entirely in the soluble fraction. Mutations affecting IB distribution as well as stability to intracellular proteolysis were detected by immunochemical screens and verified by gel assays. Immunochemical screens such as those employed here may allow identification of folding and stability mutants in heterologously expressed proteins when there is no other basis for selection or screening. These results also suggest that one solution to production problems arising from IB formation may be to identify mutations in the target protein that favor expression of soluble protein while retaining biological activity.
Learning from the immune system: laboratory methods for creating and refining molecular diversity in polypeptides.
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Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures.
Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.
Mutational analysis of the C-terminus of human interferon-gamma.
We have developed an expression/mutagenesis system and a series of screening procedures for the study of structure-function relationships in human interferon-gamma (HuIFN-gamma). Here we report a preliminary evaluation of the C-terminal portion of the molecule. An expression vector, p652trp gamma, was constructed which includes (i) the HuIFN-gamma gene under control of the trp promoter, (ii) elements controlling replication of both single- and double-stranded versions of the vector DNA; and (iii) the ampicillin resistance gene. (Other vectors using these same elements were constructed but proved to be unsatisfactory, being characterized by a rapid decline, as cells containing them were passaged, in their potential to achieve high expression levels.) A mutagenesis cassette was constructed by introduction of unique restriction sites flanking the nucleotides encoding the C-terminal 23 amino acids, and this cassette was replaced with chemically synthesized, degenerate oligonucleotides by ligation. Colonies from cells transformed with the reconstructed vector were stored in LB glycerol in microtiter plates, and these were screened by hybridization with synthetic oligonucleotides. Plates were grown in minimal medium to express the encoded interferon and lysed by an efficient, mild procedure. A polyclonal antibody specific for the C-terminal four amino acids of HuIFN-gamma was used to establish that the lysis procedure preserved the C-terminus, and to score for frame shift and nonsense mutations. Immunochemical assays also were used, with mixed results, to quantify IFN-gamma concentration in the lysate. An antiviral assay was employed to assess biological activity. Over 1000 isolates were screened and clones with properties representative of various classes of phenotypes were further characterized, in some cases after partial purification from the lysate. Three types of mutations were isolated: point mutations, nonsense mutations and frame shift mutations. The results from each type of mutation confirm earlier observations of the important role of basic residues in the 128-131 region of the molecule for biological activity. At the same time, the results suggest that most residues within the cassette can be altered without significant effects on biological activity. These results are discussed in the context of several possible mechanisms.
Mutational analysis of the C-terminus of human gamma-interferon.
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Identification and nucleotide sequence of the sup8-e UGA-suppressor leucine tRNA from Schizosaccharomyces pombe.
Using the translation of rabbit globin mRNA in wheat germ extracts as an assay for ochre and opal suppression, a UGA suppressor tRNA from Schizosaccharomyces pombre strain sup8-e was purified by column chromatography and two-dimensional gel electrophoresis. The purified tRNA can be aminoacylated with leucine by a crude aminoacyl-tRNA synthetase preparation from a wild type S. pombe strain, and has high activity in the suppressor assay. By a combination of post-labeling fingerprinting and rapid gel sequencing methods the nucleotide sequence of this suppressor tRNA was determined to be: pG-C-G-G-C-U-A-U-G-C-C-ac4C-G-A-G-D-G-G-D-G-D-A-A-G-G-G-m22G-G-C-A-G-A-psi-U-U*-C-A-m1G-C-C-C-U-G-C-U-G-U-U-G-U-A-A-A-A-C-G-m5C-G-A-G-A-G-T-psi-C-G-m1A-A-C-C-U-C-U-C-U-G-G-C-C-G-C-A-C-C-AOH. The anticodon sequence U*CA is complementary to the UGA codon. An interesting feature of the suppressor tRNA is an expanded anticodon loop of nine nucleotides owing to an A-C nonpair at the first anticodon stem position.
The nonessential nature of sulfhydryl groups for ATPase activity in myosin. A cyanylation study.
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Aminoacyl-tRNA synthetase families and their significance to the origin of the genetic code.
A correlation of various aspects of the protein structures and substrate and mechanistic specificities of the aminoacyl-tRNA synthetases has led to the identification of at least one family of enzymes probably derived from a common ancestral synthetase. While strong correlations exist only in one part of the array of 64 codons comprising the Genetic Code, this itself may be interpreted as a meaningful pattern, most consistent with a development of the present code from earlier codes containing fewer amino acids and fewer available codons. Specifically, strong correlations in the enzymes whose cognate tRNAs respond to codons containing a central pyrimidine, including the enzyme family of Ile-, Phe-, Val-, Met-, and Leu-tRNA synthetases, suggests that these enzymes evolved last, and that, therefore, an earlier version of the Genetic Code was comprised solely of codons containing a central purine. It is suggested that further study of the historical interrelationships of these enzymes could lead to a fairly detailed picture of how the Genetic Code developed.
[Behavior of the trace elements copper, zinc and manganese in bovine rumen. 1. Trace element content of different fractions of rumen fluid and the effect of copper sulfate administration].
Two adult, rumen-fistulated steers were fed, over four consecutive 3-week periods, Cu-deficient and normal hays with and without copper sulphate supplementation. Their rumen liquor levels of copper, zinc and manganese were determined correspondingly. The rumen liquor samples were split into 3 fractions by way of centrifugation, sodium dodecyl sulphate treatment of the sediment and filtration. The fractionation of the rumen liquor showed copper to be contained mainly in the water-soluble supernatant (68%), whilst zinc and manganese are more strongly linked to the microbial fraction (57 and 46%, resp.). The plant residue did not average but 9, 24 and 22% of copper, zinc and manganese, respectively. Copper sulphate supplementation to normal hay resulted in a significantly increased copper content in the supernatant and in the microbial fraction. At the same time, the zinc and manganese content values in the microbial fraction were found increased. However, the same copper sulphate supplementation to Cu-deficient hay was found to reduce highly significantly the copper level in the microbial fraction. It is supposed that the excessively high molybdenum content of this hay (2.5 ng/kg) has prevented any effect of the copper sulphate supplementation. The positive effects of copper sulphate supplementation on the zinc and manganese levels in the bacterial fraction were maintained even when feeding Cu-deficient hay rich in Mo.
[Behavior of trace elements copper, zinc and manganese in bovine rumen. 2. Passage rate and course of trace element concentration in rumen as influenced by copper sulfate administration].
In experiments on the distribution of copper, zinc and manganese in fractions of rumen liquor (Wetzel and Menke, 1977 (1)) changes in PEG and in trace element concentration during day-time (between 8 a.m. and 6 p.m.) were measured at 2-hour intervals. Rumen volume was 61 l and passage rate 108 and 128 l/day in the two animals used. Average daily passage was calculated to be 40 mg copper, 98 mg zinc and 371 mg manganese. These values were found to be 11% higher than copper intake and 44 and 68% lower than zinc and manganese intakes, respectively. The differences indicate a remarkable endogenous secretion of copper, and a high absorption of zinc and manganese within the rumen. Concentration (mcg/g dry matter) of copper, zinc and manganese in the bacterial fraction and in total rumen liquor decreased slowly in the first hours after feeding, reaching a minimum after 4 to 6 hours and showed a slight increase thereafter. After addition of copper sulphate the curves mostly became linear. The conclusion is drawn that 1.8 ppm copper in inorganic binding does not lead to changes in trace element concentration to an extent that might be harmful to bacterial fermentation.
Analogs of methionyl-tRNA synthetase substrates containing photolabile groups.
Three photolabile analogs of substrates of methionyl-tRNA synthetase were synthesized. In one, the 4-thiouridine at the 8 position of E. coli tRNAfMet was alkylated with [14C]p-azidobromoacetanilide. In the second, [14C]p-azidobenzoic acid hydrazide was condensed with the 3'-terminal dialdehyde of periodate-oxidized Escherichia coli tRNAfMet. The modified tRNAs could be purified by chromatography on benzoylated DEAE-cellulose. The third photolabile compound was [3H]methioninyl-8-azido-adenosine 5'-phosphate, an analog of the methionyl adenylate intermediate in the aminoacylation reaction. Irradiation of each of these compounds in the presence of equimolar amounts of E. coli methionyl-tRNA synthetase of micrometer concentrations gave 5-15% crosslinking.
Psychiatric illness and non-cancer hysterectomy.
Forty-nine randomly selected women who received hysterectomy for reasons other than cancer were studied preoperatively with systematic interviews and record reviews, and were diagnosed using the explicit criteria of Feighner, et al. Fifty-seven percent were found to be psychiatrically ill, with 27% suffering from hysteria (Briquet's Syndrome), and 18% from primary depression. Recently some investigators have attributed a "post-hysterectomy syndrome" characterized by multiple psychologic and somatic symptoms to the surgery itself. However, a high pre-operative prevalence of psychiatric illness, particularly hysteria, must be considered when evaluating symptoms in a post-hysterectomy population.
Synthesis and reactions of 6-methylsulfonyl-9-beta-D-ribofuranosylpurine.
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