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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pH dependence of the reversible and irreversible thermal denaturation of gamma interferons.
Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
[The value of ultrasound examination of the rheumatoid elbow joint].
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[Possibilities for the use of elbow joint arthroscopy in rheumatology].
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[Sonography in rheumatic orthopedics. Current status and indications today].
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[Epiphysiolysis of the proximal tibia--an atypical sports injury?].
Epiphyseolysis of the proximal tibia is a most unusual injury. The case reports show that there was no adequate trauma leading to this damage. One has to argue, that disposition could be a predisposing factor. Therapy and prognosis of this injury are presented.
Disulfide bonds and thermal stability in T4 lysozyme.
Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.
Structure, function, nomenclature.
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Active immunoglobulin fragments synthesized in E. coli--from Fab to scantibodies.
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