Search PubMed⌕ Search

Biomedical subjects

R Knecht

Publications and source records attributed to R Knecht.

52 records · Page 3Linked to original sources
PubMed1985

Elastase-cathepsin G inhibitors eglin b and eglin c differ by a single Tyr----His substitution. A micro-method for the identification of amino-acid substitution.

The structures of eglin b and eglin c, both potent inhibitors of human neutral granulocytic proteinase elastase and cathepsin G, were compared by micro amino-acid analysis and peptide mapping techniques. Eglin b and eglin c differ by one amino-acid substitution in the middle of the polypeptide chain. Tyrosine residue at position 35 of eglin c was substituted by histidine in eglin b. This amino-acid substitution requires one base exchange (U----C) at the DNA level and apparently does not affect the reactive site of eglins. Though without disulfide linkages, eglins are very rigid molecules and can be effectively digested by trypsin only after rigorous acid incubation.

Amino Acids↗
PubMed1983

Sequence determination of eglin C using combined microtechniques of amino acid analysis, peptide isolation, and automatic Edman degradation.

The complete amino acid sequence of a proteinase inhibitor, eglin c (Mr 8100), has been determined with less than 150 micrograms of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.

Amino Acid Sequence↗
PubMed1983

A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.

Allergens↗
PubMed1982

A complete separation of dimethylaminoazobenzenesulphonyl-amino acids. Amino acid analysis with low nanogram amounts of polypeptide with dimethylaminoazobenzenesulphonyl chloride.

A chromatographical system has been developed to give a complete baseline separation of all dimethylaminoazobenzenesulphonyl (DABS)-amino acids for high-sensitivity amino acid analysis [Chang, Knecht & Braun (1981) Biochem. J. 199, 547--556]. The system, which uses a Merck RP-18 column with phosphate buffer (12 mM, pH 6.5)/acetonitrile mixture, allows reliable analysis of DABS-amino acids at the 1--2 pmol level. The accuracy of this new system is demonstrated by the composition analysis of two immunoglobulin light chains (214 amino acid residues) with differences at only three amino acid residue positions.

Amino Acids↗
PubMed1982

A sensitive peptide mapping method. Identification of three amino acid substitutions within two anti-azobenzenearsonate monoclonal antibody light chains.

Peptide mixtures, precolumn-derivatized with dimethylaminoazobenzene isothiocyanate, have been separated by reversed-phase high-performance liquid chromatography to generate a dimethylaminoazobenzene thiocarbamoyl peptide map. The eluted peptide derivatives are detected in the visible region with a sensitivity of 2-5 pmol and can be collected for direct structural analysis. This technique was applied to compare the sequence homology of two immunoglobulin light chains which were derived from two anti-azobenzenearsonate monclonal antibodies, namely 10K44-7A1 and 10K26-12A1. The complete variable region sequences of 10K44-7A1 and 10K26-12A1 light chains were established based on the sequence analysis of tryptic peptides, intact light chains and reference sequences obtained previously [Siegelman M. and Capra, J.D. (1981) Proc. Natl Acad. Sci. USA, 78, 7679-7683]. Altogether, three amino acid substitutions have been detected within complementary determining regions 1 and 2, and framework region 3, all requiring only a single base change at the DNA level. This new technique provides detection limits and the feasibility of analysing peptides which are not obtainable with conventional techniques.

Amino Acid Sequence↗
PubMed1981

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.

Amino Acids↗
PubMed1979

Amino acid sequences of antibody light chain variable regions of pedigreed rabbits: kappa light chain K49-501 (allotype b4 anti-streptococcal group A-variant polysaccharide antibody).

The amino acid sequence of positions 1--150 of a light chain, isolated from another monoclonal rabbit anti-streptococcal group A-variant polysaccharide antibody, was determined. The analysis was performed with 2 mumol of polypeptide chain, using a grossly modified Beckman 890B sequenator. This sequence stretch accounts for the whole variable region and a considerable part of the constant region at a total length of 218 amino acids. This allotype b4 light chain was isolated from a non-precipitating, end-group-specific antibody with a KD = 1.3 X 10(-5)M. This brings the present number of totally known rabbit VL sequences of antigen elicited antibodies to 21. A comparison of these 21 sequences reveals a building plan of ribbit VL homologous to that of human and murine VL regions. The observed variability does follow a pattern of linked amino acid substitutions, indicating that this information must be contained in the germ-line of the rabbit in the form of multiple VL region genes. This conclusion, however, does not rule out the occasional variant being due to somatic rearrangement. Finally, this comparison reveals that the joining peptide between positions 96--110 is also a separate entity in rabbit VL region sequences.

Amino Acid Sequence↗
PubMed1968

[Gastic cancer].

Explore the source record for details and available documents.

Adult↗
PubMedDate not supplied

Results of cochlear implantation in patients with severe to profound hearing loss--implications for patient selection.

In patients with some residual hearing and minor benefit from conventional hearing aids, the benefits of cochlear implantation have to be weighed carefully against eventual adverse effects. In this study, pre- and post-operative thresholds as well as functional results after cochlear implantation are reported; 17 of 44 implanted adults had residual hearing pre-operatively (mean threshold(250 to 4000 Hz): 106 dB HL) in the implanted ear. Residual hearing in the implanted ear could not, in general, be preserved post-operatively. Seventeen of 44 implanted children had some amount of residual hearing in the implanted ear pre-operatively (implanted ear: 114 dB HL; contralateral ear: 109.9 dB HL; mean thresholds(250 to 4000 Hz))). Contrary to the results in adults, residual hearing in the implanted ear remained statistically unchanged. Hearing in the contralateral ear increased significantly from 109.9 to 101.9 dB HL post-operatively. This increase was mainly attributed to maturation of the central auditory pathway. In adults with residual hearing, the monosyllable word recognition scores increased significantly from 9 per cent pre-operatively to 42 per cent post-operatively. Children with residual hearing tended to perform better on speech-related test material compared to children without prior auditory experience. Cochlear implantation is indicated in adults and children with residual hearing and minor benefit from conventional amplification. The contralateral ear in children should be considered for additional acoustical stimulation.

Adult↗
PubMedDate not supplied

Carcinomas unresponsive to either cisplatinum or anti-EGFR therapy can be growth inhibited by combination therapy of both agents.

BACKGROUND: Previously we demonstrated that the antitumor efficacy of monoclonal antibodies against the EGFR (epidermal growth factor receptor) of human tumor xenografts mainly depends on the EGFR content of tumors rather than on the tumors' entity. In this study we wanted to elucidate whether the described cumulative effect of cisplatin and Anti-EGFR therapy also depends on the EGFR expression. MATERIALS AND METHODS: Xenotransplanted carcinomas with different EGFR levels were treated with monoclonal antibodies against the EGFR (EMD 72000 and EMD 55900), cisplatinum and a combination of both. RESULTS: Each monoclonal antibody alone led to an EGFR-dependent significant tumor growth reduction. Cisplatinum alone had no growth inhibitory effects on tumors with high content in contrast to those with low EGFR content. The combination of antibodies with cisplatinum resulted in an EGFR-independent tumor growth inhibition which was stronger than observed in the case of monotherapy. DISCUSSION: The obtained results may address upcoming phase I/II trials to use Anti-EGFR/Cisplatinum therapy regardless of the EGFR content of tumors.

Adenocarcinoma↗