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Biomedical subjects

J Weidner

Publications and source records attributed to J Weidner.

29 records · Page 2Linked to original sources
PubMed1995

Quantification of a matrix metalloproteinase-generated aggrecan G1 fragment using monospecific anti-peptide serum.

Several members of the matrix metalloproteinase family have been reported to cleave aggrecan in the interglobular domain between Asn-341 and Phe-342. An antiserum was prepared against a peptide conjugate corresponding to the C-terminal sequence of the matrix metalloproteinase-generated aggrecan G1 fragment (Phe335-Val-Asp-Ile-Pro-Glu-Asn341). A quantitative radioimmunoassay, with a limit of detection of about 80 pM, was developed using this antiserum. This antiserum requires the free carboxyl group of the C-terminal asparagine for optimal recognition. If the C-terminal asparagine is excised from the sequence, replaced with closely related amino acids, or extended across the matrix metalloproteinase cleavage site, there is a 40-10,000-fold loss in detection. Using peptides cleaved from the N-terminus, it was determined that the antiserum requires the entire Phe-Val-Asp-Ile-Pro-Glu-Asn sequence for optimal recognition. The radioimmunoassay detects matrix metalloproteinase-generated G1 fragments with similar sensitivity to the Phe-Val-Asp-Ile-Pro-Glu-Asn peptide, but it does not recognize intact aggrecan. Immunoreactive aggrecan G1 fragments of molecular mass 50 kDa are generated by the matrix metalloproteinases stromelysin and gelatinase A. In contrast, under identical conditions, the closely related metalloproteinases, gelatinase B and collagenase, as well as cathepsin G, cathepsin B and human leucocyte elastase, did not generate a G1 fragment recognized by the antiserum. The anti-Phe-Val-Asp-Ile-Pro-Glu-Asn serum detects stromelysin-generated aggrecan G1 fragments from mouse, guinea pig, rabbit and human, indicating that the detection is not species-specific. This antiserum and radio-immunoassay should be useful for quantifying and characterizing matrix metalloproteinase-generated aggrecan G1 fragments in articular cartilage and synovial fluids from humans and various animal models of articular-cartilage destruction.

Aggrecans↗
PubMed1994

Inhibition of calcineurin by a novel FK-506-binding protein.

FK-506, a potent immunosuppressive drug, acts during the commitment phase of T-lymphocyte activation to block a subset of calcium-associated events necessary for transcription of certain early lymphokine genes. The drug binds to an abundant, cytosolic 11.8-kDa protein termed the FK-506-binding protein (FKBP12). The FKBP12.FK-506 complex inhibits calcineurin, a calcium-dependent phosphatase that is a component of the signal transduction pathway leading to early lymphokine gene transcription. FKBP12 is one member of a growing gene family. Prior to this report, all other FKBP family members had been irrelevant to the mechanism of action of FK-506 because no other FKBP.FK-506 complexes were able to bind and inhibit calcineurin. Here, we report the purification and characterization of a novel FK-506-binding protein, FKBP12.6. Having 85% amino acid sequence identity to FKBP12, FKBP12.6 is, among the FKBPs, most closely related to FKBP12. When complexed with FK-506, FKBP12.6 binds to and inhibits calcineurin, making it only the second FKBP discovered thus far to do so. The ability to inhibit calcineurin establishes the potential relevance of FKBP12.6 to the immunosuppressive or toxic side effects of FK-506.

Amino Acid Isomerases↗
PubMed1994

Determination of the kinetic parameters of Escherichia coli leader peptidase activity using a continuous assay: the pH dependence and time-dependent inhibition by beta-lactams are consistent with a novel serine protease mechanism.

Bacterial leader peptidase (LPase) is a potential target for the development of novel anti-infective agents, but to data only peptides based upon natural macromolecular substrates have been reported as inhibitors. In this work is described a continuous assay for Escherichia coli LPase activity, based upon Ac-WSASALAKI-AMC (I) as the substrate, that can be monitored either spectrophotometrically or spectrofluorometrically. The LPase reaction is coupled to the liberation of AMC (aminomethylcoumarin) via a nonspecific leucine aminopeptidase. LPase and a short form of the enzyme (LPase-sf) lacking the membrane spanning domains displayed saturable kinetics toward I. The second-order rate constants were approximately 2 x 10(5) M-1 h-1 at pH 7.5 and were comparable to those reported in the literature for peptide substrates based upon natural cleavage sites in preproteins. LPase was inhibited by beta-lactams. [S-(R*,S*)]-4-[(1-(((1-(5-toluoyl)butyl)amino)carbonyl)-3,3-dimethyl-4- oxo-2-azetidinyl)oxyl]benzoic acid (L-684,-248, 588 microM) inhibited the LPase-catalyzed hydrolysis of 50 microM I and 125 microM Ac-WLVP-Nleu-LSFAAEGDDPA-NH2 by 30% and 88% over 1 and 4 h, respectively. The inhibition of LPase by L-684,248 and its C-4 diasteromer was time dependent and yielded second-order rate constants (kinact/Ki) of 12 and 7.7 M-1 min-1, respectively. The process was structurally specific as the C-3 diethyl substituted beta-lactam (C-4 S-isomer) was inactive. The latter data correlate with the LPase preference for alanine at the P1 position of peptide substrates [Kuo et al. (1993) Arch. Biochem. Biophys. 303, 274-280].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Nonisotopic detection of mutations using a modified single-strand conformation polymorphism analysis.

This report describes a rapid convenient screening system with improved sensitivity to detect mutations in the cystic fibrosis transmembrane regulator (CFTR) gene based on nonisotopic SSCP analysis. Because conventional SSCP analysis is often hampered by poor yield of single-stranded DNA, we applied the well-established solid-phase technique in which streptavidin-coated magnetic beads are used to immobilize biotinylated polymerase chain reaction (PCR) products. High yield of single-stranded DNA can be eluted from the solid phase by denaturation and used for SSCP analysis. An additional advantage of this procedure is that the immobilized single strand is available without any further purification steps for solid-phase sequencing.

Base Sequence↗
PubMed1991

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha.

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.

Amino Acid Sequence↗
PubMed1990

Evaluation of an oncology curriculum.

This report describes and presents an evaluation of the undergraduate component of the Cancer Education Program at The Bowman Gray School of Medicine. New learning objectives on cancer were introduced into the second and third year of the Medical School curriculum. A twenty-five question multiple choice examination measured the accomplishment of the new objectives. Students in the class of 1985, who did not receive instruction on the new objectives as part of their formal medical education, served as a control. Students in the classes of 1986 and 1987, who respectively received partial and full exposure to the new objectives, served as experimental groups. Despite limitations in the evaluation procedure, data obtained from the study suggested the possibility of a small but meaningful increase in overall knowledge and specific gains in both students' knowledge and their perceptions of being better prepared to deal with psychosocial aspects of cancer.

Curriculum↗
PubMed1989

Human ehrlichiosis.

Ehrlichiosis is one of the latest tick-borne illnesses to be reported in humans. The authors describe two cases of this rickettsial disease that were apparently acquired in Missouri. They discuss diagnosis and treatment.

Animals↗
PubMed1978

AMTEC: a cooperative effort in medical technology education.

A committee in the St. Louis Metropolitan area has been established to promote communication and cooperation among the area's existing hospital-based programs in medical technology. Area Medical Technology Education Coordinators (AMTEC) was established three years ago primarily to facilitate the administrative functions of medical technology education and to serve as an instrument for the exchange of ideas. Its primary undertaking has been the central processing of applications to the area programs, as an aid in the admission process. In addition, a continuing education program sponsored by the committee has been established, and various "curriculum sharing" activities have been sponsored for the students enrolled in the schools. Future plans for the committee include sponsoring an on-going evaluation process of graduates by employers, and establishing a criterion-referenced question pool. The authors describe the experiences of the committee to date and plans for the implementation of future goals.

Medical Laboratory Science↗
PubMedDate not supplied

Induction of single- and double-strand breaks in plasmid DNA by monoenergetic alpha-particles with energies below the Bragg-maximum.

The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo. For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNA-layer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics. Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 +/- 0.9) x 10(-11) breaks/Gy x dalton. The ratio of ssb/dsb was 0.5 +/- 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

Alpha Particles↗