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Biomedical subjects

M J Corey

Publications and source records attributed to M J Corey.

At least 19 recordsLinked to original sources
PubMed2000

Mechanistic studies of the effects of anti-factor H antibodies on complement-mediated lysis.

We have recently reported that complement factor H, a negative regulator of complement-mediated cytotoxicity, is produced and secreted by most bladder cancers. This observation was exploited in the development of the BTA stat and BTA TRAK diagnostic assays, both of which make use of two factor H-specific monoclonal antibodies in sandwich format. Here we show that both antibodies exert interesting effects on the biochemistry of complement activation in in vitro systems. Antibody X13.2 competes with C3b for association with factor H and strongly inhibits factor H/factor I-mediated cleavage of C3b, thereby evidently inactivating a negative regulator of complement; yet, the antibody strongly inhibits complement-mediated lysis as well. Conversely, antibody X52. 1, which does not compete with C3b and has no effect on solution-phase cleavage of C3b, is capable of enhancing complement-mediated lysis of various cell types, including cancer cells, by over 10-fold. Our observations indicate that it is possible to deconvolute the biochemical roles of factor H in complement by means of appropriate inhibitors, a finding with potentially valuable implications for both basic research and cancer therapy.

Animals↗
PubMed1998

LNCaP produces both putative zymogen and inactive, free form of prostate-specific antigen.

BACKGROUND: Our objective was to investigate the presence of prostate specific antigen (PSA) and alpha-1-antichymotrypsin (ACT) mRNA and protein in prostate cancer cell lines, and the complexing characteristics of expressed PSA. METHODS: RT-PCR, immunohistochemistry, and Western blots were employed. Trypsin treatment of PSA was performed to establish the possible presence of an activatable form of PSA. RESULTS: ACT mRNA and protein were detected in LNCaP, PC-3, and DU 145 by RT-PCR and by immunohistochemistry, respectively. Only LNCaP cells were positive for PSA mRNA and protein. LNCaP expressed approximately 30% active PSA, approximately 40% putative zymogen form of PSA, and approximately 30% stably inactive PSA. CONCLUSIONS: We have shown that the majority of PSA expressed by LNCaP cells is present in free, noncomplexed forms in the conditioned media. A portion (40%) can be activated by trypsin, while the rest is stably inactive PSA. LNCaP cells may serve as a source of the "unreactive" PSA present in prostate cancer patients' serum.

Biomarkers, Tumor↗
PubMed1997

A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis.

The demand for convenient and sensitive means of measuring cytotoxicity and complement-mediated killing is likely to be increased by the recent identification of Complement Factor H, an important regulatory protein of both the classical and alternate pathways of complement, as a tumor-associated antigen. Here we describe a simple luminometric assay capable of detecting the death of approximately 0.03 nucleated human-cell equivalent or approximately 1 rabbit-erythrocyte equivalent. The assay measures the release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from dead or damaged cells by coupling its enzymatic activity to production of ATP, which in turn is measured by well-known methods involving firefly luciferase. This is accomplished by means of a reaction series in which the activity of G3PDH is coupled with that of phosphoglycerate kinase, the next enzyme in the glycolytic pathway. As described, the assay uses inexpensive, commercially available reagents. This coupled assay was used to demonstrate that an anti-factor-H antibody is capable of enhancing complement-mediated killing of the Raji cancer cell line by > 1000%.

Adenosine Triphosphate↗
PubMed1997

Characterization of 10 new monoclonal antibodies against prostate-specific antigen by analysis of affinity, specificity and function in sandwich assays.

While prostate-specific antigen (PSA) is already an invaluable marker for prostate cancer, there is continuing demand for new anti-PSA antibodies with specific characteristics, e.g., high sensitivity and specificity and equimolar binding to free PSA (f-PSA) and the PSA-alpha-1-antichymotrypsin complex (PSA-ACT), as well as the ability to distinguish between these 2 immunoreactive forms of PSA. We have therefore generated and characterized 10 anti-PSA monoclonal antibodies (MAbs). Apparent dissociation constants (Kd) of MAbs were determined by direct ELISA yielding Kd-0.2-164.0 nM. Western blots suggested that 3 of the MAbs (60-1A2, 60-8A2 and 17-1A2) bind to linear epitopes. Sandwich assays identified 5 major antigenic regions as binding targets of the MAbs. Three combinations of MAbs recognize f-PSA and PSA-ACT in equimolar fashion with high sensitivity. Two of the MAb combinations are specific for f-PSA. Physical analysis of the new antibodies has allowed us to assign the MAbs to binding classes (based on their sandwiching capabilities) and to determine accurate apparent dissociation constants.

Animals↗
PubMed1997

High-affinity peptide ligands to prostate-specific antigen identified by polysome selection.

We have used the polysome-selection method to isolate peptide ligands that bind with high affinity to Prostate-Specific Antigen (PSA), an important prostate-cancer marker. Two random libraries, each encoding approximately 10(12) random peptides, were transcribed into RNA and translated in vitro. Polysomes were panned by affinity selection of the nascent peptides against immobilized PSA. Over 30% of the selected species had significant affinity for PSA; the dissociation constant of the complex formed by the best isolate with PSA was < 10(-9) M. Formation of streptavidin conjugates of selected peptides improved their affinities and, in one case, virtually eliminated non-specific binding. The polysome-selection method can be used to produce high-affinity peptide ligands of potential use in diagnostic and therapeutic procedures.

Amino Acid Sequence↗
PubMed1997

Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies.

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with alpha 1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited > 90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.

Amino Acid Sequence↗
PubMed1996

On the failure of de novo-designed peptides as biocatalysts.

While the elegance and efficiency of enzymatic catalysis have long tempted chemists and biochemists with reductionist leanings to try to mimic the functions of natural enzymes in much smaller peptides, such efforts have only rarely produced catalysts with biologically interesting properties. However, the advent of genetic engineering and hybridoma technology and the discovery of catalytic RNA have led to new and very promising alternative means of biocatalyst development. Synthetic chemists have also had some success in creating nonpeptide catalysts with certain enzyme-like characteristics, although their rates and specificities are generally much poorer than those exhibited by the best novel biocatalysts based on natural structures. A comparison of the various approaches from theoretical and practical viewpoints is presented. It is suggested that, given our current level of understanding, the most fruitful methods may incorporate both iterative selection strategies and rationally chosen small perturbations, superimposed on frameworks designed by nature.

Catalysis↗
PubMed1989

Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.

The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% +/- 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes were 4.1 microM (D52N) and 13.4 microM (E35Q) vs. 8.6 microM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of approximately 5 mg per liter of culture by high-salt elution from the cell walls.

Animals↗