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Biomedical subjects

T C Warren

Publications and source records attributed to T C Warren.

27 records · Page 2Linked to original sources
PubMed1988

Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes.

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.

Alleles↗
PubMed1985

Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients.

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.

Amino Acids↗
PubMed1984

Epidermal growth factor (EGF) binding to membranes immobilized in microtiter wells and estimation of EGF-related transforming growth factor activity.

The binding of radiolabeled epidermal growth factor (EGF) to immobilized A-431 target cell membranes coupled to polyvinyl chloride microtiter wells is described. Saturation curves and Scatchard analysis of the data indicate that the observed binding parameters are consistent with those previously reported. Binding capacity of the membranes are approx. 6.6 pmol EGF per mg membrane protein. Kinetics of 125I-EGF binding were slower, however, than reported for binding to membranes in suspension, although binding constants were not greatly different. The high- and low-affinity binding constants for 125I-EGF were calculated to be approximately 1 X 10(12) M-1 and 2.5 X 10(9) M-1, respectively. Application of this technique in a competitive binding assay requires no more than 2.5 micrograms of membrane protein per assay, is essentially complete after 60 min, and facilitates screening of a large number of samples in a short time. Therefore, this will assist in the evaluation and quantitation of EGF and EGF-related transforming growth factor activity in physiological fluids. This technique may also be applied to analyses of other hormone-receptor systems.

Carcinoma, Squamous Cell↗
PubMed1984

Distinct high-performance liquid chromatography pattern of transforming growth factor activity in urine of cancer patients as compared with that of normal individuals.

Reverse-phase high-performance liquid chromatography (HPLC) performed on urine from cancer patients and normal controls revealed the presence of seven chromatographically distinct peaks of transforming growth factor (TGF) activity, as measured by colony formation of normal rat kidney cells in soft agar. Comparison of urines from normal donors and cancer patients showed no differences in EGF (epidermal growth factor)-dependent beta-TGF-like activity but did reveal distinct patterns of EGF-related, EGF-independent alpha-TGF-like activity. All urine samples contained at least two chromatographically distinguishable forms of EGF-dependent TGF activity, eluting from HPLC as broad peaks with 30 and 43% acetonitrile. The remaining five TGFs eluted as sharp peaks with 32, 34, 35, 37, and 38% acetonitrile, demonstrated EGF-competing activity, and thus were functionally related to EGF. Two of the five EGF-related TGFs were consistently elevated only in the urine of cancer patients and eluted with 32% (TGFA) and 37% (TGFD) acetonitrile Two of the other EGF-related TGFs, eluting with 34% (TGFB) and 35% (TGFC) acetonitrile, were commonly found in both normals and cancer patients. The fifth EGF-related TGF, TGFE, eluting with 38% acetonitrile, was found only in normal donor specimens. TGFA corresponded to the unique Mr 30,000 TGF activity previously identified only in the urine of cancer patients. These observations demonstrate that cancer patients produce high levels of EGF-related TGF activities which can be readily distinguished, using reverse-phase HPLC, from EGF-related TGFs produced by normal individuals. Using a solid-phase competitive radioreceptor binding assay for EGF, we demonstrated that quantitation of EGF-competing activity is as sensitive and effective as the soft-agar colony formation assay for distinguishing HPLC profiles of urinary TGF from cancer patients versus that from normal individuals.

Adolescent↗
PubMed1981

Isolation and characterization of an ester hydrolase active on phorbol diesters from murine liver.

An esterase which cleaves the 12-ester group of phorbol-12,13-diesters has been purified to electrophoretic homogeneity from murine liver cytosol using ammonium sulfate fractionation, Sephadex G-200 gel filtration, Con A-Sepharose chromatography, and phenyl-Sepharose chromatography. The enzyme is a single chain, hydrophobic glycoprotein with a molecular weight of 60,000 and exhibits optimum activity at pH 7.5 to 8.5. The hydrolase has an isoelectric point (pI) of 5. The enzyme is heat- and acid-labile. Zinc, cobalt, and fluoride ions inhibit its activity. Phenylmethylsulfonyl fluoride is a potent inhibitor of the hydrolase. Sarkosyl also inhibits the enzyme at millimolar concentrations. The enzyme inactivates biologically active phorbol-12,13-diesters in a dose-, time-, and temperature-dependent manner. The enzyme inhibits phorbol-12,13-dibutyrate binding to its receptor in a noncompetitive manner. The inhibition constant (KI) has been found to be 6.6 X 10(-8) M.

Animals↗
PubMed1981

Tissue and species distribution and developmental variation of specific receptors for biologically active phorbol and ingenol esters.

A new and rapid binding assay for [3H]phorbol-12,13-dibutyrate (PDBu) binding to its receptors in cell or tissue homogenates or subcellular fractions is described. PDBu binding sites are widely distributed in mouse tissues. Brain and spleen contain exceptionally high concentrations of these receptors. PDBu receptors are present in all regions of the brain. The choroid plexus and pituitary bind markedly lower amounts of PDBu than other brain regions. Brains or spleens from different murine strains bind almost the same amounts of PDBu while those organs from embryonic mice bind every little PDBu. Binding increases with age and reaches a maximum at approximately 4 weeks of age. Young and mature brains from various species contain these specific binding sites. These data suggest a structural or enzymatic role for PDBu receptors.

Aging↗