Biomedical subjects

J B Wheatley

Publications and source records attributed to J B Wheatley.

10 recordsLinked to original sources

PubMed2000

Synthesis of a statistically exhaustive fluorescent peptide substrate library for profiling protease specificity.

A statistically exhaustive, 8800 compound tripeptidal amidomethylcoumarin library was synthesized as discreet compounds using solid-phase combinatorial chemistry. A subset of the compounds was purified by HPLC and tested in a high-throughput fluorometric assay against several known serine and cysteine proteases to demonstrate the utility of this library for profiling protease substrate specificity.

Chromatography, High Pressure Liquid↗

PubMed1999

Salt-induced immobilization of affinity ligands onto epoxide-activated supports.

The immobilization of affinity ligands onto epoxy-activated stationary phases is enhanced at high concentrations of certain salts, such as ammonium sulfate and potassium phosphate. This enhancement is thought to occur because of a salt-induced hydrophobic interaction between the affinity ligand and the surface of the stationary phase. The increase in concentration of the affinity ligand near the reactive epoxy groups leads to an increase in the rate of reaction between the nucleophilic groups on the affinity ligand and the epoxide. The salt-induced enhancement is applicable to proteins and nucleotides at neutral pH and to small affinity ligands at elevated pH. In most cases, the hydrolysis of the epoxy groups does not limit the amount of affinity ligand immobilized. This review discusses the use of high salt concentrations to immobilize proteins, oligonucleotides and peptides to epoxy-activated silica and polymer supports. These modified supports can be used in affinity applications such as affinity chromatography or immunoassays.

Chromatography, Affinity↗

PubMed1996

Salt-induced immobilizations of DNA oligonucleotides on an epoxide-activated high-performance liquid chromatographic affinity support.

Synthetic oligonucleotides, possessing a recognition sequence for the transcription factor NF-kappa B, were immobilized onto an epoxide-activated hydroxyethylmethacrylate HPLC affinity support in the presence of high concentrations of potassium phosphate. The extent of immobilization increased with salt concentration in a manner analogous to that which has been reported for salt-induced immobilizations of proteins. High immobilization efficiencies were achieved, and at 2.7 M potassium phosphate, 85-90% of the DNA initially present in the reaction mixture was immobilized. Reactions were examined for double stranded DNA, one strand of which was modified with a 5'-mercaptohexyl spacer arm, and for each of the strands comprising the duplex. For double stranded immobilizations, about 85% of the non-modified strand (the d22 strand) was released from the support under melting conditions, suggesting that d22 exhibited low reactivity when organized as the duplex. For immobilizations of single stranded DNA, mild acid hydrolysis of the products was used to provide information concerning the mode of attachment. For reactions of the d22 strand alone, only about 60% each of guanine and adenine were recovered from the immobilized oligonucleotide following mild acid hydrolysis. This suggests that when d22 is immobilized as the single strand, significant attachment occurs through the purine bases, in contrast with the low reactivity exhibited by d22 in the duplex. Purified p50 protein, the DNA binding element of NF-kappa B, and nuclear extracts from phorbol ester-stimulated HeLa cells were injected onto a column packed with the double stranded product. In both cases p50 was retained on the column and was recovered upon elution with a salt gradient.

Base Sequence↗

PubMed1996

Glutathione-associated enzymes in the human cell lines of the National Cancer Institute Drug Screening Program.

The steady state expression of glutathione S-transferases (GSTs) at both the protein and mRNA level is reported for the 60 tumor cell lines that are used for the National Cancer Institute Drug Screening Program. Individual GST isozymes were separated, identified, and quantified (with reverse-phase calibration curves) through a novel high performance liquid chromatographic procedure. GSTP1 was the predominant isozyme and was found at quantifiable levels in all but two of the cell lines. This isozyme ranged from 0.03% to 2.7% of the total cytosolic protein. For the mu family, 90% of the lines had GSTM2, 68% had GSTM3, but only 28% were positive for the M1 phenotype. The M1 proportion is lower than would be expected from the standard M1 null phenotype for human populations. Isozymes of the alpha family were detected only at very low levels in 35% of the lines. Significant quantitative correlations among enzyme activity, total enzyme protein, and mRNA were shown for GSTP1. However, such relationships were not apparent for the mu or alpha families. Levels of glutathione (GSH), and the transcript levels of other enzymes involved in GSH homeostasis were determined. gamma-Glutamyl cysteine synthetase (gamma-GCS) was present in all cell lines, but did not correlate with levels of intracellular GSH. Glyoxalase-I and gamma-glutamyl transpeptidase, both involved in GSH salvage, were found in 100% and 70% of the cell lines, respectively. Using a pattern-matching computer program, COMPARE, we compared and correlated the arrays of mRNA and protein levels with the pattern of chemosensitivity or chemoresistance of the 60 cell lines with 175 agents constituting a standard agent database. This database is composed of compounds to which a putative mechanism of action has been assigned. Although Pearson correlation coefficients relating the target and drug patterns were generally modest, when the patterns for the enzyme protein and mRNA levels for GST pi were correlated to drug sensitivity patterns, the list of 30 agents most closely matching (for which P < 0.05) was enriched with alkylating agents. gamma-GCS also showed an enrichment of alkylating agents in the COMPARE correlations, indicating that high levels of gamma-GCS may be an important determinant of resistance. In contrast, none of the other enzymes or GSH had patterns of expression that resulted in an obvious correlation to the sensitivity or resistance of alkylating agents.

Antineoplastic Agents↗

PubMed1994

Variability of glutathione S-transferase isoenzyme patterns in matched normal and cancer human breast tissue.

The determination of GST levels in blood has been proposed to a marker of tumour burden in general, whereas level of the P1 isoenzyme has been identified as a prognostic factor for breast-cancer patients receiving no adjuvant chemotherapy. Particular glutathione S-transferase (GST) isoenzymes differ in their substrate specificity, however, and their presence or absence might therefore account for the resistance of tumours to particular chemotherapeutic drugs, as already established for cultured cell lines. Determination of the GST isoenzyme profile of a cancer tissue could have prognostic value in the selection of treatment if the levels of expression/activity show a degree of variation comparable with that exhibited by actual patient responses. Using reversed-phase h.p.l.c. to quantify affinity-isolated GSTs, we have analysed full isoenzyme profiles in the first large sample of matched normal and cancer human tissues (18 breast-cancer patients). In no patients did the tumour tissues express any isoenzymes that were not found in normal breast tissue. In addition to the GSTs, another enzyme, identified as enoyl-CoA isomerase, was regularly found in breast tissue cytosol following elution from a hexyl-glutathione affinity column. In most cases, the average level of GST was substantially elevated in the cancer tissues above the levels in normal breast tissue from the same patient. Furthermore, the relative levels of the isoenzymes were substantially more variable in the cancer samples than in the normal breast tissue, providing a plausible mechanism for the well established variable response to treatment.

Adult↗

PubMed1994

Coupled affinity-reversed-phase high-performance liquid chromatography systems for the measurement of glutathione S-transferases in human tissues.

HPLC affinity and reversed-phase modes were coupled for the direct measurement of glutathione S-transferases (GSTs) in cytosol extracts. Two coupling designs were examined. In the sequential configuration the affinity column served to extract the isoenzymes which were then eluted directly onto the reversed-phase column as a single fraction. Subsequent separation in the reversed-phase mode provided a GST profile based on the subunit composition of the isoenzymes as a whole. In the second configuration (rapid sampling configuration), gradient elution was performed in the affinity mode resulting in resolution of the intact isoenzymes. The eluate from the affinity separation was sampled in continuous, repetitive intervals and automatically subjected to ongoing reversed-phase analysis. This multidimensional approach provided information on the GST subunit content and also gave information about the distribution of the subunits among individual isoenzymes, thereby forming a basis for the determination of the actual isoenzymatic composition of the GSTs. In both configurations, events were automated and co-ordinated through the use of computer and multiport switching valves. Examples of GST separations from these procedures are shown for human lung and liver tissues. A comparison of the GST subunit analyses from normal and cancer lung tissue excised from the same patient showed substantial elevations of GSTs in the cancer sample. Two-dimensional affinity-reversed-phase analysis of a human liver sample illustrates the utility of the technique for determining the isoenzymatic organization of GST subunits. The criteria for extending two-dimensional analysis to more complex GST mixtures are discussed.

Chromatography, Affinity↗

PubMed1994

Examination of glutathione S-transferase isoenzyme profiles in human liver using high-performance affinity chromatography.

A method for the examination of the glutathione S-transferase isoenzyme profiles in human liver using a new HPLC affinity support is described. Liver cytosol was injected directly onto an HPLC column (5 x 0.46 cm) containing a support with a covalently bound affinity ligand (S-octylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted with a linear gradient of a different affinity ligand in the mobile phase. Coinciding with the affinity ligand gradient, a salt gradient (0-200 mM sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers. The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reversed-phase chromatography. For one liver three Alpha class isoenzyme subunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-performance affinity chromatography method reduced analysis time by a factor of ten when compared to other affinity analysis methods for the glutathione S-transferases.

Chromatography, Affinity↗

PubMed1992

Multiple ligand applications in high-performance immunoaffinity chromatography.

A mixture of antibodies specific for albumin and transferrin, immobilized onto a single support, was used for the simultaneous extraction of albumin and transferrin from immunoglobulin G by high-performance immunoaffinity chromatography. The affinity column was coupled to a strong cation exchanger in order to monitor the success of the extraction and to demonstrate the compatibility of the two chromatographic modes. Coupling of non-affinity chromatography with multiple ligand affinity chromatography is discussed as an alternative to positive affinity chromatography for protein purification.

Chromatography, Affinity↗

PubMed1991

Effect of antigen size on optimal ligand density of immobilized antibodies for a high-performance liquid chromatographic support.

The antigen binding capacities for purified polyclonal antibodies immobilized onto a silica-based high-performance liquid chromatographic (HPLC) affinity support are described for three serum proteins over a range of antibody ligand densities. The rate of decline in the specific activity of the immobilized antibodies with respect to increasing ligand density was found to increase with the molecular weights of the antigens. The antibodies used were purified from whole antiserum using high-performance affinity chromatography and were examined using HPLC on an SCX stationary phase. Conditions are also described for efficient coupling of the ligand to the support.

Animals↗

PubMed1986

Thyrotoxic myopathy in mice: accentuation by a creatine transport inhibitor.

To demonstrate the importance of creatine and phosphocreatine in skeletal muscle during periods of metabolic stress, thyrotoxicosis was induced in mice fed the creatine transport inhibitor, beta-guanidinopropionic acid (beta-GPA). Adding 2% of beta-GPA to the diet of normal mice inhibited weight gain and caused a 75% reduction of creatine and phosphocreatine concentrations in skeletal muscle. Addition of 0.25% or 2% of thyroid powder to the diet of normal mice was associated with hyperactivity, cardiomegaly, and a high mortality rate. Superimposing thyrotoxicosis on mice already depleted of creatine and phosphocreatine resulted in degeneration of muscle fibers. These results indicate that high concentrations of creatine and phosphocreatine are essential for the maintenance of muscle integrity during periods of metabolic stress.

Animals↗