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Biomedical subjects

A D Hamilton

Publications and source records attributed to A D Hamilton.

84 records · Page 5Linked to original sources
PubMed1995

Burkitt lymphoma Daudi cells contain two distinct farnesyltransferases with different divalent cation requirements.

Farnesylation is a lipid posttranslational modification required for the biological function of several signaling proteins including the Ras oncoprotein where this modification is required for malignant transformation. Here we report the identification of two distinct farnesyltransferases (FTases) in Burkitt lymphoma Daudi cells. Separation of Daudi cell cytosolic fractions by ion exchange chromatography resulted in two peaks (FTases I and II) that, on gel filtration, show molecular masses of 90 000 and 250 000 Da, respectively. Immunoblotting experiments showed that FTase I is composed of an alpha/beta-heterodimer of about 50 000 Da each. FTase II contained a beta-subunit that is immunologically indistinguishable from the beta-subunit of FTase I and the previously reported human and rat brain FTase but contained an alpha-subunit that reacted poorly with a rat brain anti-alpha-antibody. As in rat brain FTase, Daudi FTases I and II both required magnesium for enzymatic activity. However, their zinc requirements differed. In the absence of Zn2+, FTase I had little activity (10%) whereas FTase II had 30% of its maximum activity (maximum activity obtained in the presence of Zn2+). Furthermore, whereas both FTases I and II were potently inhibited by KB-Ras C-terminal Cys-Val-Ile-Met tetrapeptide mimics, only FTase I but not FTase II required zinc for peptide binding and inhibition.

Alkyl and Aryl Transferases↗
PubMed1995

CAAX peptidomimetic FTI-244 decreases platelet-derived growth factor receptor tyrosine phosphorylation levels and inhibits stimulation of phosphatidylinositol 3-kinase but not mitogen-activated protein kinase.

Cysteine farnesylation of the Ras carboxyl terminal tetrapeptide CAAX motif (where C = cysteine, A = leucine, isoleucine, or valine, and X = methionine or serine) is required for Ras biological activity. In this report, we describe the effects of inhibitors of farnesyltransferase (FTase), the enzyme responsible for this lipid modification, on platelet-derived growth factor (PDGF) signaling in NIH-3T3 cells. In vitro, the CAAX peptidomimetic FTI-232 exhibits potent inhibition of FTase activity (IC50 = 150 nM) and its carboxyl-methylated counterpart, FTI-244, inhibits Ras processing in vivo. Treatment of NIH-3T3 cells with FTI-244 inhibits PDGF-induced DNA synthesis but not stimulation of mitogen-activated protein kinase (MAPK). However, FTI-244 significantly reduces PDGF-induced tyrosine phosphorylation levels of PDGF receptor (PDGFR) as well as its association with, and activation of, phosphatidylinositol-3-kinase (PI-3-K), a key enzyme in PDGF-induced mitogenesis.

3T3 Cells↗
PubMed1995

Stabilization of helical domains in short peptides using hydrophobic interactions.

The contribution of hydrophobic interactions in the stabilization of helical structure was compared for a series of short peptides that incorporated two epsilon-(3,5-dinitrobenzoyl)Lys residues at various positions. Results showed that in aqueous/organic mixtures, methanol induced helical stability over a wider range and at higher concentration than trifluoroethanol (TFE); a similar degree of stability was seen in low mole fraction mixtures of TFE in water. Solvent mixture titrations in TFE/water demonstrated that helical stability was highest for the peptide having a pair of modified residues spaced by three other residues. Solvent mixture titrations in TFE/water appear to be useful in indicating the degree of hydrophobic stabilization.

Amino Acid Sequence↗
PubMed1995

A non-peptide mimetic of Ras-CAAX: selective inhibition of farnesyltransferase and Ras processing.

Cysteine farnesylation of the carboxyl-terminal tetrapeptide CAAX (C = Cys, A = Leu, Ile, or Val, X = Met or Ser) of the oncogene product Ras is required for its malignant transformation activity. As a consequence farnesyltransferase (FTase), the enzyme responsible for this lipid modification, has become one of the most sought-after targets for anticancer drug development. We have recently designed peptide mimics of the COOH-terminal Cys-Val-Ile-Met of KB-Ras where the dipeptide Val-Ile was replaced by aminobenzoic acid derivatives. Although these peptidomimetics are potent inhibitors of FTase in vitro, they retain several undesirable peptide features that hamper their use in vivo. We report here the design, synthesis, and biological activity of the first non-peptide mimetics of CAAX where the tripeptide AAX was replaced by biphenyl derivatives. (R)-4-[N-(3-mercapto-2-aminopropyl)]amino-3'- carboxybiphenyl, where the cysteine is linked to the biphenyl derivative through a secondary amine, contains no amino acids, lacks peptidic features, and has no hydrolyzable bonds. This peptidomimetic is a potent inhibitor of FTase in vitro (IC50 = 50-150 nM) and disrupts Ras processing in whole cells. Furthermore, this non-peptide mimetic of CAAX is highly selective for FTase (666-fold) relative to the closely related geranylgeranyltransferase I. This selectivity is also respected in vivo since the processing of Ras but not the geranylgeranylated Rap1A was disrupted in whole cells. Structure activity relationship studies revealed that FTase recognition and inhibitory potency of CAAX peptidomimetics require free thiol and carboxylate groups separated by a hydrophobic moiety, and that precise positioning of these functional groups must correspond to that of the parent CAAX. The true CAAX peptidomimetic described in this manuscript has several desirable features for further development as a potential anticancer agent. It is not metabolically inactivated by FTase, does not require a pro-drug strategy for inhibition in vivo, and is selective for farnesylation relative to geranylgeranylation.

Alkyl and Aryl Transferases↗
PubMed1994

Enhanced extraction of phenobarbital from serum with a designed artificial receptor.

The primary goal of this work was to determine whether artificial receptors that function on the basis of molecular recognition have analytical capabilities. As an example of such a receptor, we have chosen one directed toward barbiturates. Chloroform enriched with this artificial receptor (1 mM) can extract more than 90% of the phenobarbital from a 20 microM phenobarbital solution in human control serum using a volume ratio (organic/serum) as small as 0.5. In the absence of this receptor, the volume ratio must be greater than 10 to achieve similar extraction efficiencies. In addition to volume ratio, the role of pH, receptor concentration, and solvent type are discussed. The experimental results are found to be in good agreement with predictions based on chemical equilibria. Through the use of this and other similar receptors, the amount of organic solvent used in extractions can be minimized.

Barbiturates↗
PubMed1994

Design and structural requirements of potent peptidomimetic inhibitors of p21ras farnesyltransferase.

Cysteine farnesylation of the ras oncogene product, p21ras, on its carboxyl-terminal CA1A2X box (C = cysteine, A = aliphatic, and X = methionine or serine) is required for its transforming activity. p21ras farnesyltransferase (FTase), the enzyme responsible or this important posttranslational modification can be inhibited by simple CA1A2X peptides. We have synthesized a family of CA1A2X peptidomimetics where the central 2 aliphatic amino acids are replaced by a variety of spacer groups with different shapes and conformational characteristics to investigate the structural requirements of these inhibitors. The biological activities of CA1A2X peptidomimetics, where the dipeptide "A1A2" is replaced by 3- or 4-aminomethylbenzoic acid (AMBA) and 3- or 4-aminobenzoic acid (ABA), are evaluated in a p21ras FTase inhibitory assay. Peptidomimetics Cys-4-ABA-Met and Cys-3-AMBA-Met contain spacers that provide good distance correspondence of the carboxylate and ammonium separation with that of the parent KB p21ras tetrapeptide, Cys-Val-Ile-Met, and are as potent FTase inhibitors (IC50 values of 50 and 100 nM, respectively). In contrast, replacing the central dipeptide with 4-AMBA reduced FTase inhibitory activity by 17-fold whereas replacement by 3-ABA reduces inhibitory activity of the peptidomimetics by 43-fold. Cys-4-ABA-Met (IC50 = 50 nM) is 128 times more potent as a p21ras FTase inhibitor than Cys-3-ABA-Met (IC50 = 6400 nM), yet these two peptidomimetics differ only in the substitution pattern around the phenyl ring. These results coupled with computer modeling studies demonstrate that the interaction between FTase and the peptidomimetics requires precise structural and conformational characteristics; in particular, correct positioning of the Cys and Met must be respected. Furthermore, Cys-3-AMBA-Met and Cys-4-ABA-Met are true inhibitors of p21ras FTase since they are not farnesylated by this enzyme, in contrast to Cys-Val-Ile-Met, which inhibits the enzyme by acting as alternative substrate. Computer modeling studies of the potent FTase inhibitor Cys-4-ABA-Met show that a folded conformation, where the thiol and carboxylate groups are close, is not possible. Therefore a beta-turn conformation that would result in the simultaneous coordination of the Cys-thiol and Met-carboxylate to zinc ion is not important for inhibition of p21ras FTase, as previously suggested.

Alkyl and Aryl Transferases↗
PubMed1993

Potent inhibition of human tumor p21ras farnesyltransferase by A1A2-lacking p21ras CA1A2X peptidomimetics.

The ras oncogene product p21ras requires farnesylation and subsequent plasma membrane association for its transforming activity. This key post-translational modification is catalyzed by p21ras farnesyltransferase, which transfers farnesyl from farnesylpyrophosphate to the cysteine of the CA1A2X carboxyl-terminal tetrapeptide of p21ras. In the present report, we describe potent inhibition of p21ras farnesyltransferase by CA1A2X peptidomimetics containing no peptidic amide bonds. We synthesized a series of CA1A2X analogues where the 2 aliphatic amino acids A1 and A2 were replaced by a hydrophobic spacer, 3-aminomethylbenzoic acid (AMBA). The peptidomimetic Cys-AMBA-Met, inhibits p21ras farnesyltransferase from human colon carcinoma (COLO-205) and Burkitt's lymphoma (Daudi) with IC50 values of 60 and 120 nM, respectively. Cys-AMBA-Met is 3-, 8-, and 9-fold (COLO-205) and 2-, 5-, and 7-fold (Daudi) more potent than the corresponding tetrapeptides of p21KB-ras (CVIM), p21N-ras (CVVM), and p21KA-ras (CIIM), respectively. Replacing methionine at the X position with negatively charged glutamate reduces its ability to inhibit the enzyme, whereas positively charged lysine at this position abolishes the inhibitory character of the peptidomimetic. A hydrophobic moiety at the X position, as in Cys-AMBA-Phe, retains potent inhibitory activity. Leucine in the X position of CA1A2X is a post-translational signal for protein geranylgeranylation rather than farnesylation, and, as expected, Cys-AMBA-Leu does not inhibit the enzyme. Furthermore, CVIM, CVVM, and CIIM are farnesylated by human p21ras farnesyltransferases and inhibit these enzymes by serving as alternative substrates. In contrast, the peptidomimetics described here are true p21ras farnesyltransferase inhibitors since none is farnesylated by this enzyme.

Alkyl and Aryl Transferases↗
PubMed1992

Prevalence of haemoglobin variants in a diabetic population and their effect on glycated haemoglobin measurement.

The measurement of glycated haemoglobin (glycated Hb) by automated high pressure liquid chromatography enabled the prevalence of haemoglobin variants (Hb variant) to be determined in a large diabetic population within Leeds, UK. There were 16 patients showing a Hb variant amongst a total of 5300 diabetics, the majority of the variants being HbA/S traits. There were also 13 patients showing a slightly raised HbF in the range of 2% to 5%. A multi-centre study was carried out by distributing blood specimens from some of the patients with Hb traits. Significant differences in the percentage of glycated Hb were observed relating to the methodology used. However, glycated Hb is used to monitor glycaemic control and laboratories must be aware of Hb variant interferences on their methods. Also, screening for Hb variants should be considered for the at risk groups of diabetic patients.

Diabetes Mellitus↗
PubMed1989

The introduction of a quality assessment scheme for extra-laboratory blood glucose analysis.

An external quality assessment scheme is described for on-ward testing of blood glucose by nursing staff using the Glucometer II/Glucostix system. This involved the distribution of two concentrations of glucose solutions to the wards at 2-week intervals. Initially, results were examined prior to the introduction of a formal training programme for use of the blood glucose meters when the hospital CV was 25% (low glucose) and 19% (high glucose). Some improvement was shown after 6 months, with CVs of 13 and 8%, and a greater improvement after 12 months with CVs of 6 and 8%, respectively. A ward report was designed to allow easy interpretation of performance by the participants in the scheme. The introduction of this report was well received and generated enthusiasm among the ward staff.

Blood Glucose↗
PubMed1986

Synthesis of complement components (C3, C2, B and C1-inhibitor) and lysozyme by human monocytes and macrophages.

The synthesis of C3, C2, factor B (B) C1-inhibitor and lysozyme has been studied in monocytes and macrophages isolated from the synovial fluids of patients with rheumatoid arthritis. Concentrations of all 5 proteins in culture supernatants were measured by the sandwich ELISA technique. Kinetic studies showed that only lysozyme and C3 could be detected in monocyte culture supernatants on the first day of culture, whereas C2, B and C1-inhibitor were not present until the third day. In contrast all 5 proteins could be detected in the supernatants of macrophage cultures on day 1. In both monocyte and macrophage cultures synthesis of lysozyme and C1-inhibitor continued throughout the culture period, whereas synthesis of C2, B and C3 appeared to be reduced after the fifth day in culture. Quantitative studies showed that the secretion rates of lysozyme (4,700 X 10(3) molecules/cell/hr) was similar in monocytes and macrophages. Synthesis rates for all 4 complement components in monocyte cultures were less than 0.2% of that for lysozyme. Although the synthetic rates were higher in macrophages, even then they constituted less than 2% of the rate for lysozyme. Synthetic rates for complement components, but not lysozyme, were increased by BSA-anti-BSA antigen-antibody complexes and reduced by serum-treated complexes. Although the functional activity of monocyte B was similar to that for serum, the activity of monocyte C2 was 5 times that of serum C2. As C42 formed with monocyte C2 had a half-life of 13.5 min at 30 degrees C, compared with 4.5 min for the enzyme formed with serum C2, it is probable that monocyte C2 is oxidized by the oxygen products of these cells.

Antigen-Antibody Complex↗