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Biomedical subjects

D R Harding

Publications and source records attributed to D R Harding.

At least 37 records · Page 2Linked to original sources
PubMed1987

The complement component C1s catalysed hydrolysis of peptide 4-nitroanilide substrates.

The kinetic parameter kcat/Km has been determined for the hydrolysis of peptide 4-nitroanilides, catalysed by complement component C1s. Substrates based on the C-terminal sequence of human C4a (Leu-Gln-Arg) were synthesised. Replacement of the glutamine residue by glycine or serine increased kcat/Km. Substitution of valine for the leucine residue increased kcat/Km, while substitution of glycine or lysine for the leucine residue decreased kcat/Km slightly. D-Val-Ser-Arg 4-nitroanilide is the most reactive 4-nitroanilide substrate towards C1s, so far. These results are discussed in relation to the amino acid sequences near the bonds cleaved by C1s in C4, C2 and C1 inhibitor.

Amino Acid Sequence↗
PubMed1986

Determination of thermodynamic parameters for the interaction of a lipid-binding peptide and insulin with a reversed-phase column.

The effect of temperature was investigated on the reversed-phase chromatography of a synthetic lipid associating peptide (LAP), with the following sequence (LESFLKSWLSALEQALKA) and on insulin. The LAP was chromatographed on a muBondapak-alkylphenyl column with 1% aq. triethylammonium phosphate, pH 3.2-2-propanol (80:20) as the isocratic mobile phase. The insulin was separated on a Resolve-C18 column with a mobile phase that contained 0.1 M sodium phosphate, pH 2.0-acetonitrile (71.5:28.5). With the LAP the Van't Hoff plot (In k' vs. 1000/T) was linear and the value of enthalpy for association of the peptide with the reversed-phase column was large and negative. The phase ratio was estimated for the column used in the separation and then derivation of the corresponding entropic term demonstrated that the association was enthalpy and not entropy drive. By contrast the corresponding Van't Hoff plot derived or the insulin study was non-linear and with a positive slope. Further study indicated that the formation of the insulin-reversed phase complex could be attributed to a favorable entropy change. It is probable that the non-classical thermodynamics observed during the insulin chromatography could be related to conformational changes in the insulin structure during the chromatographic process.

Animals↗
PubMed1985

Selective labelling of apolipoproteins A-I and C-I at methionine residues by (3H) methyl exchange.

Apolipoproteins C-I and A-I were radioactively labelled with tritium by (3H)-methyl exchange. The methionine residues were first methylated with (3H)-methyl iodide at pH 4 and the reaction products were purified by gel filtration and cation exchange chromatography. The products were then demethylated with 2-mercaptoethanol (6 M) at pH 8.6 to regenerate the apolipoproteins in an unmodified but tritiated form. The specific radioactivity for apolipoprotein C-I and A-I was 3.5 X 10(6) and 1.5 X 10(7) dpm/pmol respectively. The properties of (3H)-apolipoprotein C-I were examined by reversed phase HPLC and by incorporation into very low density lipoproteins (VLDL).

Apolipoprotein A-I↗
PubMed1985

Synthesis of peptide labelled with p-iodophenylalanine which corresponds to the first amphipathic region of apolipoprotein C-I.

The amino terminal fragment (1-15) of apolipoprotein C-1, H-Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe14-Gly was prepared by the solid phase method. However, the phenylalanine residue at position 14 was replaced with p-iodophenylalanine in the chemical synthesis. The preparation of t-butyloxy-carbonyl-p-iodophenylalanine is described. After completion of the synthesis, the product was deprotected and cleaved from the resin with liquid HF. The peptide was purified by gel filtration on Sephadex G-25 and preparative high performance liquid chromatography. The purified material was shown to be homogeneous by amino acid analytical data and by chromatography in three different analytical reversed phase HPLC systems. The peptide was then labelled by the chloroamine-T procedure and good incorporation of 125I was obtained. After purification of the product by gel filtration on Biogel P-2, the labelled pentadecapeptide was tested for ability to bind to Very Low Density Lipoproteins (VLDL) in the following manner: VLDL was isolated from normolipemic serum by ultracentrifugal flotation and 100-microliter samples were incubated with labelled material dissolved in 200 microliter of 0.5 M NaCl, 0.02 M sodium phosphate buffer, pH 7.4 at 37 degrees for 30 min. The VLDL fraction was again isolated by ultracentrifugal flotation and the incorporation of radioactivity into the lipoprotein was measured. Under these conditions, a sample of [3H]-native apolipoprotein C-I was incorporated to an extent of 83% of the total sample, while the [125I]-pentadecapeptide exhibited an incorporation of 8.7%.

Amino Acid Sequence↗
PubMed1985

Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis.

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus.

Amino Acid Sequence↗
PubMed1984

Antisera to synthetic peptide recognize high molecular weight enkephalin-containing proteins.

Antisera to a synthetic peptide corresponding to the 95-117 sequence of proenkephalin were used to develop a sensitive radioimmunoassay. Gel-filtration of acid extracts of bovine adrenal medulla and purified chromaffin granules revealed that the antisera recognized high molecular weight material (Mr approximately 5,000-30,000). The material in peak I ( Mr 20 ,000-30,000) and peak II (Mr 10,000-20,000) was further purified by immunoaffinity chromatography. Sequential digestion of each of these fractions with trypsin and carboxypeptidase B generated immunoreactive Met-enkephalin. This study demonstrates that antisera against a synthetic peptide cross-react with high molecular weight enkephalin-containing precursors, validating the use of these antisera in studies of enkephalin biosynthesis.

Adrenal Medulla↗
PubMed1984

Hexabrix as a contrast agent for hysterosalpingography.

Hexabrix, a monoacidic dimeric iodinated contrast material, was compared with Renografin 60 (diatrizoate meglumine) for safety, tolerance, and efficacy in hysterosalpingography, in a double blind study of 52 patients. No serious adverse reactions were noted in either group of patients. Discomfort during and after the procedure was not statistically different in the two groups. No difference in radiographic quality was seen. It was concluded that Hexabrix, despite reduced osmolality and increased viscosity, offers no significant benefit over Renografin 60, but it is a safe and acceptable contrast agent for hysterosalpingography.

Contrast Media↗
PubMed1983

Occurrence of idiopathic, familial hyperchylomicronaemia in a cat.

Primary hyperlipoproteinaemia (hyperchylomicronaemia with slight very low density lipoprotein elevation) is described in two related male cats. Fasting hyperlipaemia, lipaemia retinalis and subcutaneous xanthomas were detected on clinical examination. In one cat lipoprotein lipase activity measured after heparin activation was significantly reduced compared to the response in a normal cat. The lipid and protein concentration in each of the lipoprotein classes and the lipoprotein distribution of the two hyperlipaemic cats, two normolipaemic relations and 16 normolipaemic adult cats were determined. Plasma cholesterol and triglyceride levels were elevated in the hyperlipaemic cats with the major proportion of triglyceride and cholesterol being present in chylomicrons whereas in normolipaemic cats the majority of triglyceride was contained in very low density lipoprotein. High density lipoprotein was the predominant lipid carrier in both the normolipaemic and the hyperlipaemic cats but the protein content in chylomicrons was elevated in the two affected cats. The lipoprotein distribution in normal cats in this study agrees with previously reported values. The hyperlipaemic cats showed many of the features of familial lipoprotein lipase deficiency (type I hyperlipoproteinaemia, exogenous chylomicronaemia) which is an inherited disease in man.

Animals↗
PubMed1983

Hyperbetalipoproteinaemia and hyperalphalipoproteinaemia in a single family.

Twelve members of a kindred were studied of whom six showed elevated low-density lipoprotein (LDL) cholesterol levels. Within the blood related group nine showed elevated high density lipoprotein (HDL) cholesterol levels and correspondingly elevated HDL lipoprotein apoprotein levels. The members with elevated LDL also therefore had elevated HDL. There was no family history of premature vascular disease in the kindred and the 74 year old member was clinically free of ischaemic vascular disease. It is considered that the coincidence of a familial tendency to high blood LDL and HDL was not associated with appearance of premature arterial disease in this kindred.

Adolescent↗
PubMed1982

Evaluation of digital venous angiography for the diagnosis of renovascular hypertension.

Thirty-two patients being evaluated for hypertension by angiography were also studied by digital video subtraction angiography (DVSA). Twenty-three of the 76 renal arteries were found by conventional angiography to have significant lesions. Two experienced angiographers evaluated the DVSA studies without knowledge of the angiographic results. The accuracy of DVSA for evaluation of renal arteries was 87% for Observer I and 80% for Observer II. Sensitivities were 87% and 83% and specificities 87% and 79% for the two observers. Of the 13 patients with significant lesions, Observer I identified at lest one lesion in all 13 while Observer II identified a lesion in 12 of the 13. The high false-positive rate (26% for Observer I and 37% for Observer II) was thought to be caused by subtraction artifacts, quantum noise, relatively low spatial resolution, and the Mach effect.

Angiography↗
PubMed1982

Hysterosalpingography: comparison of Conray 60 and Sinografin.

Conray 60 and Sinografin were compared as contrast agents for hysterosalpingography in 40 consecutive patients in a double-blind protocol. Emphasis was centered on the incidence and severity of symptoms during and after the procedure. Of patients receiving Conray, 36% suffered moderate or severe discomfort compared with 78% of the patients receiving Sinografin, and 13% of patients receiving Conray complained of moderate delayed discomfort compared with 28% of those receiving Sinografin. Both agents produced excellent diagnostic studies. Conray was clearly superior to Sinografin in terms of patient comfort.

Diatrizoate↗
PubMed1981

Separation of the apoprotein components of human very low density lipoproteins by ion-paired, reversed-phase high performance liquid chromatography.

A number of crude apolipoprotein samples isolated from human very low density lipoproteins (VLDL) were analyzed by reversed phase high performance liquid chromatography. The mobile phase consisted of a 1% solution of the polar ion-pairing reagent triethylammonium phosphate. A slow, nonlinear gradient of acetonitrile (37--42%) was used to elute the apolipoproteins. The order of elution was as follows: apolipoprotein CX, apolipoprotein C-I, apolipoprotein C-III2, apolipoprotein C-III1, apolipoprotein C-IIIQ and apolipoprotein C-II. This order is consistent with the known polarity of the proteins, i.e., the most nonpolar, apolipoprotein C-II, was the last to be eluted, whereas apolipoprotein C-I, with the lowest nonpolar surface area eluted first. The recovery of the individual apolipoproteins was 80--95% and the individual peaks were characterized by amino acid analysis, UV absorption spectra amd chromatography of pure protein standards.

Acetonitriles↗
PubMed1981

Use of the chromogenic p-(p-(dimethylamino)phenylazo) benzyl (AZ) ester in the synthesis of leu-enkephalin.

Solution synthesis of Leu-enkephalin has been achieved using the coloured carboxyl protecting p-(p-(dimethylamino)phenylazo)benzyl moiety throughout the synthesis. The intense colour of this protecting group allowed facile purification of protected intermediates on silica gel or ion exchange columns and visualisation on thin-layer chromatography. The chromogenic p-(p-(dimethylamino)phenylazo)benzyl ester group readily withstands successive treatments with 25% trifluoroacetic acid in dichloromethane yet is cleanly removed by catalytic hydrogenation.

Amino Acids↗
PubMed1981

Use of perfluoroalkanoic acids as volatile ion pairing reagents in preparative HPLC.

This report describes the successful use of the ether soluble, ion pairing reagents perfluoropropoinic and perfluorobutyric acid in the preparative and analytical reserved phase HPLC of underivatised peptides. The preparative separation of a 1-g sample of Pyr-His-Gly, the proposed anorexigenic peptide, is described on C18-silica which was packed in a flexible-walled cartridge and subjected to radial compression. The mobile phase consisted of an aqueous solution of perfluorobutyric acid (5 mM) and a flow rate of 100 ml/min was used. The purified peptide was simply isolated by neutralizing and freeze-drying the corresponding peak and then extracting the excess ion pairing reagent with ether. The product was then shown to be homogeneous by analytical HPLC and amino acid analysis. The tripeptide failed to show any effect on food intake, water intake or body weight in female rats. Similarly no effect was noted on the reproductive cycles of the rat.

Animals↗
PubMed1978

Reversed-phase, high-pressure liquid chromatography of peptides and proteins with ion-pairing reagents.

Reversed-phase, high-pressure liquid chromatography has been successfully applied to the analysis of peptides and proteins by the addition of hydrophilic (for example, phosphoric acid) or hydrophobic (for example, hexanesulfonic acid) ion-pairing reagents, or both, to the mobile phase. Examples described included proteins such as insulin, glucagon, and 1-24 ACTH pentaacetate (ACTH is adrenocorticotrophic hormone).

Adrenocorticotropic Hormone↗