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R C Sheppard

Publications and source records attributed to R C Sheppard.

15 recordsLinked to original sources
PubMed1991

Production and characterization of an anti-peptide antibody specific for the growth-associated protein, GAP-43.

In an attempt to raise specific anti-GAP-43 antibodies, a C-terminal tetradecapeptide was synthesized based on the predicted rat GAP-43 amino acid sequence using the F-moc polyamide solid phase procedure. The synthesized carboxy-terminal peptide was purified by reverse phase HPLC, conjugated to bovine serum albumin (BSA) and used as an immunogen. Polyclonal antipeptide antibodies raised in rabbits were affinity purified and their specificity for GAP-43 tested by Western blotting. Brain and spinal cord homogenates and GAP-43 enriched synaptosomal membrane fractions were analysed either by SDS-PAGE or reverse phase HPLC. The anti-GAP-43 antibodies detected a major immunoreactive band at 43 kDa (B-50), and a minor immunoreactive band at 38 kDa (B-60) together with an additional protein-band at 68 kDa, which was related to the peptide carrier, BSA. Immunohistochemical studies using these carboxy-terminal antipeptide antibodies revealed a widespread distribution of GAP-43 immunoreactivity throughout the adult rat brain and spinal cord, in a pattern generally consistent with earlier histochemical studies. It is concluded that the C-terminal GAP-43 specific tetradecapeptide is a potent immunogen and provides a convenient tool for the production of sequence specific anti-GAP-43 antibodies.

Aging↗
PubMed1979

Rapid chemical synthesis and circular dichroism properties of some 2'-5'-linked oligoriboadenylates.

Specific synthesis of some oligoadenylates including A2'p5'A2'p5'Ap(2'), the 2'-phosphorylated oligoribonucleotide core of the recently discovered protein synthesis inhibitor pppA2'p5'A2'p5'A is described using a novel solid-phase method. The CD spectra of A2'p5'Ap(2'), A2'p5'A2'p5'Ap(2') and A2'p5'A2'p5'A (derived by treatment of the phosphorylated synthetic trimer with E. coli alkaline phosphatase) are presented. Comparison of the latter spectrum with that of A2'p5'A2'p5'A obtained similarly from a biologically derived sample of pppA2'p5'A2'p5'A provides further evidence that this molecule is in fact the first naturally-occurring 2'-5'-linked oligoribonucleotide.

Adenosine↗
PubMed1979

Rapid synthesis of oligodeoxyribonucleotides. III. Effect of added carboxylate ion on the efficiency of internucleotide bond formation. Solid-phase synthesis of the dodecanucleotide, d(pT-A-A-C-T-G-C-T-C-A-C-T).

In solid-phase oligodeoxyribonucleotide synthesis the addition of benzoic acid to internucleotide phosphodiester bond-forming reactions lowers coupling yields by far greater amount than anticipated on a simple nucleotide-carboxylate molar ratio. High coupling yields were consistently obtained in synthesis of the dodecanucleotide, d(pT-A-A-C-T-G-C-T-C-A-C-T), using rigorously purified nucleotide starting materials.

Benzoates↗
PubMed1978

[52-Homoserine]-basic pancreatic trypsin inhibitor. Preparation and properties of a protein analog.

Treatment of basic pancreatic trypsin inhibitor (BPTI) with cyanogen bromide smoothly cleaves the polypeptide chain at the single methionyl residue. The newly formed homoserine lactone and alpha-amino functions are held in proximity by a disulfide linkage, and in neutral aqueous solution react together spontaneously to re-form the peptide chain. The resulting analog, [52-homoserine]-BPTI is very similar to the native molecule in most properties measured. The rate of formation of this analog from the chain-cleaved intermediate has been determined. It is apparent that the facility of analog synthesis is due in large part to the retention of the native protein conformation in the cyanogen bromide-cleaved intermediate.

Amino Acid Sequence↗
PubMed1977

Rapid synthesis of oligodeoxyribonucleotides. II. Machine-aided solid-phase syntheses of two nonanucleotides and an octanucleotide.

Preparation of the two nonanucleotides, d(pG-G-A-G-G-A-G-A-A) and d(pT-T-C-T-G-T-T-G-A), and the octanucleotide, d(pT-G-G-C-C-C-A-T) is described by a rapid solid-phase method on a polyamide support. The syntheses were carried out using a modified solid-phase peptide synthesiser and required one day for each nucleotide addition. A microparticulate anion-exchanger, Partisil 10SAX, was used for the efficient fractionation of synthetic, base-protected oligonucleotides.

Base Sequence↗
PubMed1977

Rapid synthesis of oligodeoxyribonucleotides: a new solid-phase method.

A method is described that makes use of a new polyamide resin for the rapid synthesis of short oligodeoxyribonucleotides. The method is illustrated by the preparation of two heptadeoxyribonucleotides, d(pT6-C) and d(pC-A-G-T-G-A-T) using a phosphodiester approach. A further development involved use of phenyl isocyanate as an in situ drying agent, which obviated the need for solvent co-evaporation prior tothe internucleotidic coupling steps. Improved fractionation of thymidyl oligonucleotides was obtained by use of a new microparticulate, silica-based anion-exchanger.

Amides↗
PubMed1977

Studies on the partial synthesis of protein analogs by direct coupling to terminal homoserine lactone derivatives. Experiments with basic pancreatic trypsin inhibitor.

Treatment of basic pancreatic trypsin inhibitor (BPTI, I) with cyanogen bromide smoothly yields the chain cleaved derivative II. The utility of the seco-lactone (II) in the partial synthesis of protein analogues has been investigated. It is shown that the homoserine lactone ring is sufficiently reactive to combine directly with the radiolabelled synthetic peptide glycylglycylanine t-butyl ester in both aqueous and non-aqueous solution, leading to a BPTI analogue which has been purified and characterized.

Cyanogen Bromide↗
PubMedDate not supplied

An acquired interatrial fistula secondary to para-aortic abscess documented by transesophageal echocardiography.

Para-aortic ring abscess and resulting fistulous communication between adjacent structures frequently occur in prosthetic aortic valve endocarditis but are rarely diagnosed preoperatively. We report a patient who had an abscess involving the aortic-mitral intervalvular fibrosa that eroded into the interatrial septum, causing an interatrial communication with a left-to-right shunt. The abscess was detected by transthoracic echocardiography, but the fistula was only seen by the subsequent transesophageal echocardiogram. To our knowledge, this is the first report of an interatrial fistula secondary to a para-aortic valve abscess and its diagnosis preoperatively. Transesophageal echocardiography should be performed in any patient suspected to have complicated aortic endocarditis.

Abscess↗
PubMedDate not supplied

DBU as an N alpha-deprotecting reagent for the fluorenylmethoxycarbonyl group in continuous flow solid-phase peptide synthesis.

The versatility and efficiency of the N alpha-fluorenylmethoxycarbonyl method of solid-phase peptide synthesis have been further enhanced by recent and continuing refinements in side-chain protecting group, linker and amino acid-coupling technology. In contrast, comparatively little work has been carried out on studying and further improving the N alpha-deprotection step. This report demonstrates that, in low concentrations, the non-nucleophilic amidine 1,8-diazabicyclo[5.4.0]undec-7-ene is a preferred alternative to the more commonly employed piperidine for the rapid and efficient cleavage of the N alpha-fluorenylmethoxycarbonyl group in the continuous flow method of solid-phase peptide synthesis. In the cases studied, use of this base did not cause cyclization of Asp(OBut)-Gly or Asn-Gly sequence to succinimide derivatives. At the recommended concentration of 2% 1,8-diazabicyclo[5.4.0]undec-7-ene in dimethylformamide, racemization of resin-bound C-terminal S-trityl cysteine was substantially reduced compared with standard piperidine-mediated deprotection conditions. Additionally, use of the base appears to reduce the extent of broadening of ultraviolet fluorenylmethoxycarbonyl-deprotection peaks occasionally observed when piperidine is employed, indicating that its reaction kinetics and mode of action are different.

Amino Acid Sequence↗