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Biomedical subjects

J T Potts

Publications and source records attributed to J T Potts.

At least 181 records · Page 10Linked to original sources
PubMed1978

Human parathyroid hormone: synthesis and chemical, biological, and immunological evaluation of the carboxyl-terminal region.

The carboxyl-terminal region of human parathyroid hormone (hPTH) was synthesized by the solid phase method. The 32-amino acid fragment, hPTH-(53-84), was prepared for two reasons: 1) to produce antisera directed exclusively against the carboxyl-terminal region of hPTH, which represents the predominant form of the hormone in blood; and 2) to determine whether a portion of the hormone other than the amino-terminal region has any of the biological activity of intact hormone or can bind to PTH receptors. The synthetic fragment was evaluated for heterogeneity by amino acid composition and by several high resolution analytical systems (thin layer electrophoresis, polyacrylamide gel isoelectric focusing, and Edman sequence analysis). Synthetic hPTH-(53-84) lacked PTH-like activity in both in vitro (renal) and in vivo (bone) assays and failed to inhibit the action of native PTH in vitro, indicating lack of receptor binding by the carboxyl-terminal region of the molecule. Because the fragment lacked biological activity, nonchemical evaluation was performed to confirm that the peptide represents the carboxyl terminus of hPTH. Immunological comparison of synthetic hPTH-(53-84) to native hPTH was undertaken using an RIA that employed an antibovine PTH antiserum whose antigenic recognition was limited to the carboxyl-terminal region of the hormone. Synthetic hPTH-(53-84) was more immunoreactive than native hPTH. The immunological findings provide further evidence that the synthetic peptide accurately represents the carboxyl-terminal region of native hPTH.

Antigen-Antibody Reactions↗
PubMed1978

Purification of human parathyroid hormone: recent studies and further observations.

During the isolation of human parathyroid hormone there is an extensive loss of immuno-assayble hormone over the successive extraction steps, due in part to the presence of fragments that are soluble in 4% trichloroacetic acid. These fragments are derived from both the amino- and carboxyl-terminal regions of the hormone. The hormonal fractions precipitated with trichloroacetic acid were further purified by gel filtration and ion-exchange chromatography. At the final ion-exchange purification step, some preparations of the hormone eluted in multiple fractions. When the various components were characterized separately by immunoassay, amino acid composition, enzymic cleavage and partial sequence analysis, they were found to be closely comparable, although the most acidic fraction contained a blocked terminal amino group. Extraction of a number of batches of tissue permitted revision of the amino acid composition of human parathyroid hormone. Biosynthetic studies with labelled amino acids confirmed the absence of tyrosine and the presence of phenylalanine and threonine and localized these residues to definite regions of the molecule.

Amino Acid Sequence↗
PubMed1977

DNA complementary to parathyroid mRNA directs synthesis of pre-proparathyroid hormone in a linked transcription-translation system.

DNA complementary in sequence to the messenger RNA for pre-proparathyroid hormone was synthesised using reverse transcriptase. In a linked transcription-translation system using RNA polymerase and cell-free extract from wheat germ, the DNA directed the synthesis of a protein identified as pre-proparathyroid hormone by N-terminal sequencing and by electrophoretic and immunologic criteria.

Amino Acid Sequence↗
PubMed1977

Synthesis, purification, and chemical characterization of the amino-terminal 1-34 fragment of bovine parathyroid hormone synthesized by the solid-phase procedure.

Peptides prepared by solid-phase synthesis during a systematic study of structure-activity relations in parathyroid hormone have been subjected, after sequential purifications, to rigorous analysis of purity by a variety of analytical techniques including sequence analysis by the Edman procedure. The present paper undertakes a critical appraisal of the utility of different tests of peptide purity and the use of the procedures in guiding and monitoring optimal synthesis strategies. Sequence analysis of a peptide representing the amino-terminal 34 residues of bovine parathyroid hormone (bPTH-(1-34)) revealed the presence of at least 30% of contaminating error peptides which were undetected by other analytical procedures. The major contaminant was identified as a peptid in which glutamine at position 29 was deleted. A repeat synthesis using fluorescamine rather than ninhydrin to monitor the coupling reaction resulted in a preparation that lacked the contaminant resulting from deletion. These findings demonstrate the particular value of sequence analysis in the evaluation of purity of peptides synthesized by the solid-phase technique.

Adenylyl Cyclases↗
PubMed1977

Biosynthesis and secretion of calcitonin by avian ultimobranchial glands.

The biosynthesis and release of calcitonin from chicken ultimobranchial glands were evaluated both in vivo and in vitro. Changes in the content of calcitonin in plasma and ultimobranchial glands of chicks during embryonic development were measured using a radioimmunoassay for salmon calcitonin that readily detects chicken calcitonin. Calcitonin content of the ultimobranchial glands underwent a striking rise during the late-embryonic and newborn periods, and a transient phase of brisk calcitonin secretion into blood was observed at the time of hatching. Ultimobranchial glands from late-embryo and newborn chicks, when incubated in vitro with radioactive leucine, incorporated labeled leucine into protein and released radioactive protein into the culture medium linearly over a period of 30 h. Analyses, by electrophoresis on polyacrylamide gels and by reactivity with anti-calcitonin sera, of radioactive protein released into the media revealed two radioimmunoassayable and immunoprecipitable peaks of chicken calcitonin. The two peaks could not be distinguished by their molecular weights upon electrophoresis on urea-SDS gels. These observations are most consistent with the existence of two isohormones of chicken calcitonin. No evidence for a biosynthetic precursor of calcitonin was obtained. Changes in calcium concentrations between 1.0 and 3.5 mM is incubation media in vitro did not affect release or storage of calcitonin, and changes in dietary calcium intake did not produce observable in vivo alterations in calcitonin content of ultimobranchial glands.

Animals↗
PubMed1977

Renal effects of native parathyroid hormone and synthetic biologically active fragments in pseudohypoparathyroidism and hypoparathyroidism.

To gain further insight into the biological significance of parathyroid hormone (PTH) metabolism, native parathyroid hormone and synthetic peptides, similar to PTH metabolites generated in vivo, have been given intravenously to human subjects. The resultant changes in renal excretion of adenosine 6':5' monophosphate (cyclic AMP) and inorganic phosphate have been measured in five pseudohypoparathyroid, four hypoparathyroid, and one pseudopseudohypoparathyroid patient. As anticipated, native PTH promptly increased urinary cyclic AMP and phosphate excretion in the hypoparathyroid and pseudo-pseudohypoparathyroid patients, and had little or no effect on their excretion in the pseudohypoparathyroid patients. Synthetic bovine parathyroid hormone 1-34 and synthetic human parathyroid hormone 1-34 had effects essentially identical to each other and to native PTH. We conclude that the PTH resistance of pseudohypoparathyroidism is probably not caused by a defect in PTH metabolism. We further conclude that synthetic human or bovine parathyroid hormone 1-34 could be used for diagnostic evaluation of patients.

Creatinine↗
PubMed1977

The metabolism of [6-3H]1alpha, hydroxycholecalciferol to [6-3H]1alpha,25-dihydroxycholecalciferol in a patient with renal insufficiency.

To evaluate whether 1alpha-hydroxycholecalciferol is metabolized to 1alpha,25-dihydroxycholecalciferol in man, [6-3H]1alpha-hydroxycholecalciferol was given intravenously to a patient with renal failure who was maintained daily on 100,000 IU vitamin D and calcium supplements. Using Sephadex LH-20 and high-pressure liquid chromatography, it was clearly demonstrated that 1alpha-hydroxycholecalciferol rapidly disappears from the blood and is metabolized to 1alpha,25-dihydroxycholecalciferol.

Dihydroxycholecalciferols↗
PubMed1977

Design and synthesis of parathyroid hormone analogues of enhanced biological activity.

Three analogues of bovine parathyroid hormone (bPTH), [Tyr-34]bPTH-(1--34) amide, [Nle-8,Nle-18,Tyr-34]bPTH-(1--34)amide, and [Nle-8,Nle-18, o-NPS-Trp-23,Tyr-34]bPTH-(1--34)amide were synthesized by the solid-phase method. The synthetic peptides were found to be homogeneous in multiple analytical systems. These analogues represent the NH2-terminal one-third of the hormone, previously shown to contain all the structural requirements necessary for biological activity, but containing several structural modifications associated with enhancement and stabilization of biological activity. When tested in the in vitro renal adenylylcyclase assay, in which unsubstituted bPTH-(1--34) has a potency of 5400 MRC Units/mg, [Tyr-34]bPTH-(1--34)amide had a potency of 16,1000 MRC Units/mg, [Nle-8,Nle-18,Tyr-34]bPTH-(1--34)amide was 10,100 MRC Units/mg, and [Nle-8,Nle-18,o-NPS-Trp-23,Tyr-34]bPTH-(1--34)amide was 10,600 MRC Units/mg. The diverse structural features incorporated in these hormone analogues, resulting in a several-fold increase in biological activity in vitro, demonstrate additive effects on biological activity and should prove valuable in certain biological applications, as well as in the design of other parathyroid hormone analogues of enhanced potency.

Adenylyl Cyclases↗