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D Goltzman

Publications and source records attributed to D Goltzman.

178 records · Page 10Linked to original sources
PubMed1978

Electron and immunoelectron microscopic study of thyroidal medullary carcinoma.

A correlated light, immunofluorescent, electron and immunoelectron microscopic study on the histogenesis and immunochemical nature of the tumor amyloid in medullary carcinoma of thyroid (MCT) was performed. The light microscopic finding of cytoplasmic inclusions in tumor cells was related ultrastructurally to a unique form of cellular degeneration composed of particulate and fibrillar matters, which could be the precursor of amyloid deposits. The immunofluorescent staining revealed calcitonin in tumor cells and in portions of amyloid. Ultrastructurally, the storage granules were morphologically heterogeneous and contained variable amount of immunoreactive calcitonin. Immunoelectron microscopy demonstrated disintegration of the granules with apparent discharge of calcitonin into the matrix of the above-mentioned particulate and fibrillary mass. Immunoreactive calcitonin was not shown on the mature amyloid fibrils nor in most of the granular matters closely associated with the amyloid. The results indicate that the amyloid in MCT, though related to the secretory products of the tumor cells, may not represent the hormonally active calcitonin molecule.

Amyloid↗
PubMed1978

Characterization of the immunochemical forms of calcitonin released by a medullary thyroid carcinoma in tissue culture.

Immunoreactive calcitonin released by a medullary thyroid carcinoma in tissue culture has been found to exhibit heterogeneity when analyzed by gel chromatography and radioimmunoassay, in a pattern analogous to that seen in the circulation of the patient from whom the neoplasm was removed. To examine the cause of the heterogeneity, the immunoreactive material released by the tumor into tissue culture medium was further analyzed by gel electrophoresis in the presence of the protein denaturant 8 M urea, by gel chromatography after reduction and alkylation, by affinity chromatography on concanavalin A-agarose, and by bioassay in a renal adenylyl cyclase system of enhanced sensitivity. The results suggest that the larger immunochemical forms of calcitonin described in the circulation of patients with medullary thyroid carcinoma may be released directly from the neoplasm and need not derive from peripheral metabolism of the monomer. It could be demonstrated that a major proportion of the immunochemical enlargement is dependent upon intermolecular disulfide bridge formation whereas aggregation or non-convalent protein binding account for a smaller component of the heterogeneity. In view of the absence of binding of the immunoreactive material to the lectin agarose, carbohydrate side chains, at least of the alpha-d glucosyl variety, do not seem to contribute significantly to calcitonin enlargement. Additionally, the studies indicate that, at least by in vitro assay, the larger immunochemical forms of calcitonin, representing the majority of the immunoreactivity released by a medullary thyroid carcinoma, are biologically inactive.

Adenylyl Cyclases↗
PubMed1978

Parathyroid hormone and hyperparathyroidism: current concepts.

Our expanding knowledge of the physiologic and biochemical factors governing calcium homeostasis has improved our capacity to evaluate hypercalcemia, of which an increased incidence is being discovered through the widespread use of multiphasic screening. Consequently, we are diagnosing mild forms of hyperparathryoidism more often than formerly. The enhanced recognition of virtually symptom-free hyperparathyroidism presents a problem in patient management because the natural history of this form of hyperparathyroidism is unknown and the pathologic lesion underlying the condition may be ambiguous, resulting in difficult decisions in surgical management. Further study is required to resolve this issue in order to provide optimal care for the affected individual.

Animals↗
PubMed1977

Medullary carcinoma of the human thyroid in monolayer culture: morphological and cytochemical correlations.

Mechanically or enzymatically dissociated cells from three human medullary thyroid carcinomas (MTC) were grown in monolayer culture for periods up to seven months. Cultures of each tumor contained clusters of small epithelial-like cells which were readily identified by phase contrast microscopy. Immunocytochemical studies and electron microscopy showed that these cells contained abundant calcitonin and numerous secretory granules. Amine-storing mechanisms were also demonstrable in these cells by formaldehyde-induced fluorescence. Homogeneous cultures of epithelial-like cells showed no evidence of transitions into fibroblast-like cells. Addition of thyroxin to the tissue culture medium appeared to promote survival of epithelial-like cells in cultures of one tumor. The ability to morphologically recognize cultured cells with endocrine activity should facilitate establishment of human MTC lines for biochemical and physiological studies.

APUD Cells↗
PubMed1976

Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin.

The conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (w/w) resulted in release of a peptide comigrating with intact hormone marker in systems resolving both on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone, ProPTH-(-6 + 34), consisting of the prohormone hexapeptide covalently bonded to the NIH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by: (i) thin-layer chromatography and amino acid analysis of digestion products; (ii) comparison of the biological activity of the prophormone substrate and the products of digestion; and (iii) peptide end-group analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. It is concluded that the specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule; inasmuch as conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is, therefore, no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.

Adenylyl Cyclases↗
PubMed1976

Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.

Adenylyl Cyclases↗
PubMed1976

Metabolism and biological activity of parathyroid hormone in renal cortical membranes.

Recent studies from several laboratories have documented the presence of fragments of parathyroid hormone in blood or peripheral tissues or in both. Inasmuch as amino-terminal fragments are known to be biologically active, it has been suggested that fragments, rather than the intact polypeptide of 84 amino acids, might be the active molecular species in tissue fluids. Accordingly, the metabolism of native bovine parathyroid hormone, bPTH-(1-84), was studied in purified renal cortical membranes from several species and correlated with hormonal stimulation of adenylyl cyclase in these membranes in vitro. Analysis of whole incubation mixtures or membrane-bound hormone by gel electrophoresis and gel chromatography after incubation of [3H]bPTH-(1-84) or 125-I-labeled bPTH-(1-84) or unlabeled biologically active bPTH-(1-84) with purified canine renal cortical membranes revealed no evidence of proteolysis, and yet the uncleaved hormone readily stimulated adenylyl cyclase. Kinetic studies of hormone-stimulated adenylyl cyclase activity revealed no difference in rate of onset of activity between bPTH-(1-84) And the active synthetic amino-terminal tetratriacontapeptide bPTH-(1-34), and hence there was no evidence of precursor-product relationship between the native hormone and an active amino-terminal fragment. The results suggest, insofar as the activity detected in these membranes reflects the biological response of the hormone in vivo, that the native hormone is indeed biologically active at the receptor level directly without the requirement for cleavage into active fragments.

Adenylyl Cyclases↗
PubMed1976

Role of 5'-guanylylimidodiphosphate in the activation of adenylyl cyclase by parathyroid hormone.

We have studied the effects of guanylylimidodiphosphate (Gpp(NH)p), an analogue of GTP, on the stimulation of renal cortical adenylyl cyclase by bovine parathyroid hormone (bPTH, or bPTH-(1-84)). Incubation of canine renal membranes with bPTH-(3-34), a specific antagonist of parathyroid hormone, in either the presence or absence of Gpp(NH)p, prevented subsequently added bPTH-(1-84) from stimulating adenylyl cyclase. The addition of the antagonist to a cyclase system previously activated by both bPTH-(1-84) and Gpp(NH)p, however, produced no inhibition of enzyme activity. Removal of bPTH by washing the membranes virtually abolished activity, but washing after addition of bPTH plus Gpp(NH)p did not prevent continued accumulation of cAMP. The persistence of the activity of the enzyme brought about by the addition of Gpp(NH)p plus bPTH, despite washing or addition of specific inhibitor of bPTH action, indicates that the activity of the hormone-specific adenylyl cyclase in membrane suspensions is independent of cintinuous occupancy of the peptide-hormone receptor by bPTH in the presence of the guanyl-nucleotide analogue.

Adenylyl Cyclases↗
PubMed1975

Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhibiting analogues.

Two synthetic analogues of bovine parathyroid hormone (PTH) with NH2-terminal modifications, PTH-(3-34) and [desamino-Ala-1]PTH-(1-34), were found to lack agonist activity but to demonstrate antagonist properties when tested in the rat renal cortical adenylyl cyclase assay in vitro against the native hormone or against PTH-(1-34), the active synthetic NH2-terminal tetratriacontapeptide. The inhibition exhibited by these analogues was proportional in degree to the dose of inhibitor, abolished by oxidation of the analogue, reversible by addition of an excess of active hormone, and specific for parathyroid hormone-stimulated renal adenylyl cyclase. No inhibition of basal or sodium fluoride-stimulated renal adenylyl cyclase could be demonstrated. Two other synthetic bovine analogues, PTH-(13-34) and PTH-(1-26), were devoid of agonist and antagonist properties. The over-all results suggest that the requirements for receptor binding of parathyroid hormone are rather broad. Conformational factors or binding interactions involving specific residues, or both seem to require the entire sequence from residue 3 to residue 27 for receptor binding to occur. A dichotomy between receptor binding and adenylyl cyclase activation was demonstrated only by alterations or deletions involving the first 2 NH2-terminal residues of the hormone and emphasizes the importance of these residues in eliciting the biological activity of parathyroid hormone. The two antagonists, [desamino-Ala-1]PTH-(1-34) and PTH-(3-34), should be useful in further analysis of the initial steps in hormone action.

Adenylyl Cyclases↗
PubMed1975

Metabolism and biological activity of proparathyroid hormone and synthetic analogues in renal cortical membranes.

The metabolism of natural and synthetic analogues of bovine proparathyroid hormone (Pro-bPTH) and the biological activity of the synthetic fragments were evaluated in an in vitro assay employing renal cortical membranes (adenylyl cyclase bioassay). Apparent biological activity of the prohormone analogue was correlated with the cleavage of prohormone to hormone by the membranes. Analysis by electrophoresis on polyacrylamide gels and by ion-exchange chromatography on carboxymethyl cellulose (CMC) of the synthetic analogue Pro-bPTH-(-6 leads to +34) labelled with 125I, after incubation with renal cortical membranes, revealed conversion of the analogue to a fragment co-migrating or co-eluting with bPTH (1-34). Similar electrophoretic analyses using biosynthetic Pro-bPTH-(-6 leads to +84) internally labelled with [3H] leucine revealed degradation of Pro-bPTH-(-6 leads to +84) to smaller fragments. Proteolysis of both native prohormone and prohormone analogue was markedly reduced in incubations performed in the presence of benzamidine, a competitive inhibitor of trypsin and trypsin-like enzymes. Inclusion of benzamidine in incubations with purified renal cortical membranes from rat or dog in the in vitro renal adenylyl cyclase bioassay resulted in a ten-fold lowering of the potency of the fragment Pro-bPTH-(-6 leads to +34) relative to that of bPTH-(1-34); the potency of Pro-bPTH-(-6 leads to +34) was reduced from 5.4-3.6% to 0.5-0.3%, on a molar basis, of the potency of bPTH-(1-34). There was no effect of benzamidine on the intrinsic activity of bPTH-(1-34). These studies indicate that most if not all of the apparent biological activity of Pro-bPTH-(-6 leads to +34), at least in vitro, is dependent upon prior enzymic conversion to bPTH-(1-34).

Adenylyl Cyclases↗
PubMedDate not supplied

Studies of the effects of 1,25-dihydroxyvitamin D on skeletal and calcium homeostasis and on inhibition of tumor cell growth.

Vitamin D, parathyroid hormone (PTH), and parathyroid hormone-related peptide (PTHrP) are major regulators of calcium metabolism and vitamin D can also reduce the growth of normal cells and tumor cells. PTHrP and PTH act via a common membrane receptor (PTHR). The mouse PTHR is regulated by a kidney-selective upstream promoter P(1) and ubiquitous downstream promoter P(2). In vitro and in vivo 1,25(OH)(2)D can inhibit PTHR expression in bone but not cartilage by downregulating transcription via P(2). Gene transcription of PTHrP per se can also be downregulated by 1,25(OH)(2)D and by low calcemic vitamin D analogs. This inhibitory effect may reduce the hypercalcemia caused by overproduction of PTHrP by tumor cells. In a malignant keratinoctye cell line, phosphorylation of the retinoid X receptor alpha occurs through the activated Ras-MAP kinase pathway and results in attenuated trans-activation by the vitamin D receptor, its heterodimeric partner. This decreases the growth-inhibitory efficacy of 1,25(OH)(2)D. Studies of the capacity of vitamin D to alter PTHrP production and action and of its anti-proliferative effects can, therefore, shed important light on basic mechanisms controlling these events, and may also have major implications for clinical medicine and therapeutics.

Animals↗