[Massive rectal bleeding in diverticulosis of the colon].
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Aged↗
Biomedical subjects
M Rosenblatt
Publications and source records attributed to M Rosenblatt.
Differential effects of parathyroid hormone responsive cultured human cells on biological activity of parathyroid hormone and parathyroid hormone inhibitory analogues.
We have employed parathyroid hormone (PTH) responsive human cells cultured from dermis or giant cell tumors of bone (GT) to evaluate the biological properties of a newly developed in vivo PTH inhibitor, [Tyr34]bPTH-(7-34)-amide (PTH-Inh). Short periods of incubation of cells from dermis or GT with maximal stimulatory concentrations of PTH in the presence of increasing concentrations of PTH-Inh resulted in a dose-dependent inhibition of the adenosine cyclic 3',5'-phosphate (cAMP) response (Ki = 3 X 10(-7) M and 4.2 X 10(-7) M for GT and dermal cells, respectively). In both cell cultures, PTH-Inh alone did not increase cAMP levels, and in desensitization experiments, preincubation with PTH-Inh alone did not desensitize cells to PTH. Hence, the analogue displayed no agonist properties. Unexpectedly, when PTH-Inh was incubated with dermal cells in the presence of PTH, the PTH-Inh failed to block desensitization, suggesting a loss of biological effectiveness of the inhibitor. When medium containing PTH-Inh alone was removed from dermal cells and tested for inhibition of the acute PTH response in untreated cells, there was apparent loss of inhibitory efficacy (t1/2 = 20 h). In contrast, incubation of native PTH or bPTH-(1-34) with cells did not affect the biological activity of these ligands. Unlike the dermal cells, the PTH-Inh did block desensitization to PTH in GT, and there was no loss of inhibitor efficacy when medium containing PTH-Inh was incubated with GT (48 h) and then tested in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Biological Assay↗
N-cyclo-[Leu5]enkephalin: a rational approach for the synthesis of conformationally restricted cyclic pentapeptides.
The enkephalins are neuropeptides belonging to the class of endogenous opioids. The conformationally restricted analog, N-cyclo-[Leu5]enkephalin, was recently synthesized. Since the synthesis of cyclic pentapeptides which lack proline and contain amino acids with bulky side chains is problematic, the synthesis, purification, and analytical characterization of N-cyclo-[Leu5]enkephalin is described in detail. This conformationally restricted cyclic pentapeptide was prepared from H-Gly-Phe-Leu-[O-(2,6-dichlorobenzyl)-Tyr]-Gly-NHNH2, which was synthesized by the solid-phase method of peptide synthesis. Cyclization was accomplished through an azide intermediate at high dilution, using high-pressure liquid chromatography to monitor the reaction. The desired cyclic monomer was isolated and purified by semipreparative HPLC. The structure of the purified cyclic product was confirmed by multiple chemical techniques including amino acid analysis, lack of an amino terminus (as assessed by reaction with ninhydrin and Edman reagent), and mass spectroscopy.
Amino Acids↗
Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH.
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Animals↗
Thoracic wounds in Israeli battle casualties during the 1982 evacuation of wounded from Lebanon.
During the first 3 weeks of the operation "Peace for Galilee" (O.P.G.) 938 wounded soldiers were evacuated from Lebanon to the Rambam Medical Center in Haifa, Israel. Of these, 64 (6.8%) had chest wounds with or without other wounds. The characteristics of these wounds, the treatment given in the field and at the hospital and their results, are examined and a review of the literature of chest war wounds are summarized. A rapid and efficient in-field medical aid was the most important contributing factor to the low mortality and morbidity rates achieved. As far as we know, this is the first time computerized tomography was used in the diagnosis of war wounds of the chest.
Adolescent↗
Formation of water-soluble complex between the 1-34 fragment of parathyroid hormone and dimyristoylphosphatidylcholine.
Two biologically active, 34 amino acid fragments of parathyroid hormone interact with dimyristoylphosphatidylcholine to form lipoprotein particles. In the lipid-bound form these parathyroid hormone peptides exhibit an increased amount of folded secondary structure and the tryptophan residue of [Nle8, Nle18, Tyr34] b PTH (1-34) amide appears to become buried in a more hydrophobic environment. The lipoprotein particle which is formed has dimensions of approximately 65 X 7 nm but aggregates to larger structures with increasing temperature. Above the phase transition of the phospholipid the peptides no longer affect the morphology of the lipid and the spectral properties of the peptide are not perturbed by the lipid. This is similar to the behavior of glucagon with dimyristoylphatidylcholine. The results indicate that several nonhomologous peptide hormones have common features which allow them to fold into an amphipathic helix and solubilize phospholipid.
Circular Dichroism↗
Endogenous opioids and ventilatory responses to hypercapnia in normal humans.
Though administration of opioid peptides depresses ventilation and ventilatory responsiveness, the role of endogenous opioid peptides in modulating ventilatory responsiveness is not clear. We studied the interaction of endogenous opioids and ventilatory responses in 12 adult male volunteers by relating hypercapnic responsiveness to plasma levels of immunoactive beta-endorphin and by administering the opiate antagonist naloxone. Ventilatory responsiveness to hypercapnia was not altered by pretreatment with naloxone, and this by itself suggests that endogenous opioids have no role in modulating this response. However, there was an inverse relationship between basal levels of immunoactive beta-endorphin in plasma and ventilatory responsiveness to CO2. Furthermore, plasma beta-endorphin levels rose after short-term hypercapnia but only when subjects had been pretreated with naloxone. We conclude that measurement of plasma endorphin levels suggests relationships between endogenous opioid peptides and ventilatory responses to CO2 that are not apparent in studies limited to assessing the effect of naloxone.
Adult↗
Endogenous opioids and ventilatory responses to hypoxia in normal humans.
We studied the putative role of endorphins in modulating hypoxic ventilatory responsiveness. In 12 healthy men, minute ventilation (VE)and mouth occlusion pressure (P0.1) responses to progressive isocapnic hypoxia were determined before and after the intravenous administration of the opioid antagonist naloxone (10 mg) or placebo. Plasma levels of beta-endorphin were measured before and after hypoxia. Naloxone did not affect the slopes or x-intercepts of the relationships between either VE or P0.1 and arterial O2 saturation. There was no correlation between the baseline plasma level of beta-endorphin and any measure of responsiveness to hypoxia. Plasma beta-endorphin levels were not affected by either short-term hypoxia or naloxone alone; however, when hypoxia followed naloxone administration, mean +/- SD beta-endorphin increased from 8.0 +/- 8.9 pg/ml to 20.2 +/- 16.6 pg/ml (p less than 0.005). We concluded that endogenous opioids do not have an important modulating influence on hypoxic ventilatory responsiveness in adult human volunteers.
Adult↗
Evaluation of an in vitro parathyroid hormone antagonist in vivo in dogs.
A potent competitive inhibitor of PTH-stimulated biological responses in vitro, [Nle8,Nle18,Tyr34] bovine PTH (bPTH)-(3-34)amide, was evaluated in vivo in dogs. These studies confirm observations in vitro, suggesting that positions 1 and 2 of the peptide are critical to its biological activity. However, unlike the results from studies in vitro, this PTH analog is a weak agonist with effects on parathyroid target tissues that produce hypercalcemia and phosphaturia and increase urinary cAMP excretion. Assessed by these three parameters of hormonal action in vivo, the estimated potency of this analog is less than 1% of that of the intact hormone. In addition, PTH-induced biological responses were not inhibited by relatively large doses of the bPTH-(3-34) analog. These results emphasize the need for a systemic, integrated approach, combining chemical with biological studies, to design effective inhibitors of hormonal action in vivo. Although the rationale for introducing particular modifications into the peptide structure is most frequently based on bioassays performed in vitro, the success of the strategy chosen must rely, ultimately, upon the demonstration of specific biological properties in vivo.
Animals↗
Effects of synthetic analogs and fragments of bovine parathyroid hormone on adenosine 3',5'-monophosphate level, ornithine decarboxylase activity, and glycosaminoglycan synthesis in rabbit costal chondrocytes in culture: structure-activity relations.
Previously, we demonstrated that PTH increases the level of cAMP, the activity of ornithine decarboxylase (ODC; EC 4.1.1.17; which is a rate-limiting enzyme in polyamine biosynthesis), and glycosaminoglycan (GAG) synthesis (which is characteristic of the chondrocyte phenotype) in rabbit costal chondrocytes in culture. These studies suggested that the accumulation of cAMP and the induction of ODC by PTH are good markers of the differentiated phenotype of cultured chondrocytes. In the present study, the biological effects of a series of bovine PTH (bPTH) fragments and analogs on these three parameters of PTH action were examined. bPTH-(1-34), [Nle8,Nle18,Tyr34]bPTH-(1-34) amide and bPTH-(1-27) amide increased cAMP levels, ODC activity, and GAG synthesis in a dose-dependent manner over the concentration range of 10(-9)-10(-5) M. The order of decreasing potency was: bPTH-(1-34), [Nle8,Nle18,Tyr34]bPTH-(1-34) amide, and bPTH-(1-27) amide. On the other hand, [Nle8,Nle18,Tyr34]bPTH-(3-34) amide, bPTH-(5-27) amide, and [Tyr34]bPTH-(20-34) amide failed to increase cAMP levels, ODC activity, or GAG synthesis when present in concentrations up to 10(-5) M. However, [Nle8,Nle18,Tyr34]bPTH-(3-34) amide, bPTH-(5-27) amide, and [Tyr34]bPTH-(20-34) amide inhibited bPTH-(1-34)-stimulated increases in cAMP and ODC activity. These results partially define the principal structural determinants within the PTH molecule required for biological activity and expression of the differentiated phenotype of chondrocytes.
Animals↗
Rat parathyroid hormone-(1-34) fragment: renal adenylate cyclase activity and receptor binding properties in vitro.
The rat PTH molecule contains five sequence differences from either the bovine or the human hormone within the biologically active 1-34 region. A synthetic rat 1-34 peptide was tested for activity by in vitro activation of canine and rat renal adenylate cyclase and binding to canine renal membrane receptors. The mean potency of 21,400 Medical Research Council units/mg in the canine adenylate cyclase system and 24,900 in the rat system was 8- to 10-fold higher than human 1-34 and 2- to 4-fold greater than bovine 1-34. These values represent the highest potency we have observed to date for a PTH preparation by these assay systems. In contrast, receptor binding of the rat fragment was comparable to that of bovine and human 1-34. Half-maximal inhibition of radioligand binding occurred at 1.7- 2.0 X 10(-9) M with all synthetic hormones. Hence, the amino acid substitutions in rat 1-34 appear to affect the cyclase-activating sequence domain without increasing avidity for the receptor. Analogs combining the rat sequence with modifications known to enhance receptor binding and/or retard enzymatic degradation offer a promising approach to the preparation of still more potent parathyroid agonists.
Adenylyl Cyclases↗
Adherent stem cells: frequency in mouse marrow and terminal clone sizes in long-term culture.
Adherent stem cells (ASC) are hematopoietic stem cells of the mouse bone marrow that are equivalent to CFU-S in pluripotency (they produce CFU-S) and proliferative ability (they go through up to 25 doublings). They can be assayed in vitro by a limiting dilution overlay technique in which stromal bone marrow long-term cultures serve as underlayers. In this article we estimate the occurrence rates of ASC in fresh and adherent bone marrow cells and report the distribution of population doublings estimated for 152 ASC clones.
Animals↗
Photoaffinity labeling of parathyroid hormone receptors: comparison of receptors across species and target tissues and after desensitization to hormone.
Cells derived from human giant cell tumors of bone and fibroblasts derived from human neonatal foreskin respond to parathyroid hormone (PTH) by increasing the intracellular and extracellular levels of adenosine cyclic 3',5'-phosphate (cAMP). Using photoaffinity labeling methods, we examined these cells for the presence of a PTH receptor or a binding subunit of a receptor complex. A previously designed biologically active and photolabile radioligand analogue of PTH was reacted with these intact cells. After photolysis, the cells were extracted, and the proteins were denatured, reduced, and separated by electrophoresis on sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels followed by autoradiography. A single membrane component, Mr 70 000, was labeled specifically in intact cells cultured from skeletal and dermal tissue. By mixing, in pairs, photolabeled proteins from (a) intact human cells derived from giant cell tumors of bone, (b) intact human fibroblasts, and (c) canine renal cortical membranes, the receptors (or their binding subunits) for PTH were compared directly and found to be identical in terms of molecular size (as determined by the migration position on NaDod-SO4-polyacrylamide gels) across species (dog and human) and target tissue (bone, skin, and kidney). Preincubation of cells cultured from giant cell tumors of bone with PTH resulted in loss of the PTH-induced cAMP response (desensitization). Preincubation with PTH was accompanied by a marked decrease in photoaffinity labeling of the PTH binding component and suggests that the loss of hormone response in cells preincubated with PTH was related to a decrease in the number or availability of PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
An in vitro clonal assay of adherent stem cells (ASC) in mouse marrow.
Hematopoietic stem cells with high proliferative capacity can be assayed when stromal bone marrow cultures are overlaid with limiting dilutions of marrow samples. This leads to hematopoietic growth after 4 weeks in a fraction of cultures, consistent with expectations based on Poisson statistics. It will be shown that monoclonal cultures are obtained that last from 2 to 15 weeks and that can generate up to several million mature granulocytes. The originating clone-forming cell is named adherent stem cell (ASC) because of its adherence to plastic or stromal surfaces. The ASC is comparable to the CFU-S in frequency, proliferative capacity and in its ability to give rise to CFU-S. As an unexpected additional finding we report that a mode of "clonal succession" was apparent in cultures which expressed more than one clone.
Animals↗
Parathyroid hormone: biochemical aspects of biosynthesis, secretion, action, and metabolism.
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Adenylyl Cyclases↗
Evaluation of the biological properties of parathyroid hormone and analogues in a vascularly isolated parotid gland-based assay.
Infusion of bovine parathyroid hormone (bPTH) preparations into the arterial blood supply of the vascularly isolated parotid gland in anaesthetized sheep increases salivary phosphate concentration and gland blood flow rate with rapid onset and offset of action. These responses have been used as a bioassay for PTH and PTH analogues and for assessing the properties of an in-vitro inhibitory analogue [Nle-8, Nle-18, Tyr-34]bPTH-(3-34)amide. [Nle-8, Nle-18, Tyr-34]bPTH-(1-34)amide at 10(-9) to 10(-8) mol/l was four to five times more potent than bPTH(1-34) on both salivary phosphate and blood flow assays. Human PTH(1-34) was not significantly more potent than bPTH(1-34). The [Nle-8, Nle-18, Tyr-34]bPTH-(3-34)amide analogue had very slight agonist activity at 3 X 10(-7) mol/l and at a 100:1 ratio of analogue to PTH it completely inhibited the action of bPTH(1-34) on phosphate secretion and gland blood flow. It caused partial inhibition at 10:1 and had no evident effect at 1:1. These results differ from previous in-vitro results and indicate that the preparation may be valuable for evaluation of agonist and antagonist analogues of PTH. The vascularly isolated parotid gland of the sheep permits repeated random testing of analogues in a control-test-control sequence and the results indicate high sensitivity to PTH in a rapidly reactive in-vivo system with two responding parameters.
Animals↗
Difference between 1-84 parathyroid hormone and the 1-34 fragment on renal tubular calcium transport in the dog.
Acute clearance studies were performed in chronically thyroparathyroidectomized dogs to determine similarities and differences between the effects of 1-34 and 1-84 parathyroid hormone (PTH) on urinary calcium excretion. In a small (physiological) dose, 1-84 PTH caused no mean change in percentage calcium excretion, while the 1-34 fragment was frankly calciuric. At this dose, the two hormone preparations were equally phosphaturic. Fragments of PTH in the range 25-84 did not alter calcium or phosphate transport. We conclude that the entire molecule is required in order for the distal nephron effect of the hormone to enhance net calcium transport in the kidney to become manifest.
Adenylyl Cyclases↗
Semi-preparative high-performance liquid chromatographic purification of a 28-amino acid synthetic parathyroid hormone antagonist.
Structure-activity studies of parathyroid hormone (PTH) over the last decade have led to the design and synthesis of a peptide hormone inhibitor of PTH action in vivo, [Tyr-34]bPTH-(7-34)amide. To evaluate the biological properties of this 28-amino acid hormone analogue, sufficient amounts of the peptide needed to be prepared in a high state of purity to permit continuous infusion in groups of animals for periods of several hours. For this purpose, gel chromatography followed by semi-preparative, reversed-phase high-performance liquid chromatography (HPLC) using a lyophilizable solvent gradient system of acetonitrile in 0.1% trifluoroacetic acid was utilized. In a parallel purification scheme, gel filtration followed by ion-exchange chromatography was employed. Comparison of yields and product purity were determined by a battery of chemical analytical techniques. Semi-preparative HPLC generated a highly purified product; substantial heterogeneity was detected in the ion-exchange-purified material. The HPLC-purified product retains the biological activities of binding to PTH receptors in vitro and inhibiting PTH action in vivo.
Amino Acid Sequence↗