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Biomedical subjects

M Rosenblatt

Publications and source records attributed to M Rosenblatt.

At least 109 records · Page 6Linked to original sources
PubMed1990

Removal of partial agonism from parathyroid hormone (PTH)-related protein-(7-34)NH2 by substitution of PTH amino acids at positions 10 and 11.

PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2 have previously been shown to be shown to be more potent antagonists than the corresponding PTH peptide, [Tyr34]bPTH(7-34)NH2. However, these peptides also display partial agonism for adenylate cyclase activity in ROS 17/2.8 cells. In this study, design of a pure potent antagonist of PTH and PTHrP by removal of agonism from PTHrP(7-34)NH2 with retention of antagonist potency was accomplished. Since [Tyr34]bPTH(7-34)NH2 lacks agonist activity, we introduced two amino acids native to the PTH sequence into their respective positions in PTHrP and the potent D-Trp12 analog. [Asn10Leu11]- and [Asn10,leu11,D-Trp12]-PTHrP(7-34)NH2 were found to be 23- and 26-fold more potent as antagonists in ROS cells than PTHrP(7-34)NH2 and [D-Trp12]PTHrP(7-34)NH2, respectively. In addition, these peptides did not display partial agonism, even in an assay based on highly responsive cells pretreated with dexamethasone and pertussis toxin. In contrast, when the PTHrP sequence Asp10,Lys11 was inserted into [Tyr34]hPTH(7-34)NH2, antagonist potency declined by more than 6-fold and PTH-like agonist activity was installed. These results demonstrate that the activation domain of both PTH and PTHrP can be extended to include the 1-12 region and that the 10-12 region, in addition to the N-terminal hexapeptide, is important not only for receptor binding but also for hormonal signal transduction.

Adenylyl Cyclases↗
PubMed1990

Treatment of bone-derived ROS 17/2.8 cells with dexamethasone and pertussis toxin enables detection of partial agonist activity for parathyroid hormone antagonists.

In the design and biological evaluation of PTH antagonists, certain analogs, although antagonists in vitro, possess partial agonist properties in vivo that preclude their utility as antagonists. In an effort to identify weak agonism of PTH analogs, an attempt was made to enhance the responsiveness of the widely employed rat osteosarcoma (ROS 17/2.8) cell adenylate cyclase assay. Because responsiveness to PTH in these cells is enhanced upon treatment with dexamethasone (dex) or pertussis toxin (PT), we have evaluated their use to aid in detection of partial agonism for PTH and PTH-related protein (PTHrP) antagonist analogs. Treatment of cells with dex alone (30 nM for 3 days) or with PT alone (40 ng/ml for 1 day) increased basal adenylate cyclase activity by 27%. However, combination of the dex and PT treatments increased basal cAMP production 70%. The in vivo partial agonist [Nle8,18,Tyr34]bPTH(3-34)NH2 increased cAMP production 3-fold over basal levels in untreated cells, nearly 5-fold in PT-treated cells, 8-fold in cells treated with dex, and 10-fold in cells treated with dex plus PT. Similar results were obtained with PTHrP(7-34)NH2: the 6-fold stimulation observed in control cells was converted to 14-fold in cells treated with dex plus PT. Agonist activity undetectable in the conventional assay was observed in the dex plus PT system: [Tyr34]- and [D-Trp12,Tyr34]bPTH(7-34)NH2, which exhibit no agonist activity under control conditions, stimulated cAMP production 2.6- and 2.1-fold, respectively, under dex plus PT treatment. In contrast, the antagonist analogs [Asn10,Leu11]- and [Leu11,D-Trp12]PTHrP(7-34)NH2, hybrid peptides of PTH and PTHrP, had no agonist activity under any conditions. Because of increased responsiveness, this assay should occupy an important step in the pathway for evaluation of PTH antagonists and permit identification of weak partial agonist activity before extensive in vivo testing.

Adenylate Cyclase Toxin↗
PubMed1989

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone.

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.

Amino Acid Sequence↗
PubMed1989

Pleural block: technique for regional anesthesia during percutaneous hepatobiliary drainage.

A new technique was used to achieve safe regional anesthesia in seven patients undergoing percutaneous hepatobiliary drainage. The pleural block was administered, and pain was assessed during and after the procedure by means of sensitivity to pin prick, verbal response, electrocardiographic tracings, and blood pressure measurements. In all patients the technique produced relief of somatic pain and did not alter hemodynamic or respiratory status.

Anesthesia, Conduction↗
PubMed1988

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers.

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M], as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2-3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.

Amino Acid Sequence↗
PubMed1988

Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene.

A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.

Amino Acid Sequence↗
PubMed1988

Direct action of the parathyroid hormone-like human hypercalcemic factor on bone.

Peptides containing residues 1-34 of the human parathyroid hormone (PTH)-like hypercalcemic factor (hHCF), termed hHCF-(1-34)-NH2, produce effects similar to those of PTH in several biological systems in vitro and in vivo. However, there is conflicting evidence regarding the potency of hHCF on bone and, by implication, its role in calcium mobilization and the skeletal contribution to tumor-associated hypercalcemia. To resolve this conflict, the effects of infusing either hHCF-(1-34)-NH2 or a peptide containing residues 1-34 of bovine PTH [bPTH-(1-34)] into unrestrained thyroparathyroidectomized rats on a low calcium diet were compared. Direct effects on bone histology and serum calcium levels, which are totally dependent on calcium mobilization from bone in these animals, were examined. bPTH-(1-34) and hHCF-(1-34)-NH2 were equipotent in producing dose-dependent calcium mobilization from bone. At an infusion rate of 0.1 nmol/hr, both peptides produced hypercalcemia and extensive nephrocalcinosis. Histomorphometric analysis of tibiae from these animals after 48 hr of peptide infusion showed a dose-related increase in osteoclast number from 3-5 cells per mm2 at 0.01 nmol/hr to approximately equal to 32 cells per mm2 at 0.1 nmol/hr of hHCF or bovine PTH. These findings indicate that hHCF has a direct PTH-like effect on bone and, in this model system, the hHCF-(1-34)-NH2 is equipotent to bPTH-(1-34).

Animals↗
PubMed1988

A tumor-secreted protein associated with human hypercalcemia of malignancy. Biology and molecular biology.

This investigation addresses a theoretical concept of tumor pathogenesis proposed over 40 years ago, namely that malignancy-associated hypercalcemia can result from endocrine secretion by tumors of a PTH-like factor. These studies demonstrate that a fragment of hHCF alone, without added or tumor-secreted cofactors or hormones, can produce hypercalcemia and other biochemical abnormalities associated with HHM. The hypercalcemia can be generated by hHCF-(1-34)NH2 action on bone, although kidney and gut could contribute to the HHM syndrome when it occurs naturally. No other tumor-secreted peptide displays this biological profile. These studies establish one (PTH-like) mechanism by which human tumors could produce hypercalcemia. Furthermore, the finding that hHCF-(1-34)NH2 is more potent than PTH in some systems is of considerable interest for the future design of hormone analogs. A broad spectrum of biological properties of hHCF-(1-34)NH2, including production of components of the HHM syndrome, can be inhibited by a PTH antagonist. Because [Tyr-34]bPTH-(7-34)NH2 selectively and competitively occupies PTH receptors, our studies demonstrate formally that hHCF-(1-34)NH2 mediates some (and perhaps all) of its actions via receptors conventionally regarded as intended for interaction with PTH, but which actually may be present to allow for expression of bioactivity of both secreted proteins. Although some structural homology is shared by the two hormones and many contribute to interaction with receptors, the disparity in structure, especially within the 1-34 domains responsible for bioactivity in both hormones, is more pronounced. The similarity in biological profiles despite structural differences between hHCF and PTH is emphasized by the inhibitory action of [Tyr-34]bPTH-(7-34)NH2 against the tumor peptide even in the absence of much of the homologous region in the PTH antagonist. This investigation provides impetus for designing more potent antagonists, which must now be regarded more appropriately as inhibitors of both PTH and hHCF. Such antagonists may best be generated from hybrid structures of the two hormones. In any case, these studies establish a promising new approach to therapy of tumor-associated hypercalcemia.

Base Sequence↗
PubMed1988

Comparison of postreceptor effects of 1-34 human hypercalcemia factor and 1-34 human parathyroid hormone in rat osteosarcoma cells.

A tumor-derived factor believed to cause hypercalcemia by acting on the parathyroid hormone (PTH) receptor was recently purified, cloned, and found to have NH2-terminal sequence homology with PTH. The 1-34 region of this protein was synthesized, evaluated for its postreceptor effects on the ROS 17/2.8 cell line, and its properties were compared to 1-34 PTH. Both 1-34 human humoral hypercalcemia factor (HCF) and 1-34 PTH stimulated adenylate cyclase with an effective concentration (EC)50 of approximately 1 nM. The extent of stimulation by both peptides was equally enhanced by dexamethasone. They both had a pronounced inhibitory effect on growth in the presence of dexamethasone, with an EC50 of approximately 0.1 nM, reduced alkaline phosphatase (AP) activity by approximately 70% in the absence of dexamethasone and by approximately 80% in the presence of dexamethasone with an EC50 of 0.03 nM, and when present at a concentration of 10 nM, reduced AP mRNA levels (estimated by Northern analysis) by approximately 80% in the presence or absence of dexamethasone. Thus, in addition to similar dose-response curves for adenylate cyclase stimulation, both HCF and PTH produced identical postreceptor effects in ROS 17/2.8 cells. These effects of HCF are probably mediated by the interaction of the tumor-derived factor with the PTH receptor.

Animals↗
PubMed1988

Clearance and early hydrolysis of atrial natriuretic factor in vivo. Structural analysis of cleavage sites and design of an analogue that inhibits hormone cleavage.

This study examines the clearance and early hydrolysis of atrial natriuretic factor (ANF) in vivo. Radiolabeled ANF was cleared from the circulation of the rat with biphasic kinetics; the majority (90%) of ANF cleared with a t1/2 of 15 s, the remaining peptide was cleared with a t1/2 of 5 min. Microsequence analysis of ANF peptides recovered from the circulation of rats revealed five major degradation products of the intact hormone. The first cleavage occurred between amino acids 12 and 13 of the hormone and would inactivate ANF. Over time, additional fragments of the hormone were generated, including fragments of 6, 7, 21, and 24 amino acids in length. Whole body radioautography of rats injected with [123I]-ANF revealed the kidney as a predominant organ involved in clearance of ANF. Subsequent amino acid sequence analyses of radiolabeled ANF exposed to the kidney in vivo indicated that this organ generated four of the five major hydrolysis products observed in circulation, namely, the 6, 7, 16, and 21 amino acid fragments of the hormone. In an attempt to stabilize ANF in vivo, a synthetic analogue of the hormone was prepared that contained the amino acid analogue, aminoisobutyric acid, substituted at position 13. This analogue completely abolished the in vivo cleavage of ANF at this site. These studies demonstrate the usefulness of a protein chemistry approach in characterizing hormone metabolism in vivo and designing analogues with enhanced in vivo stability to cleavage.

Amino Acid Sequence↗
PubMed1988

The 7-34-fragment of human hypercalcemia factor is a partial agonist/antagonist for parathyroid hormone-stimulated cAMP production.

The N-terminal fragment of human hypercalcemia factor (hHCF), hHCF-(1-34)NH2, has bioactivities similar to PTH in vitro and in vivo. Because it interacts with PTH receptors and is more potent than PTH in some systems, the hHCF sequence may provide interesting leads for the design of potent and selective PTH and hHCF antagonists. Based on the antagonist activity of [Tyr34]bovine PTH-(7-34)NH2 [( Tyr34]bPTH-(7-34)NH2), we synthesized the corresponding fragment of hHCF, hHCF-(7-34)NH2 and examined its properties in vitro. In the bone-derived rat osteosarcoma cell line ROS 17/2.8, hHCF-(7-34)NH2 and [Tyr34]bPTH-(7-34)NH2 were equipotent for inhibition of radiolabeled PTH-binding. In contrast, hHCF-(7-34)NH2 was 8-fold more potent that [Tyr34]bPTH-(7-34)NH2 for inhibiting PTH-stimulated cAMP production. hHCF-(7-34)NH2 also inhibited PTH-binding and PTH-stimulated adenylate cyclase activity in bovine renal cortical membranes: hHCF-(7-34)NH2 and [Tyr34]bPTH-(7-34)NH2 were equipotent in this system. In addition, hHCF-(7-34)NH2 antagonized hHCF-(1-34)NH2 action in both systems with similar inhibition constants. However, unlike the PTH analogue, hHCF-(7-34)NH2 (8 microM) was a weak partial agonist, producing a 2.4-fold increase in cAMP (5% of the maximal response) in ROS cells. This same system also detects agonism for [Nle8, 18Tyr34]bPTH-(3-34)NH2, another PTH partial agonist/antagonist. These results demonstrate that hHCF-(7-34)NH2 interacts with PTH receptors based in large part on the region which is not homologous to PTH, and suggest the utility of the ROS 17/2.8 cell system for identifying weak agonism of PTH and hHCF analogues in vitro.

Adenylyl Cyclases↗
PubMed1988

Evaluation of novel parathyroid hormone analogs using a bovine renal membrane receptor binding assay.

A PTH membrane receptor binding assay based on a stable hormone analog radioligand was refined and used with bovine renal cortical membranes to evaluate PTH reference peptides and novel analogs of the hormone. Systematic studies were performed to optimize several aspects of the receptor binding assay. The sulfur-free agonist analog [Nle8,18,Tyr34]bovine PTH-(1-34)NH2 was iodinated using Iodogen. The monoiodinated derivative was purified and isolated by reverse phase HPLC. Immediate dilution of the purified radioligand in albumin-containing buffer and cold storage of aliquots yielded a tracer that was stable for at least 2 months. When used in the binding assay, the radioligand displayed specific, high affinity, and saturable binding to both bovine and canine renal cortical membranes. The Kd values were 0.47 +/- 0.07 and 0.63 +/- 0.08 nM for bovine and canine membranes, respectively. Maximum binding values were 475 +/- 46 and 395 +/- 48 fmol/mg protein for bovine and canine membranes, respectively. Furthermore, when used on a routine basis, this assay system proved reliable and reproducible. A series of previously characterized PTH agonists and antagonists was tested as reference peptides to validate this assay. Inhibition of binding by agents was dose dependent, parallel concentration-dependent binding curves were observed, and a close correlation between binding affinity and adenylate cyclase activity was obtained. In addition, a series of novel analogs designed to determine the consequences of sequence elongation at the N- and/or C-terminus or incorporation of amino acid substitutions to enhance resistance to enzymatic degradation were examined. With the exception of C-terminal extension from position 34 to position 38, which diminished receptor affinities, all modifications retained full biological activity. This refined receptor binding assay should facilitate future studies of newly designed PTH analogs.

Adenylyl Cyclases↗
PubMed1988

A new highly potent parathyroid hormone antagonist: [D-Trp12,Tyr34]bPTH-(7-34)NH2.

Based upon N-terminal parathyroid hormone (PTH) analog structure-activity relationship studies, position 12 was found to possess a wide structural latitude and was chosen as a site for single amino acid substitutions. Replacement of the naturally-occurring Gly with D-Trp at position 12 in the PTH antagonists [Tyr34]bPTH-(7-34)NH2 and [Nle8,18,Tyr34]bPTH-(7-34)NH2 increased in vitro receptor affinity. The D-Trp12 containing analogs were 12-fold more potent than their unsubstituted counterparts as inhibitors of PTH binding to renal and bone PTH receptors and 13-27-fold more potent as inhibitors of PTH-stimulated renal and bone adenylate cyclase activity. Based upon Scatchard analyses of saturation binding experiments and Schild analyses of adenylate cyclase experiments, [D-Trp12,Tyr34]bPTH-(7-34)NH2 was shown to interact with PTH receptors in a competitive manner. These studies demonstrate, therefore, that D-Trp12 substitution in PTH antagonists improves inhibitory properties in vitro and is compatible with a helical conformation at this position as a new direction for the design of PTH antagonists.

Adenylyl Cyclases↗
PubMed1988

A chemically synthesized radiolabeled signal peptide: design, preparation, and biological evaluation of an iodinated analog of preproparathyroid hormone.

The chemically synthesized signal peptide (native-sequence signal peptide) of preproparathyroid hormone exhibits signal sequence-like activity by inhibiting the translocation/processing of precursor proteins to their mature forms in an in vitro translation system. In order to prepare a biologically functional radiolabeled form of this peptide, we undertook structure-function studies of the native-sequence signal peptide. Since conventional iodination of peptides is performed under oxidizing conditions, chemical design efforts were focused on the oxidation-labile residues, methionine and cysteine, present in the native sequence. Substitution of the three methionines with norleucine and the single cysteine with alanine yielded a surfur-free analog, [Nle-(-25), Nle-(-21),Nle-(-18),Ala-(-14),D-Tyr-(+1)]pre-proPTH-(-29-+1)amide, which is resistant to oxidation and active in the inhibition of processing assay. An interaction between the signal region and one of the components of the intracellular secretory apparatus, signal recognition particle (SRP), was demonstrated: iodinated sulfur-free analog was cross-linked (using the homo-bifunctional reagent disuccinimidyl suberate) to the 54 kilodalton (kDa) subunit of SRP. The 68 kDa and 72 kDa subunits of SRP were also labeled, but to a lesser extent, by the iodinated peptide.

Amino Acid Sequence↗
PubMed1988

Permanent loss in stem cell self renewal capacity following stress to the marrow.

A technique of irradiating the entire mouse except for one hind limb was developed to provide repeated proliferative demand on the stem cell pool. Animals received 200 cGY weekly for a total dose of 3,400 to 4,000 cGy. During irradiation, shielded bone marrow cellularity was similar to that of unirradiated controls. Shielded marrow colony-forming unit (CFUs) content increased while marrow CFUs self renewal capacity decreased as compared with unirradiated age-matched controls. Following irradiation experimental animals were monitored monthly for 10 to 12 months for marrow cellularity, CFUs content, and self renewal capacity. Shielded marrow cellularity and CFUs content remained elevated over age-matched controls throughout the period of observation. These findings are compatible with the requirement of the shielded hind limb to provide hematopoietic support for the remainder of the animal. Shielded marrow self renewal capacity, a measurement reflecting primitive hematopoietic stem cell function, remained depressed and did not recover with time. These experiments provide evidence for there being limitations on the self renewal capacity of the stem cell compartment. While the small amount of shielded marrow had sufficient capacity to support the animal its average self renewal capacity was permanently reduced.

Animals↗