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M Rosenblatt

Publications and source records attributed to M Rosenblatt.

At least 19 recordsLinked to original sources
PubMed1995

Probing the bimolecular interactions of parathyroid hormone with the human parathyroid hormone/parathyroid hormone-related protein receptor. 1. Design, synthesis and characterization of photoreactive benzophenone-containing analogs of parathyroid hormone.

Parathyroid hormone (PTH) regulates calcium and phosphate metabolism through a G-protein-coupled receptor which is shared with PTH-related protein (PTHrP). Therefore, structure-activity studies of PTH and PTHrP with their common receptor provide an unusual opportunity to examine the structural elements in the two hormones and their common receptor which are involved in the expression of biological activity. Our approach to studying the nature of the bimolecular interface between hormone and receptor is to use a series of specially designed photoreactive benzophenone- (BP-) containing PTH analogs in "photoaffinity scanning" of the PTH/PTHrP receptor. In this report we describe a series of BP-containing agonists and antagonists which have been synthesized by solid-phase methodology and characterized physiocochemically and biologically. Each of the 12 analogs contains a single BP moiety at a different defined position. Examples of BP-containing agonists prepared and studied in human osteogenic sarcoma Saos-2/B-10 cells are [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13)(Kb = 13 nM; Km = 2.7 nM) and [Nle8,18,L-Bpa23,Tyr34[bPTH(1-34)NH2(L-Bpa23) (Kb = 42 nM; Km = 8.5 nM). Another BP-containing analog, [Nle8,18,D-2-Nal12,Lys13(epsilon-pBZ2),L-2-Nal23 ,Tyr34]bPTH(7-34)NH2, was a potent antagonist (Kb = 95 nM; Ki = 72 nM). The amino acids substituted by residues carrying the BP moiety span the biologically active domain of the hormone (Phe7, Gly12, Lys13, Trp23, and Lys26). Analysis of photo-cross-linked conjugates of PTH/PTHrP receptor with BP-containing PTH analogs should help to identify the "contact points" between ligand and receptor.

Adenylyl Cyclases↗
PubMed1995

Probing the bimolecular interactions of parathyroid hormone and the human parathyroid hormone/parathyroid hormone-related protein receptor. 2. Cloning, characterization, and photoaffinity labeling of the recombinant human receptor.

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and cross-linking of a benzophenone-substituted PTH analog, [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of approximately 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of 125I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.

Affinity Labels↗
PubMed1995

Conformation of parathyroid hormone antagonists by CD, NMR, and molecular dynamics simulations.

The conformation of two highly potent parathyroid hormone (PTH) antagonists was investigated in water/2,2,2-trifluoroethanol mixtures. The two peptides are derived from the sequence (7-34) of PTH and of PTH-related protein (PTHrP) and have a D-Trp replacing Gly in position 12. In the analogue derived from PTHrP, Lys11 was replaced by Leu to remove the residual agonist activity. The study was conducted by CD and two-dimensional proton magnetic resonance spectroscopy, and the nuclear Overhauser effects found were utilized in restrained distance geometry and molecular dynamics simulations. Both peptides adopt a helical C-terminal conformation, which seems more stable in the case of the PTHrP analogue. A type II' beta-turn centered around D-Trp12 and Lys13 is present in both structures.

Amino Acid Sequence↗
PubMed1995

Crush injury in pregnancy: an unusual experience in obstetrics.

BACKGROUND: Crush injury is the result of prolonged pressure to the limbs when individuals are trapped under the debris of earthquakes, bombings, and other disasters. Muscle integrity is compromised, and the local and systemic manifestations, including rhabdomyolysis, hyperkalemia, renal failure, and disseminated intravascular coagulation may be fatal. CASE: A 32-year-old woman, gravida 2, para 1, was trapped under fallen masonry at 6 weeks' gestation for approximately 6 hours. On initial examination, she showed early signs of hypovolemic shock and crush injury, including hyperkalemia. Prompt management with fluid therapy and careful monitoring led to a rapid recovery. Antenatal follow-up was routine and concluded in spontaneous delivery of a healthy infant. CONCLUSION: The unique physiology of the pregnant woman may affect the outcome of crush injury, and it presents a particular challenge to the trauma team. The obstetrician should be aware of its dangers and should be an active participant in the prompt management of pregnant victims of crush injury to ensure a satisfactory outcome for both mother and fetus.

Adult↗
PubMed1995

Retrieval of intravascular foreign bodies: experience in 32 cases.

OBJECTIVE: Recent reports suggest that both the nature of intravascular foreign bodies and the tools available to retrieve them have changed substantially in the past decade. We reviewed our recent experience with percutaneous retrieval of intravascular foreign bodies to determine the efficacy and safety of the procedure using currently available devices. MATERIALS AND METHODS: Between 1990 and 1994, we attempted retrieval of 35 intravascular foreign bodies in 32 patients. Twelve patients (38%), including all five with intraarterial foreign bodies, were treated for complications of transcatheter interventional procedures that resulted in embolization of seven coils, four intravascular stents, an inferior vena cava filter, and a valvuloplasty balloon fragment. From procedure records, we reviewed the types of retrieval devices and methods used; the medical record was studied to determine the occurrence and treatment of any procedure-related complications. RESULTS: Retrieval was successful in 31 (97%) of 32 patients. All five intraarterial and 29 of 30 IV objects were removed. Nitinol goose-neck snares were used in 28 of 32 cases, but more than one retrieval system was required in eight cases (25%), often using grasping forceps, tip-deflecting wires, or stone baskets to move the foreign body into a more favorable position for snaring. In the single failure, the tip of a largely extravascular catheter fragment lay in a venous valve and could not be snared in a patient who refused surgery. Two of five patients with arterial foreign bodies suffered occlusive arterial spasm, reversible with local administration of nitroglycerine. Two large objects were repositioned to the femoral vein and removed by surgical cutdown. No other procedural complications occurred, and none of the patients required additional compression, transfusion, or surgical intervention. CONCLUSION: We conclude that use of preformed nitinol goose-neck snares facilitates retrieval of intravascular foreign bodies in most cases, although interventional radiologists must be familiar with a variety of techniques to deal with the expanding spectrum of foreign bodies currently encountered.

Adolescent↗
PubMed1995

Distribution and pharmacokinetics of a potent peptide antagonist of parathyroid hormone and parathyroid hormone-related protein in the rat.

Humoral hypercalcemia of malignancy results from the production by cancer cells of parathyroid hormone related protein that activates receptors in bone. Peptide antagonists that block parathyroid hormone receptors in vivo would be instrumental in the clinical treatment of humoral hypercalcemia of malignancy. We report the in vivo whole body distribution and blood plasma pharmacokinetics of the parathyroid hormone receptor antagonist [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide to determine parameters that are likely to affect its administration regimen. A single intravenously injected dose of [Nle8,18,D-Trp12,monoiodinated Tyr34]bPTH(7-34)amide was rapidly cleared from blood plasma. The plasma concentration reaches a maximum at 10 min (Cmax = 1.93 +/- 0.27% of total injected CPM/ml), and the intact PTH derivative was detectable in plasma by HPLC analysis at this time. In vivo binding to plasma proteins was noncovalent. The peptide was rapidly cleared from blood by the liver, and more slowly by the kidney. Radiolabel was detected in excreted feces at 8 hr, but the preferred route of excretion was renal as judged by significant counts in excreted urine. Absorption of labeled peptide by skin and bone was sustained. Strong and sustained absorption also occurred in the vas deferens, seminal vesicle and hypothalamus. Given the rapid clearance of antagonist, multiple or sustained dosing schemes might be necessary to achieve the desired pharmacological effect. The high counts in liver at early time points after i.v. injection suggest that other routes of administration that do not bypass the hepatic first-pass effect would result in very low blood levels of drug.

Amino Acid Sequence↗
PubMed1992

Stem cell factor, interleukin-3, and interleukin-6 promote retroviral-mediated gene transfer into murine hematopoietic stem cells.

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6-prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.

3T3 Cells↗
PubMed1992

Synthesis of fully active biotinylated analogues of parathyroid hormone and parathyroid hormone-related protein as tools for the characterization of parathyroid hormone receptors.

The synthesis, purification, and characterization of biotinylated analogues of parathyroid hormone (PTH) and PTH-related protein (PTHrP) are described. A novel methodology was developed which allowed the selective biotinylation during solid-phase synthesis of either the Lys13 or Lys26 residue in PTH/PTHrP sequences. Incorporation of orthogonally protected N alpha-Boc-Lys(N epsilon-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the epsilon-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle8,18,Tyr34]bPTH(1-34)NH2 (analogue 1a) were prepared by modification of Lys13 with a biotinyl group (analogue 1) or a biotinyl-epsilon-aminohexanoyl group (analogue 2) or at Lys26 with a biotinyl-epsilon-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu11,D-Trp12,Lys13(N epsilon-(biotinyl-beta-Ala))]PTHrP(7-34)NH2 (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys35]PTHrP(1-35)NH2, in solution, with N-biotinyl-N'-(6-maleimidohexanoyl)hydrazide, resulted in analogue 4. The high affinities of the biotinylated analogues for PTH receptors present in human osteosarcoma B-10 cells or in porcine renal cortical membranes (PRCM), were comparable to those of the underivatized parent peptides. The analogues were also highly potent in stimulation of cAMP formation (analogues 1-4) or inhibition of PTH-stimulated adenylyl cyclase (analogue 5) in B-10 cells. The most potent analogue (analogue 1) had potencies in B-10 cells (Kb = 1.5 nM, Km = 0.35 nM) and in porcine renal membranes (Kb = 0.70 nM) identical or similar to those of its parent peptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1992

Concentrations of parathyroid hormone-related protein in rat milk change with duration of lactation and interval from previous suckling, but not with milk calcium.

PTH-related protein (PTHrP) is found in high concentrations in the milk of various mammals. However, little is known about the regulation of PTHrP production or the physiological role(s) of PTHrP in the mammary glands. To address these questions, we examined in lactating rats 1) the longitudinal changes in PTHrP concentrations in milk and PTHrP mRNA levels in the mammary glands throughout lactation, 2) the effects of the nonsuckling interval on milk PTHrP concentration, and 3) the correlation between PTHrP and calcium concentrations in milk. PTHrP concentrations in milk, measured by RIA and in vitro bioassay, increased with the duration of lactation. The maximal concentrations of PTHrP (observed between days 19-21 of lactation in rats milked serially) were 4.8- to 8.0-fold higher than the concentrations on day 7. PTHrP mRNA levels in the mammary glands also increased during the late stages of lactation. The longitudinal changes in calcium concentrations in milk were small and did not parallel the changes in PTHrP. When pups were removed from the mother for 4-24 h, milk PTHrP decreased while calcium increased in a time-dependent manner. As a whole, calcium concentrations in milk did not correlate with PTHrP throughout lactation. These data suggest that the production and secretion of PTHrP into milk are regulated independently of the other major milk proteins by a factor(s) that changes with progression of lactation and in relation to suckling status.

Animals↗
PubMed1992

A pharmacological comparison of parathyroid hormone receptors in human bone and kidney.

While abundant information is available characterizing PTH receptor properties in other species, data on human PTH receptors is very limited. We have been interested in the possibility that tissue-specific differences among human PTH receptors (i.e. bone vs. kidney) might exist. We have, therefore, compared pharmacological profiles for a wide array of PTH and PTH-related peptide (PTHrP) analogs in human osteoblast-like cells (SaOS-2) and human renal cortical membranes (RCM) using radioiodinated (Tyr36)hPTHrP(1-36)NH2 as a probe for PTH receptor function. The rank order of receptor affinity for 10 PTH/PTHrP receptor agonists tested was very similar in the bone and kidney assay systems. Binding affinity for these peptides was greater in human (h)RCMs and SaOS-2 membranes than in SaOS-2 intact cells. The relative binding affinities for (Tyr36)hPTHrP(1-36)amide, hPTH(1-34), bovine (b)PTH(1-34), and rat PTH(1-34) were similar in human RCMs, SaOS-2 membranes, and SaOS-2 cells. bPTH(1-84) and hPTHrP(1-74) both manifested lower receptor affinity than the amino-terminal analogs. Seven PTH/PTHrP receptor antagonists were also studied in this homologous human assay system. The binding affinity for hPTHrP(7-34)NH2 was 2- to 3-fold greater than that for (Tyr34)bPTH(7-34)NH2 in RCM and SaOS-2 membranes. However, in SaOS-2 cells, a striking reversal in the relative binding affinities was observed; the PTHrP(7-34) analog was nearly 3-fold less potent than (Tyr34)bPTH(7-34)NH2, underscoring a difference between intact and broken cell preparations. Two hybrid PTH-PTHrP receptor antagonists demonstrated similar relative affinity to each other in the human bone and kidney assay systems. Affinity cross-linking of receptors in human renal and skeletal tissues demonstrated an indistinguishable dominant 85-kilodalton receptor protein. We conclude that the binding and bioactivity profiles of a broad array of PTH and PTHrP peptides are very similar or identical in human renal and skeletal tissues. Differences relating to intact vs. broken cell preparations accounted for some variation in potency. These studies emphasize the importance of employing homologous assay systems to study PTH receptor function and the existence of interspecies differences among PTH receptors. The results support the possibility that PTH receptors in human bone and human kidney are very similar if not identical.

Affinity Labels↗
PubMed1992

A novel, mild, specific and indirect maleimido-based radioiodolabeling method. Radiolabeling of analogs derived from parathyroid hormone (PTH) and PTH-related protein (PTHrP).

In an effort to design a mild, non-oxidative and site-specific means of radiolabeling bioactive molecules we have employed maleimido-sulfhydryl chemistry to produce bioactive hormone radioligands. We have prepared two novel radioiodolabeled reagents, 3'-maleimidopropanoyl-3-125I-tyramide and its retro analog, N-maleoyl-N'-3-(4-hydroxy-3-125I-phenyl)propanoyl ethylenediamide, by either oxidative radioiodination of the precursors or radiolabeling of the phenolic component prior to its incorporation into the radiolabeling reagents. These reagents were then used to radiolabel analogs of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) in an efficient way, yielding reaction mixtures which were easily purified. The radioligands obtained are stable upon storage and bind in a reversible manner to a single population of binding sites displaying affinity in the low nanomolar range. The potencies of these analogs are comparable to the non-modified PTH and PTHrP analogs. This study demonstrates the utility of the novel maleimido-based indirect radioiodination approach and highlights some of its advantages over either direct oxidative procedures or acylation using the Bolton-Hunter reagent.

Adenylyl Cyclases↗
PubMed1991

Cyclic parathyroid hormone related protein antagonists: lysine 13 to aspartic acid 17 [i to (i + 4)] side chain to side chain lactamization.

Cyclization of parathyroid hormone related protein (7-34)amide [PTHrP(7-34)NH2] via covalent bond formation between the epsilon-amino of Lys13 and the beta-carboxyl of Asp17 yielded a 20-membered ring lactam. This analogue, [Lys13,Asp17]PTHrP(7-34)NH2, was 5-10-fold more potent than the linear parent peptide (Kb = 15 and 18 nM in PTH receptor binding assays, and Ki = 130 and 17 nM in PTH-stimulated adenylate cyclase assays in bovine renal cortical membrane and in human bone derived B10 cells, respectively). In contrast, a linear analogue in which charges in positions 13 and 17 were eliminated and other stereoisomers of the above-mentioned lactam in which either Lys13 and/or Asp17 were replaced by the corresponding D-amino acids were much less potent with regard to antagonist bioactivity than the parent peptide. The rationale for the design of the lactam as well as the conformational implications for the PTHrP sequence in light of reported models suggested for the 1-34 peptide are described. The potential use of conformationally constrained analogues for elucidating the "bioactive conformation" of antagonists and for the design of substantially simplified molecular structures for antagonists is discussed.

Adenylyl Cyclases↗
PubMed1991

Utero-vaginal hypoplasia. Sonographic, embryologic and clinical considerations.

Congenital absence of hypoplasia of the uterus is a cause of primary amenorrhea in approximately 15% of cases. Ultrasound is often employed as an early imaging modality in the evaluation of patients with primary amenorrhea. Demonstration of total absence or marked hypoplasia of the uterus in the presence of normal ovaries during pelvic ultrasound examination, suggests the diagnosis of congenital uterine aplasia or hypoplasia. Sonography may obviate the need for laparoscopy and for other imaging modalities. A case report of uterovaginal hypoplasia in association with anal atresia and recto-vaginal fistula is presented, and the value of ultrasound in the diagnosis of this entity is discussed. The embryology and clinical features of this anomaly are also reviewed.

Abnormalities, Multiple↗
PubMed1991

Correlation of secondary structure with biological activity for a leader peptide: circular dichroism-derived structure and in vitro biological activities of preproparathyroid hormone peptide and its analogs.

Leader or signal sequences are specialized domains within precursor proteins which serve an essential role in interacting with the cellular secretory apparatus to enable intracellular transport and secretion of proteins. Despite many differences in primary amino acid sequences, signal domains interact with a common set of intracellular components, presumably because the signal sequences share an overall conformational similarity. In a few instances, mutant signal peptides from prokaryotes have been studied and their structures correlated with function (export) in vivo. A series of analogs of the precursor-specific region of preproparathyroid hormone have been prepared which contain substitutions of either proline or a charged amino acid within the hydrophobic core. These synthetic "mutants" have previously been evaluated in several in vitro assays to determine their functionality with regard to protein secretion and suitability as substrates for signal peptidase. The secondary structural content of each peptide, as well as the native sequence and sulfur-free analog, was determined in aqueous and nonaqueous conditions by circular dichroism (CD) as a function of time. The structures obtained were correlated with in vitro bioactivities. Unlike the findings or previous CD studies, all the peptides examined here had low to undetectable alpha-helical content in both aqueous and nonaqueous buffers. The unsubstituted and sulfur-free analogs had high (80-85%) beta-structure in aqueous conditions which was reduced to approximately 30% in nonaqueous solvent. The proline- and charged-substituted peptides contained about half the beta-structure content (35-55%) in aqueous buffer; in nonaqueous solvent their structure was similar to the unsubstituted peptides. The structure-activity correlates found were as follows: a high degree of structure (aqueous conditions) correlated with interaction with signal recognition particle and substrate suitability for signal peptidase; a low degree of structure (nonaqueous environment) correlated with activity in the translocation assay.

Amino Acid Sequence↗