PubMed Health⌕ Search

Biomedical subjects

M R Lerner

Publications and source records attributed to M R Lerner.

At least 55 records · Page 3Linked to original sources
PubMed1994

Discovery and structure-function analysis of alpha-melanocyte-stimulating hormone antagonists.

Structure-function relationships of alpha-melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) were investigated and novel alpha-MSH receptor antagonists were identified. Based on the alpha-MSH-[5-13] peptide sequence, a multi-use peptide library consisting of 31,360 structurally different candidates was generated, and approximately 40% of the peptides were individually screened for their ability to block receptor function. This led to the identification of antagonists with a range of potencies and revealed structural requirements necessary for receptor inactivation. The most potent antagonist Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2 has an IC50 value of 11 +/- 7 nM. Analysis revealed that D-Trp5 and Phe6 were crucial to its antagonistic properties which could be potentiated by D-Phe3. This study demonstrates that residues in positions 5-6, 7-9, and 10 of the alpha-MSH sequence constitute crucial determinants for potent antagonist activity.

Amino Acid Sequence↗
PubMed1994

Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family.

Amino Acid Sequence↗
PubMed1994

Creation and functional screening of a multi-use peptide library.

Studies of functional interactions between transmembrane proteins such as G-protein-coupled receptors and ligands would benefit from the ability to utilize synthetic molecule libraries. This is realized here by the construction and application of a multi-use combinatorial peptide library (MUPL). Peptides are liberated from their supports in a dry state so that the problem of signal interference due to mixing of peptide molecules, particularly agonists and antagonists, is avoided. In addition, the peptides are released from their supports in a controlled manner so that fractions are available for multiple independent tests, thus eliminating the need for iterative library analysis and resynthesis. The MUPL concept was validated with a functional screen which detects agonists to G-protein-coupled receptors and led to the discovery of new ligands. It is expected that combining MUPLs with functional assays will enhance both basic scientific research and the rates of drug discovery and development.

Amino Acid Sequence↗
PubMed1994

Comparison of Staphylococcus aureus and Escherichia coli infusion in conscious rats.

The purpose of this study was to compare the pathophysiology of bacteremia produced by intravenous infusion of either a Gram-positive (Staphylococcus aureus) or a Gram-negative (Escherichia coli) organism. Conscious, unrestrained, instrumented rats received S. aureus, E. coli, or sterile saline over 120 min, followed by a 240-min monitoring period. The infusates produced 90% (S. aureus,) 80% (E. coli), and 0% (saline) mortality at 24 hr. Neither bacterial group produced hypotension during the entire 360-min study period. E. coli produced early tachycardia and increased glucose, followed by decreased stroke volume and increased lactate and pO2. S. aureus caused early tachycardia followed by decreased pH, stroke volume, and cardiac output and increased lactate and systemic vascular resistance. Respiration rate and central venous pressure were not affected by either bacterial infusion. Compared to E. coli, S. aureus produced decreased pH, glucose, pO2, heart rate, and cardiac output and increased lactate, hematocrit, pCO2, and systemic vascular resistance. These data document quantitative differences in the acute response of the conscious rat to bacteremia caused by these isolates of E. coli and S. aureus.

Animals↗
PubMed1994

Characterization of a serotonin receptor endogenous to frog melanophores.

The response of a cell line of Xenopus laevis melanophores to serotonin was examined. Serotonin increased intracellular levels of cAMP and induced pigment dispersion in the cells. The responses depended on both the concentration of serotonin applied and on the time for which the cells were exposed to serotonin. Using a recently described, microtiter-plate-based bioassay, a series of serotonin receptor ligands were evaluated as agonists or antagonists at the melanophore serotonin receptor. The pharmacological profile suggests the presence of a receptor which shares some properties with but appears different from other previously described serotonin receptors.

Animals↗
PubMed1994

Tools for investigating functional interactions between ligands and G-protein-coupled receptors.

A general assay for evaluating functional interactions between ligands and G-protein-coupled receptors within minutes has been developed. The system uses the principles employed by animals such as reptiles, amphibians and fish to control their colors. In nature, activation of G-protein-coupled receptors expressed by skin cells called chromatophores effects pigment redistribution within the cells to change an animal's coloration. The in vitro 'chameleon in a dish' equivalent can use essentially any cloned G-protein-coupled receptor.

Animals↗
PubMed1994

A rapid bioassay for platelet-derived growth factor beta-receptor tyrosine kinase function.

We have extended a melanophore-based bioassay for G-protein coupled receptors to include the functional expression of the murine platelet-derived growth factor (PDGF) beta-receptor. The homodimeric ligand PDGF-BB induced activation of the transiently expressed receptor in melanophore cells. This led to dose dependent pigment dispersion whereas it did not induce pigment dispersion in wild type cells. The effective concentration of PDGF-BB giving half-maximal pigment dispersion (EC50) was 1nM after 30 minutes exposure. PDGF-AA had no ability to induce pigment dispersion in melanophore cells transiently expressing the beta-PDGF receptor. PDGF-BB-induced pigment dispersion could be blocked by the bis-indolylmaleimide Ro 31-8220 which is an inhibitor of protein kinase C isoenzymes. Functional expression of the PDGF beta-receptor extends the use of the pigment translocation assay to include transmembrane signaling receptor tyrosine kinases. It opens the opportunity for the discovery of potent agonists and antagonists through massive drug screening and investigations of functional ligand-receptor interactions for single transmembrane domain receptors.

Animals↗
PubMed1994

Renal interleukin-1 expression during endotoxemia and gram-negative septicemia in conscious rats.

Endotoxin-induced cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) are thought to contribute to the proinflammatory effects of endotoxin in gram-negative infections. Using a conscious rat model of sepsis, induced by intravenous challenge with LD95 doses of endotoxin (n = 24) or live Escherichia coli (E. coli) (n = 24), we examined frozen sections of kidney at various intervals for evidence of IL-1 alpha and TNF alpha expression. A transient glomerular endothelial IL-1 alpha expression was demonstrated at 30 and 90 min after initiation of the sepsis in both endotoxin and E. coli-treated animals using immunohistochemistry. The endothelial IL-1 alpha expression as determined by immunohistochemistry occurred at the same time as IL-1 alpha mRNA expression, as determined by Northern blot analysis. The glomerular endothelial IL-1 alpha expression coincided with a slight but significant increase in the number of the glomerular polymorphonuclear leukocytes as identified by naphthol AS-D chloroacetate esterase enzyme histochemical reaction. Glomerular endothelial IL-1 alpha expression was virtually absent by 180 and 360 min. No TNF alpha expression was detected in the renal tissues at any time interval. Neither alpha-naphthyl acetate esterase-positive nor acid phosphatase-positive monocytes/macrophages were identified in the glomeruli. Our findings provide direct in vivo evidence that the IL-1 alpha gene product is expressed locally in the kidney by glomerular endothelial cells in this septic rat model.

Animals↗
PubMed1994

Functional expression and characterization of human D2 and D3 dopamine receptors.

Functional characteristics of human D2 and D3 receptors (DRs) were examined using a new bioassay suited for the study of Gi-protein-coupled receptors (GiRs). The bioassay utilizes pigment granule aggregation within cultured Xenopus laevis melanophores for the quantitative evaluation of ligands as agonists or antagonists upon particular GiRs. Initial feasibility studies were performed by analyzing a melanocyte receptor endogenous to the melanophores. In dose-dependent manners, melatonin inhibited melatonin-stimulating hormone-induced cAMP accumulation and caused pigment aggregation that could be monitored over time. Next, melanophores were transiently transfected with cDNAs coding for the human D2BR (short form) and D3R. Expression of either receptor conferred upon the cells the ability to aggregate their melanosomes in response to selective dopaminergic agonists. The same ligands also inhibited cAMP accumulation within the transfected melanophores, and the agonist-induced pigment aggregation was shown to be sensitive to pertussis toxin. EC50 and IC50 value determinations revealed that agonists activated the D2R and D3R at similar concentrations, while each of the antagonists displaying an effect was more potent upon the D2R. The results reveal functional similarities and differences between the D2R and D3R.

Animals↗
PubMed1994

Lack of suppressed renal thrombomodulin expression in a septic rat model with glomerular thrombotic microangiopathy.

BACKGROUND: The thrombomodulin-dependent protein C anticoagulant pathway plays a major physiologic role in the down-regulation of the coagulation process. In cell culture, inflammatory cytokines or endotoxin can down-regulate endothelial thrombomodulin (TM) suggesting that suppressed TM expression may contribute to thrombotic complications noted in Gram-negative sepsis. EXPERIMENTAL DESIGN: In the present study, we have examined TM expression in the kidneys of septic rats utilizing indirect immunofluorescence and have quantified TM antigen and TM activity in extracts of the same kidneys by enzyme-linked immunosorbent assays and protein C activation assays, respectively. Conscious Sprague-Dawley rats were injected intravenously with LD95 doses of live E. coli (N = 30), or endotoxin (N = 30). Control animals (N = 30) were injected with equivalent volumes of saline. The rats were killed 30, 90, 180, 360, and 720 minutes after the initiation of sepsis. RESULTS: Glomerular capillary thrombosis developed by 180 minutes in approximately half of the animals after the initiation of sepsis. We failed to demonstrate suppressed TM expression in the kidneys of septic animals using immunofluorescence. Neither enzyme-linked immunosorbent assays, nor protein C activation assays showed decreased levels in TM antigen expression or activity at different time points during the sepsis. CONCLUSIONS: These results indicate that suppressed TM expression does not contribute to the development of the glomerular capillary thrombosis in this septic rat model.

Animals↗
PubMed1994

Dose- and time-dependent cardiovascular responses induced by ethanol.

Four groups of male Sprague-Dawley rats (10/group) were instrumented to permit access to venous and arterial blood and comprehensive cardiovascular measurements for 4 h after intragastric administrations of water or 2, 4 or 6 g/kg of ethanol. Maximum blood concentrations for each increasing dose occurred within 45 min after ethanol attaining peaks of 63 +/- 8, 103 +/- 11 and 221 +/- 33 mg/d, respectively. Cardiac outputs were significantly lower than the control group beginning at 15 min for 4 and 6 g/kg and at 120 mins for 2 g/kg with similar effects on stroke volume for the three doses. Systemic vascular resistance and heart rate were consistently increased throughout the 4 h for the 6 g/kg group. There was a delayed mean arterial blood pressure decrease in the 6 g/kg group beginning at 120 min after ethanol with significant effects in the 4 g/kg group at 60 and 120 min. Decreased central venous pressure and respiration rate prevailed during the 4 h after 6 g/kg. Blood glucose concentrations were elevated at 60 min after 6 g/kg and at 240 min for all doses. However, blood temperatures were consistently lower than the control group after all three doses beginning 45 min after the drug. These data demonstrate that ethanol evokes cardiovascular, respiratory and metabolic changes that are both dose- and time-dependent. Cardiac output and systemic vascular resistance are significantly affected suggesting a marked alteration of peripheral blood flow.

Animals↗
PubMed1993

Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores.

We report here the presence of a receptor specific for endothelin-3 (termed ETc receptor or ETcR) on Xenopus laevis dermal melanophores. Activation of ETcR causes the dispersion of the pigment granules within the melanophores. The EC50 for ET-3 to induce the pigment dispersion is 24 +/- 7 nM, compared to greater than 10 microM for both ET-1 and -2. This effect desensitizes in a manner that is dependent on both time and the concentration of ET-3 used to stimulate the cells. A cDNA encoding for ETcR was isolated by a polymerase chain reaction-mediated DNA amplification strategy using degenerate oligonucleotides prepared based on conserved regions of other known G-protein-coupled receptor sequences and by the subsequent screening of a frog melanophore cDNA library. The cloned cDNA consists of 2,240 nucleotides, with an open reading frame coding for 444 amino acids containing an initial 20-amino acid signal sequence. The predicted mature peptide consists of 424 amino acids with a heptahelical structure common to the G-protein-coupled receptor surperfamily. Its deduced amino acid sequence is 47 and 52% identical to ETA and ETB receptors, respectively, while ETA and ETB are 48% identical to each other. Expression of cDNA in HeLa cells, which do not contain endothelin receptors, enables the cells to specifically bind [125I]ET-3. Competition binding experiments performed on HeLa cells transiently expressing pETc show that the apparent Ki values for ET-3 and ET-1 to displace [125I]ET-3 are 45.5 +/- 16 and 114 +/- 22 nM, respectively.

Amino Acid Sequence↗
PubMed1993

Pigment dispersion in frog melanophores can be induced by a phorbol ester or stimulation of a recombinant receptor that activates phospholipase C.

Pigment dispersion in frog melanophores is classically mediated by receptors that activate protein kinase A via an elevation of intracellular cyclic AMP. Here, 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, is found to induce pigment dispersion. To demonstrate that an increase in cAMP is not required for the melanosome movement, a murine bombesin receptor was expressed in the melanophores. When these cells were treated with bombesin, they accumulated intracellular inositol phosphates but not cAMP and dispersed their pigment. Four agonists, one partial agonist, and two antagonists for the bombesin receptor were compared for their ability to induce or block bombesin-induced pigment dispersion. In all cases, the degree of pigment dispersion followed simple equilibrium reactions. The resulting dose-response curves allowed for the determination of the effective concentration for half-maximal pigment dispersion (EC50) or half-maximal inhibition of bombesin-stimulated pigment dispersion (IC50) for the peptides. As the pigment dispersion assay can rapidly evaluate chemicals for their effects on receptors that activate phospholipase C via a functional assay, it has potential utility for investigations of ligand-receptor interactions and for massive drug screening.

Animals↗