Biomedical subjects

R Drozdz

Publications and source records attributed to R Drozdz.

23 records · Page 2Linked to original sources

PubMedDate not supplied

Synthesis and iodination of human (phenylalanine 13, tyrosine 19) melanin-concentrating hormone for radioreceptor assay.

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19]-MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD) was 1.18 x 10(-10) M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as alpha-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.

Amino Acid Sequence↗

PubMedDate not supplied

Binding sites for melanin-concentrating hormone (MCH) in brain synaptosomes and membranes from peripheral tissues identified with highly tritiated MCH.

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in the brain of all vertebrate species. In chromatophores of teleost fishes it induces pigment granule aggregation. In mammals, however, its physiological function is not yet clear. Attempts to identify the site(s) of its action by binding analysis failed because radioiodinated MCH with the natural sequence was devoid of biological activity. We have now synthesized an analogue of rat/human MCH, [Pra4,8,12,19]-MCH, containing four L-propargylglycine (Pra) residues in positions 4, 8, 12, and 19 for catalytic tritiation to norvaline ([3H4]Nva) residues, each of which containing four tritium atoms. The resulting [3H]-MCH ([(3H4)Nva4,8,12,19]-MCH) had a specific radioactivity of approx. 12,200 GBq/mmol (330 Ci/mmol) and retained a biological activity of 10% as compared to rat/human MCH when tested in the carp scale assay. A series of qualitative binding studies performed with rat crude membranes from brain and peripheal tissues as well as with rat brain synaptosomes using the [3H]-MCH radioligand provided the first evidence for the presence of MCH receptors in mammalian tissues. The data showed that specific binding is present in the hypothalamus, hippocampus and in the adrenal gland while none was detected in the brain cortex or spleen. Owing to the tendency of [3H]-MCH to non-specific binding to tissue, glass and plastic surfaces, a saturation binding analysis with this radioligand was not possible.

Amino Acid Sequence↗

PubMedDate not supplied

Interactions of alpha-melanotropin and agouti on B16 melanoma cells: evidence for inverse agonism of agouti.

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between alpha-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7 nmol/l) and alpha-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both alpha-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by alpha-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by alpha-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.

Agouti Signaling Protein↗

PubMedDate not supplied

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor.

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.

Animals↗

PubMedDate not supplied

Receptor-specific antibodies by immunization with "antisense" peptides?

Synthetic peptides whose sequences are specified by RNA complementary to the mRNA coding for peptide hormones have been reported to be useful antigens for the generation of receptor-specific antibodies. We have synthesized an eikositetrapeptide whose sequence corresponds to the complementary strand of the mRNA coding for the sequence of human ACTH(1-24). This "antisense" ACTH(1-24) peptide, "HTCAh," was coupled to bovine serum albumin or thyroglobulin prior to injection into rabbits. The complex proved to be very antigenic, inducing antisera of high titer and specificity. The antisera were tested in ACTH and MSH binding and bioassays, with or without prior purification of IgG molecules. None of the antisera displayed any effect in these assays, nor did they bind to blotted MSH/ACTH receptor protein from Cloudman S91 melanoma cells or to ACTH antibodies. The HTCAh peptide itself did not display measurable association to tritiated or iodinated ACTH(1-24), nor did it displace ACTH(1-24) in a receptor binding assay. However, the peptide bound to a low affinity site of mouse B16 melanoma cells which was independent of the MSH/ACTH binding site and induced melanin formation in these cells, but only at relatively high peptide concentration. Thus, in our hands, the antisense peptide approach using HTCAh as antigen did not lead to receptor-specific antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗