Biomedical subjects

A M Castrucci

Publications and source records attributed to A M Castrucci.

At least 73 records · Page 4Linked to original sources

PubMed1985

Cyclic melanotropins. 9. 7-D-Phenylalanine analogues of the active-site sequence.

The cyclic melanotropin Ac-Ser1-Tyr2-Ser3-Cys4-Glu5-His6-Phe7-Arg8 -Trp9-Cys10-Lys11-Pro12-Val13-NH is a highly potent agonist as determined in several melanocyte bioassays. In linear melanotropins, a D-Phe7 substitution leads to increased potency and often prolonged biological activity. In order to determine if this substitution would have the same effect in cyclic melanotropins, we have prepared a series of these analogues. The D-Phe7-substituted cyclic melanotropins Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 and Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-11-NH2 were both more potent than their cyclic L-Phe7-containing counterparts in either the frog or lizard skin bioassay by more than a factor of 10. Neither peptide, however, exhibited prolongation of biological activity in either assay. Substitution of D-Phe7 into the cyclic 4-12 and 4-13 sequences led to a slight or no increase in potency in both assays relative to the L-Phe7 counterparts, but the activity of the melanotropins was ultraprolonged in each assay. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-12-NH2 was about equipotent to Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-13-NH2, again demonstrating, as with certain linear and cyclic L-Phe7-containing melanotropins, that the C-terminal amino acid valine is not required for biological activity or for superpotency. Similar to the linear D-Phe7 analogues that possessed ultraprolonged melanotropic activity, the 4-12 and 4-13 cyclic D-Phe7 analogues also displayed the phenomenon of superagonism, which is a time-dependent increase in efficacy over that produced by an equipotent concentration of the native hormone. Cyclization of certain linear melanotropins resulted in analogues with increased resistance to biological degradation by serum enzymes or purified proteolytic enzymes. Further, incorporation of a D-Phe7 into in the cyclic analogues led to melanotropins that were totally resistant to enzymatic inactivation by trypsin.

Animals↗

PubMed1985

Microtubule--and microfilament--disrupting drugs and melanosome migration in melanophores of Papiliochromis ramirezi (Cichlidae, Teleostei).

We report here the effects of cytochalasin B, colchicine and vinblastine on melanosome migration in Papiliochromis ramirezi melanophores. Melanosome migration was not affected by 2 X 10(-5) M cytochalasin B. On the other hand, the aggregation, usually elicited by 10(-6) M norepinephrine, was completely inhibited by simultaneous treatment with 10(-3) M colchicine and cold (0-3 degrees C). Same results were obtained with 10(-3) M vinblastine. Lower concentrations of colchicine had no effect on granule aggregation. However, the aggregation in 50% of the melanophores was still blocked by 10(-5) M vinblastine, whereas 10(-7) M vinblastine was ineffective. Melanosome dispersion usually elicited by 10(-3) M theophylline was significantly accelerated by 10(-3) M colchicine, but delayed by 10(-3) M vinblastine. Lower concentrations of both agents had no effect on pigment dispersion. Therefore our results strongly suggest that microtubule structural integrity is essential to P. ramirezi melanosome displacements. As cytochalasin B had no effect on pigment migration, actin-like microfilaments might be either non-related to granule motion or absent in these chromatophores.

Actin Cytoskeleton↗

PubMed1984

Synthesis and biological actions of melanin concentrating hormone.

A melanin (melanosome) concentrating hormone, MCH, was synthesized and the methodology for its synthesis is detailed. This heptadecapeptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , stimulated melanosome concentration (centripetal aggregation) within melanophores of all species of teleost fishes studied. Melanosome aggregation in response to MCH was not blocked by Dibenamine as was the response to norepinephrine (NE), demonstrating that melanosome aggregating responses to MCH and NE are mediated through separate receptors. Melanosome aggregation in response to MCH was reversed by an equimolar concentration of alpha-melanocyte stimulating hormone (alpha-MSH). In contrast, MCH stimulated melanosome dispersion (centrifugal movement) within melanophores of a frog (Rana pipiens) and a lizard (Anolis carolinensis). Therefore, MCH exhibits both melanosome concentrating and dispersing actions depending upon the species studied.

Amino Acid Sequence↗

PubMed1984

Synthesis of a cyclic melanotropic peptide exhibiting both melanin-concentrating and -dispersing activities.

A putative melanin-concentrating hormone was synthesized. This peptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , stimulates melanin granule aggregation within teleost melanocytes at nanomolar concentrations as does the natural purified teleost pituitary preparation. In addition, this peptide stimulates melanin granule dispersion within melanocytes of frogs and lizards. The peptide has about one six-hundredth of the activity of alpha-melanocyte-stimulating hormone on frog and lizard melanocytes and is a full agonist.

Animals↗

PubMed1984

Synthesis of tritium labeled Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2: a superpotent melanotropin with prolonged biological activity.

Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2 an octapeptide, is a melanotropin analogue (Ac-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH2), which is a superpotent agonist of frog and lizard skin melanocytes and mouse S 91 (Cloudman) melanoma cells. This melanotropin possesses ultraprolonged activity on melanocytes, both in vitro and in vivo, and the peptide is resistant to inactivation by serum enzymes. The tritium-labeled congener was prepared by direct incorporation of [3H]-labeled norleucine into the peptide. The melanotropic activity of the labeled peptide is identical to the unlabeled analogue. This labeled peptide should be useful for studies on the localization and characterization of melanotropin receptors.

Animals↗

PubMed1984

Melanotropin bioassays: in vitro and in vivo comparisons.

A reflectance method was utilized to compare the in vitro responses in three species of frogs (Rana pipiens, R. berlandieri forrei, and R. catesbeiana) and a lizard (Anolis carolinensis) to alpha- and beta-melanotropins (alpha- and beta p-MSH). The integumental chromatic response of the three ranid species was identical, in that alpha-MSH was about 2 times more potent than beta p-MSH. The melanotropins were equipotent in the lizard skin bioassay. A remarkable feature of the Anolis skin assay is that skins from this lizard can be utilized repeatedly many times in one day with an extremely high degree of precision. The reflectance method was also used to determine the in vivo potencies of alpha-MSH and beta p-MSH in the frog, R. pipiens. Surprisingly, the melanotropins were more active in the in vivo assay than in the in vitro bioassay. The darkening response of the frogs to alpha-MSH was reversed by 6 hr, but the response to beta p-MSH persisted for more than 8 hr. When alpha-MSH was incubated in frog serum, the melanotropic activity was almost totally abolished by 30 min, whereas the melanotropic activity of beta p-MSH was evident much longer (4 hr) in the presence of the serum. In light of the observation that the melanotropic activity of alpha-MSH is rapidly lost by incubation in frog serum, it is unclear why the hormone was more active as measured in vivo and why the darkening response in vivo persisted so long.

Animals↗

PubMed1984

Enzymological studies of melanotropins.

The relative stability of natural melanotropins and related synthetic analogues to serum and purified proteolytic enzymes was studied. Both alpha- and beta-MSH were rapidly inactivated by frog serum, but much more slowly by rat serum. beta-MSH was more stable than alpha-MSH to serum inactivation. Both alpha- and beta-MSH were rapidly inactivated by alpha-chymotrypsin and trypsin. The synthetic analogues, [Nle4, D-Phe7]-alpha-MSH and [Cys4, Cys10]-alpha-MSH, were totally resistant to inactivation by frog and rat serum enzymes. [Nle4, D-Phe7]-alpha-MSH was resistant to inactivation by alpha-chymotrypsin and trypsin, whereas [Cys4, Cys10]-alpha-MSH was partially resistant to these enzymes under similar conditions. Melanotropin analogues resistant to inactivation by serum enzymes may prove useful in a variety of physiological studies wherein natural melanotropins would be rapidly inactivated.

Animals↗

PubMed1984

A study on melanophore receptors of Papiliochromis ramirezi (Teleostei, Cichlidae).

Melanophores of Papiliochromis ramirezi aggregate their melanosomes in the presence of catecholamines. Their order of potency are: at 10(-4) M, norepinephrine greater than isoproterenol = epinephrine; at 10(-6) and 10(-8) M, norepinephrine = isoproterenol greater than epinephrine. These effects are antagonized not only by phentolamine but also by propranolol. The catecholamines are unable to induce pigment dispersion. Melanosome dispersion is obtained with cholinergic drugs and the order of potency is nicotine greater than acetylcholine = pilocarpine. Their effects are inhibited by atropine and also by d-tubocurarine and potentiated by physostigmine. The evidences suggest the presence of undifferentiated adrenoceptors, related to the melanosome aggregation and undifferentiated cholinoceptors related to the melanosome dispersion.

Acetylcholine↗

PubMed1984

Cyclic melanotropins. 5. Importance of the C-terminal tripeptide (Lys-Pro-Val).

In previous work we reported that [Cys4,Cys10]-alpha-MSH (II) and Ac-[Cys4,Cys10]-alpha-MSH4-13-NH2 (III) were superpotent melanotropins. Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 (VI), which constitutes the cyclic analogue of the putative active site sequence -Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10- of alpha-MSH, was much less active. In the present investigation the contribution of the Lys11 and Pro12 residues of the C-terminal carboxamide tripeptide -Lys11-Pro12-Val13-NH2 to the potency of Cys4,Cys10 containing cyclic melanotropins was studied. Ac-[Cys4,Cys10]-alpha-MSH4-11-NH2 (V) was less potent than alpha-MSH in the frog and lizard skin bioassays and the mouse S-91 (Cloudman) melanoma adenylate cyclase assay but more potent than Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 in the three assays studied. Ac-[Cys4,Cys10]-alpha-MSH4-12-NH2 (IV) was considerably more potent than the cyclic 4-11 melanotropin and was, in fact, equipotent or even slightly more potent than [Cys4,Cys10]-alpha-MSH and Ac-[Cys4,Cys10]-alpha-MSH4-13-NH2 over the linear portion of the dose-response in all three bioassays. These results demonstrate that Lys11 and Pro12 but to a lesser extent Val13 of the C-terminal tripeptide sequence contributes to the potency of the cyclic melanotropins. The further substitution of a D-Phe7 for the L-Phe7 residue into the cyclic 4-12 analogue resulted in a highly potent compound Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-12-NH2 (VII) that exhibited highly prolonged biological activity.

Adenylyl Cyclases↗

PubMed1984

Synthesis and biological actions of highly potent and prolonged acting biotin-labeled melanotropins.

Biocytin derivatives of a superpotent analogue of alpha-melanotropin, [Nle4,D-Phe7]-alpha-MSH, were prepared. [N alpha-Bct-Ser1, Nle4,D-Phe7]-alpha-MSH and [12-Bct-N alpha-dodecanoyl-Ser1,Nle4,D-Phe 7]- alpha-MSH were synthesized by solid-phase techniques, and the coupling of biotin and 12-aminododecanoic acid was achieved through their succinimido esters. These melanotropins possessed almost identical actions to [Nle4,D-Phe 7]- alpha-MSH as determined by several melanocyte bioassays. Both biocytin derivatives were highly potent agonists and exhibited prolonged biological activity as determined in the frog and lizard skin bioassays. Both biotinylated peptides were at least equipotent to alpha-MSH in stimulating Cloudman S91 mouse melanoma tyrosinase activity. The analogues were resistant to inactivation by alpha-chymotrypsin.

Animals↗

PubMed1984

Cyclic melanotropins. Part VII: Modified ring structures--synthesis and biological activity.

The highly potent cyclic analogue of alpha-MSH, Ac-[Cys4,Cys10]-alpha-MSH4-13-NH2, was structurally modified in position 4. Four analogues were prepared and their biological activities in the in vitro frog and lizard skin bioassays were determined. It was shown that removing the terminal acetylamino group to give [Mpa4,Cys10]-alpha-MSH4-13-NH2 resulted in little change in the biological activity, but a change in the stereochemistry of cysteine in position 4 to give Ac-[D-Cys4,Cys10[-alpha-MSH4-a3-NH2 led to a small decrease of activity in both bioassays. Decreasing the size of the intramolecular ring by removing one methylene group to give [Maa2,Cys10]-alpha-MSH4-13-NH2, resulted in an analogue with lower activities in both assays (about 3 times in the lizard and 500 times in the frog), and increasing the size of the righ by methylene group to give Ac-[Hcy4,Cys10]-alpha-MSH4-13-NH2 led to much lower activities in the lizard system and similar effects were seen upon decreasing the ring size in the frog skin assay.

Amino Acid Sequence↗

PubMedDate not supplied

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates.

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.

Animals↗

PubMedDate not supplied

Melanin concentrating hormone (MCH): synthesis and bioactivity studies of MCH fragment analogues.

Nineteen analogues of melanin concentrating hormone (MCH) were synthesized and tested for their skin-lightening activities in the in vitro eel skin (Synbranchus marmoratus) bioassay. All the analogues synthesized were fragments of the native sequence: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val with sequential elimination of substituents from both the carboxy- and amino-termini. All the analogues that contained tryptophan in position 15 were found to be full agonists and equipotent to MCH. In the absence of Trp15, full agonist activity was maintained but potency was reduced ten-fold or more. The minimal fragment analogue possessing equipotency to the parent peptide, MCH, was the MCH(5-15) sequence. These observations coupled with results from work reported previously by our laboratories suggest the importance of the Trp15 residue for interaction with the MCH receptor in this assay system.

Amino Acid Sequence↗

PubMedDate not supplied

Melanin concentrating hormone (MCH): structure-function aspects of its melanocyte stimulating hormone-like (MSH-like) activity.

Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the brain and secreted from the pars nervosa of teleost fish. This hormone stimulates melanosome (melanin granule) aggregation within integumental melanocytes of fishes but, in contrast, stimulates melanosome dispersion within tetrapod (frog and lizard) melanocytes. We determined the message sequence of the primary structure of MCH which is responsible for its MSH-like component of activity. Removal of the N-terminal amino acid results in an almost total loss of MSH-like activity. The C-terminal amino acid is also essential for full MSH-like activity since the analogue, MCH(1-16), is about 100 times less active than MCH. Therefore, the entire heptadecapeptide sequence of MCH appears to contribute to the MSH-like activity of MCH. Ring-contracted analogues (e.g., [Ala5, Cys10]MCH) of MCH are almost devoid of any melanosome aggregating (MCH-like) activity but generally possess considerable or as great an MSH-like activity as MCH. Racemization of MCH by heat-alkali treatment drastically reduces the MCH-like activity of MCH, but does not enhance the MSH-like activity of the hormone.

Amino Acid Sequence↗

PubMedDate not supplied

Alpha-melanocyte stimulating hormone message and inhibitory sequences: comparative structure-activity studies on melanocytes.

We investigated the structure-activity relationships of alpha-MSH (alpha-melanocyte stimulating hormone) fragment derivatives of the generic formulae Ac-alpha-MSH(x-13)-NH2 and Ac-alpha-MSH(6-x)-NH2. The minimal C-terminal sequences required for melanotropic activity were 8-13 and 7-13, respectively, in the frog and lizard skin bioassays. The Arg8-Trp9 sequence appears to be a fundamental component of the minimal message sequences found to date such as alpha-MSH(6-9), alpha-MSH(8-13) and alpha-MSH(7-13). We discovered that Ac-alpha-MSH(7-10)-NH2 was a weak and selective alpha-MSH antagonist on the lizard skin bioassay. Analysis of alpha-MSH(7-10) analogues of the generic formula Ac-Xaa-Arg-Trp-Yaa-NH2 led to Ac-[D-Trp7,D-Phe10]alpha-MSH(7-10)-NH2, a moderately potent, specific and competitive inhibitor of alpha-MSH in both the frog and the lizard skin bioassays.

Amino Acid Sequence↗

PubMedDate not supplied

Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores.

Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.

Animals↗

PubMedDate not supplied

Linear and cyclic alpha-melanotropin [4-10]-fragment analogues that exhibit superpotency and residual activity.

Two analogues of alpha-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2), Ac-[Nle4, Asp5, D-Phe7, Lys10]alpha-MSH4-10NH2 and Ac-[Nle4, Asp5, D-Phe7, Lys10] alpha-MSH4-10-NH2, were synthesized, and the melanotropic activities of the peptides were compared in several bioassays. Potencies were determined in the in vitro frog and lizard skin bioassays and in the S91 melanoma cell tyrosinase assay. Both analogues were equipotent or more potent than alpha-MSH in all bioassays, and the activities of the analogues were prolonged compared to alpha-MSH. The two analogues were very resistant to inactivation by purified proteolytic enzymes (alpha-chymotrypsin, trypsin, and pepsin). The two peptides could be topically applied and transdermally delivered across the skin of mice in vivo, resulting in a shift from pheomelanogenesis to eumelanogenesis within follicular melanocytes. The cyclic analogue exhibited greater potency, prolonged activity, and stability against enzyme inactivation than did the linear peptide. The significance of the findings for the further design of melanotropin analogues is discussed, as in the possible relevance of these melanotropin analogues for use in biomedical studies.

Animals↗

PubMedDate not supplied

Quantitative in vitro assay for crustacean chromatophorotropins and other pigment cell agonists.

An in vitro crustacean (freshwater shrimp, Macrobrachium potiuna) erythrophore bioassay for chromatophorotropins and other pigment cell agonists is described. The present assay is a quantitative method that determines the pigment responses with the aid of an ocular micrometer. The pigment granules within the erythrophores are dispersed out into the dendritic processes of the cells when the isolated carapace is placed in physiological solution. This bioassay provides, therefore, a method for measuring the response of the pigment cells to aggregating agents such as pigment concentrating hormone (PCH). This bioassay is sensitive to PCH at a concentration as low as 3 x 10(-12) M. Calcium ionophore A23187 mimics the actions of PCH, but, unlike the hormone, the ionophore-induced pigment aggregation is irreversible after physiological solution rinses. Therefore, chromatophorotropic activities of pigment dispersing agents, such as pigment dispersing hormones (PDH), can be determined on ionophore-treated erythrophores. The potencies of alpha-PDH and beta-PDH show a threefold difference (not significant). Because of its convenience and its ability to make an objective determination of the bidirectional pigment movements within erythrophores, this bioassay is a suitable method for further structure-activity studies of the various chromatophorotropins and their analogs.

Amino Acid Sequence↗