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Biomedical subjects

M E Hadley

Publications and source records attributed to M E Hadley.

At least 91 records · Page 5Linked to original sources
PubMed1985

Synthesis and biological evaluation of the superagonist [N alpha-chlorotriazinylaminofluorescein-Ser1,Nle4,D-Phe7]-al pha-MSH.

The fluorescein-labeled melanotropin [N alpha-chlorotriazinylaminofluorescein-Ser1,Nle4,D-Phe 7]-alpha-MSH, was prepared by solid-phase techniques of peptide synthesis. The biological actions of this analogue were determined in several melanocyte bioassays and were compared with the parent peptide [Nle4,D-Phe7]-alpha-MSH and the native hormone alpha-MSH. The fluorescein compound was a superpotent agonist with approximately 10 times more activity than alpha-MSH in both the frog and the lizard skin bioassays. Murine S91 melanoma cells assayed in vitro (tyrosinase bioassay) were as responsive to the fluorescein analogue as to alpha-MSH. The analogue exhibited ultraprolonged biological activity and the biological activities were unaffected by treatment of the analogue with alpha-chymotrypsin. The fluorescein-labeled melanotropin should prove useful for melanotropin receptor characterization.

Animals↗
PubMed1985

[Nle4, D-Phe7]-alpha-MSH: a superpotent melanotropin with prolonged action on vertebrate chromatophores.

The in vitro and in vivo responses of integumental chromatophores to alpha-MSH and a related analogue, [Nle4, D-Phe7] -alpha-MSH, were studied in a number of vertebrate species: the teleost, Lebistes reticulatus; the amphibians, Rana pipiens, R. catesbeiana, Xenopus laevis, Bufo alvarius, and B. cognatus; the lizard, Anolis carolinensis; the rattlesnake, Crotalus atrox. The alpha-melanotropin analogue was a superpotent agonist in the in vitro frog (R. pipiens, R. catesbeiana) and lizard (A. carolinensis) skin bioassays. In all species studied, the analogue exhibited ultraprolonged melanotropic activity, both in vitro and in vivo. This melanotropin and related analogues should prove useful in the study of numerous physiological processes, particularly when prolonged melanotropic activity is desired.

Animals↗
PubMed1985

Stimulation of S91 melanoma tyrosinase activity by superpotent alpha-melanotropins.

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin), [Nle4,D-Phe7]-alpha-MSH and related fragment analogues, Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 and Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, were studied for their ability to stimulate tyrosinase activity in Cloudman S91 mouse melanoma cells in tissue culture. All of the melanotropins stimulated tyrosinase activity in a dose-dependent manner. [Nle4,D-Phe7]-alpha-MSH was about 100 times more active than alpha-MSH as determined from the minimal effective dose (MED) required to activate the enzyme above control (basal) levels. The MED of this analogue to significantly stimulate tyrosinase activity at 24, 48 and 72 h of incubation was 10(-11) M whereas the MED of alpha-MSH was 10(-9) M at each of these times. The maximum tyrosinase activity achieved from the time of initial incubation in the presence of [Nle4,D-Phe7]-alpha-MSH was approximately 3-, 5- and 6-fold greater than control levels at 24, 48 and 72 h, respectively. The 2 [Nle4,D-Phe7]-substituted fragment analogues were at least as active as the tridecapeptide analogue and therefore at least 100-fold more active than alpha-MSH in stimulating enzyme activity. These [Nle4,D-Phe7]-substituted analogues were more active in the melanoma tyrosinase assay than in the melanoma adenylate cyclase assay or other normal melanocyte (frog and lizard skin) bioassays.

Amino Acid Sequence↗
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

[Nle4, D-Phe7]-alpha-MSH: a superpotent melanotropin that "irreversibly" activates melanoma tyrosinase.

The superpotent and ultraprolonged melanotropic properties of an alpha-melanotropin analog, [Nle4, D-Phe7]-alpha-MSH, were investigated in a Cloudman S91 (CCL 53.1) melanoma cell line. [Nle4, D-Phe7]-alpha-MSH is 100-fold more effective than the native hormone, alpha-melanocyte stimulating hormone (alpha-MSH), in stimulating melanoma cell tyrosinase activity, as determined from their minimum effective doses (10(-11)M and 10(-9)M, respectively). [Nle4, D-Phe7]-alpha-MSH also exhibits a more sustained effect than alpha-MSH on tyrosinase after removal of the melanotropins from the incubation medium. When cells were exposed to alpha-MSH (10(-7)M) for 24 h, residual activity after removal of the hormone was minimally significant. In contrast, under the same experimental conditions [Nle4, D-Phe7]-alpha-MSH treatment induced tyrosinase activity 2-3 fold above basal level, and maintained remarkable stimulatory effects up to 72 h following melanotropin removal. When the exposure time to melanotropins was reduced to 4 h, alpha-MSH failed to elicit significant tyrosinase activity, whereas [Nle4, D-Phe7]-alpha-MSH stimulated significant tyrosinase activity during the first 24 h subsequent to melanotropin removal. Interestingly, this stimulation by the analog increased at 48 h, reached a maximum at 72 h following removal of the melanotropin analog, and remained significantly stimulated for 6 consecutive days in the absence of the analog.

Adenylyl Cyclases↗
PubMed1985

Prolonged stimulation of S91 melanoma tyrosinase by [Nle4, D-Phe7]-substituted alpha-melanotropins.

alpha-Melanotropin (alpha-melanocyte stimulating hormone, alpha-MSH) stimulates tyrosinase activity in Cloudman S91 murine melanoma cells. Three [Nle4, D-Phe7]-substituted alpha-melanotropin analogues, [Nle4, D-Phe7]-alpha-MSH, Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2, and Ac-[Nle4, D-Phe7]-alpha-MSH4-10-NH2, are at least 100-fold more effective than alpha-MSH in stimulating melanoma tyrosinase, the rate-limiting enzyme in melanin biosynthesis. These [Nle4, D-Phe7]-substituted melanotropin analogues induce tyrosinase activity in melanoma cells with shorter contact times than required by the native hormone, alpha-MSH. [Nle4, D-Phe7]-substituted melanotropins also induce a prolonged (residual) stimulation of melanoma tyrosinase. Following incubation of melanoma cells in the presence of [Nle4, D-Phe7]-alpha-MSH for 24 h, tyrosinase activity is maintained for up to 6 days in the absence of the melanotropin. The shorter 4-10 and 4-11 fragment analogues also exhibit residual melanotropic activity. The prolonged stimulation of tyrosinase in the absence of the analogues is maintained even though melanoma cells continue to divide about every 24 h. These results suggest that melanoma cells possess spare melanotropin receptors and that [Nle4, D-Phe7]-substituted analogues bind almost irreversibly to these receptors or to some other component of the adenylate cyclase enzyme complex responsible for enhancing tyrosinase activity and melanin production.

Animals↗
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

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

Comparative biological activities of potent active-site analogues of alpha-melanotropin. Effect of tyrosine substitution at position-4.

We have prepared several alpha-melanotropin (alpha-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr4]-alpha-MSH4-10-NH2 and Ac-[Tyr4]-alpha-MSH4-11-NH2 were compared with alpha-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr4]-alpha-MSH4-10-NH2 was found to be less active than Ac-[Tyr4]-alpha-MSH4-11-NH2 on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr4]-alpha-MSH4-10-NH2 exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr4]-alpha-MSH4-11-NH2 was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr4]-alpha-MSH4-10-NH2 over Ac-[Tyr4]-alpha-MSH4-11-NH2 on frog melanocytes may be related to the fact that the shorter 4-10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of alpha-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle4]-alpha-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
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↗