Aiding phase unwrapping by increasing the number of residues in two-dimensional wrapped-phase distributions.

In phase unwrapping residues are points of locally inconsistent phase that occur within a wrapped-phase map, which are usually regarded as being problematic for phase-unwrapping algorithms. Real phase maps typically contain a number of residues that are approximately proportional to the subsequent difficulty in unwrapping the phase distribution. This paper suggests the radical use of the discrete Fourier transform to actually increase the number of residues in 2D phase-wrapped images that contain discontinuities. Many of the additional residues that are artificially generated by this method are located on these discontinuities. For example, in fringe projection systems, such phase discontinuities may come from physical discontinuity between different parts of the object, or by shadows cast by the object. The suggested technique can improve the performance of path independent phase-unwrapping algorithms because these extra residues simplify the process of setting the branch cuts in the wrapped image based on the distance to the nearest residue. The generated residues can also be used to construct more reliable quality maps and masks. The paper includes an initial analysis upon simulated phase maps and goes on to verify the results on a real experimental wrapped-phase distribution.

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.

Synthesis and biological activities of fatty acid conjugates of a cyclic lactam alpha-melanotropin.

Four fatty acid conjugates of a cyclic lactam-bridged alpha-MSH fragment analogue were synthesized and their potencies and biological activities compared in several melanotropin bioassays. Palmitoyl, myristoyl, decanoyl, and hexanoyl conjugates of H-Asp-His-D-Phe-Arg-Trp-Lys-NH2 were prepared. In the in vitro mouse melanoma cell assay, each of the conjugates was 10-100 times more potent than alpha-MSH or the substrate peptide in elevating tyrosinase activity. The shorter conjugates of hexanoic and decanoic acid were as potent as alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogues were about 100 times less potent. The potency of the myristoyl and palmitoyl conjugates increased with time in contact with the skins. These observations may be related to the more lipid-like nature of these peptide-fatty acid conjugates. Each of the conjugates exhibited prolonged melanotropic activity in the lizard skin bioassays and in the mouse S91 melanoma tyrosinase bioassay, since the biological response continued following removal of the conjugates from the incubation media. The prolonged residual melanotropic activity resulted from conjugation of the fatty acids to the MSH fragment analogue since the analogue itself did not exhibit prolonged activity.

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.

Psoralens stimulate mouse melanocyte and melanoma tyrosinase activity in the absence of ultraviolet light.

Psoralens (8-methoxypsoralen, 5-methoxypsoralen and 4,5,8-trimethylpsoralen) stimulate mouse melanoma cell (S91 and B16/F10) tyrosinase activity in vitro in a dose-related manner. Stimulation of enzyme activity by the psoralens was evoked in the presence or absence of light. In the presence of a melanotropin the actions of the psoralens were generally at least additive compared to the individual actions of the two agonists. The actions of the psoralens were acute and depended upon the constant presence of the agents to maintain enhanced melanoma tyrosinase activity. Tyrosinase activation by the psoralens, like that of alpha-melanotropin, was blocked by actinomycin-D or cycloheximide demonstrating that the actions of the drugs may have involved both transcriptional and translational events in the stimulation of melanogenesis. Psoralens also stimulated an immediate darkening of frog skins in vitro. Topically applied psoralens were transdermally delivered to the systemic circulation resulting in a conversion from pheomelanogenesis to eumelanogenesis within follicular melanocytes throughout the entire skin of mice (C57BL/6JAy maintained in the dark. Taken together, these results demonstrate that psoralens activate processes within melanocytes resulting in both an immediate translocation of melanosomes within the cell (frog) or in a slower genomic event involving tyrosinase activation (melanoma cells) and eumelanin formation (mouse follicular melanocytes).

[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.

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.

Comparative in vivo expression of beta(+)-thalassemia alleles.

Double heterozygotes who inherit one abnormal though stable beta-globin variant in association with a molecularly identified beta(+)-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular beta(+)-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the beta(+)-thalassaemia allele expressed as the MCH-beta(A) in pg beta(A)-globin/beta(+)-thalassemia allele/RBC = 0.5 MCH x Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the beta(+)-87 (C-->G) = 3.8 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-1 (G-->A) = 0.2 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-6 (T-->C) = 2.9 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 7); the beta(+) IVS-I-110 (G-->A) = 1.1 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 13), and the beta(+) IVS-II-745 (C-->G) = 1.74 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other beta(+)-thalassemia alleles from the literature. It can be seen that the MCH-beta(A) value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in beta-globin genes on pathophysiologically meaningful beta-globin gene expression.