Melanophores are dark brown pigment cells located in the skin of fish, amphibia, reptiles, and many invertebrates. The color of the animal can change via rearrangement of pigment granules, melanosomes, in the cells. The dark melanophores can either hide colorful cells so that the animal appears dark, or let through colors from underneath. The animal regulates its colors and patterns via communicating nerve cells and hormones in the blood stream. It is nowadays well established that melatonin-stimulation of melanophores results in aggregation of melanosomes to the cell center and that the evident outcome is more transparent cells. It has previously been shown that the activity of serine and threonine kinases as well as phosphatases regulates the distribution of melanosomes in the cells. We wanted to study if tyrosine phosphorylations were involved in the regulation of melanosome aggregation. Melatonin-stimulated signaling in the African clawed frog, Xenopus laevis, melanophores was examined. Melansome aggregation was accompanied by tyrosine phosphorylation as shown by immunoblots. Inhibition of tyrosine phosphorylation reduced melanosome aggregation by melatonin, and the phosphorylation most likely regulated pigment aggregation. Tyrosine phosphorylation of the protein was mediated via a Gi/o protein coupled receptor, probably the melatonin receptor Mel1c. The phosphorylation was most likely not a result of the classical Gi/o protein pathway, as Src-kinase and mitogen-activated protein kinase seemed required for phosphorylation and melanosome aggregation. Two candidates for the phosphorylated protein were presented, talin and β-spectrin. The possible involvement of nitric oxide in melanosome aggregation by melatonin was investigated. Nitric oxide appeared to be necessary for melanosome aggregation. The effect of nitric oxide synthase inhibition on melanosome aggregation was not mediated via changes in the tyrosine-phosphorylated protein. We speculated that nitric oxide could affect melanosome distribution via modifications of the actin cytoskeleton. The use of recombinant melanophores as a biosensor has also been examined. A human G protein coupled receptor, opioid receptor 3, was inserted into melanophores by electroporation. The transfected melanophores responded dose-dependently to opioids and an inhibitor of opioid receptors reduced the aggregation response. Future melanophore biosensors migh detect a variety of substances, such as narcotics, pheromones, odors, and tastes.

Several chemically-modified analogues of α-melanotropin (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) were prepared by solid-phase peptide synthesis, including [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH₄₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH₁₋₆-NH₂, Ac-α-MSH₇₋₁₀-NH₂, Ac-α-MSH₁₁₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH(,4-10)-NH₂, Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂, [Nle⁴, D-Phe⁷]-α-MSH, Ac-α-MSH₄₋₁₀-NH₂, Ac-[Tyr⁴]-α-MSH₄₋₁₀-NH₂ and [half-Cys⁴, half-Cys¹⁰]-α-MSH. The synthetic strategy involved: (1) p-methylbenzhydrylamine resin as a solid support, (2) N,N'-dicyclohexylcarbodiimide as a coupling reagent, (3) acetylation of the N-terminus and HF cleavage and deprotection (except for Nⁱ-For-Trp) of the fully assembled peptide-resin and (4) alkaline hydrolysis to deformylate Nⁱ-For-Trp. In the preparation of [half-Cys⁴, half-Cys¹⁰]-α-MSH, oxidative-cyclization provided formation of an intramolecular disulfide bridge. A comparative biological analysis in vitro of these above structural, stereoisomeric and conformationally-restricted analogues of α-MSH on several different vertebrate pigment cell systems provided the following results: (1) The [Nle⁴, D-Phe⁷]-α-MSH effected high melanotropic potency (> 60 times relative to α-MSH), ultralong biological activity and unprecedented metabolic stability. (2) Utilizing [Nle⁴, D-Phe⁷]-α-MSH as a molecular probe, two melanotropic receptor types were demonstrated which were mechanistically different in terms of calcium dependency and apparent hormone-receptor complex reversibility. (3) The Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂ was a highly potent active site (Met-Glu-His-Phe-Arg-Trp-Gly) analogue of α-MSH (ranging from 0.2- to 10-times relative to α-MSH) without the ultralong melanotropic activity possessed by the parent stereostructural tridecapeptide. (4) The [half-Cys4, half-Cys10]-α-MSH exhibited superpotency on frog (Rana pipiens) melanophores (≥ 10,000 times relative to α-MSH), and provided experimental evidence that a pseudocyclic conformation of the native hormone containing a β-turn structural requirement at His-Phe-Arg-Trp might be related to its biological activity at the pigment cell receptor. The [Nle⁴, D-Phe⁷]-α-MSH may be suitable for use as a radio-labeled tracer or drug-delivery agent for the localization or treatment of human melanoma in vivo.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

<p><em>Panax ginseng</em> is a traditional herb that has been used for over 2000 years to promote health and longevity. Active components of ginseng include ginsenosides, polysaccharides, flavonoids, polyacetylenes, peptides, vitamins, phenols and enzymes, of which the ginsenosides are considered to be the major bioactive constituents. Although widely used, the exact mechanisms of ginseng and its compounds remain unclear. In this thesis we use melanophores from <em>Xenopus laevis</em> to investigate the effects of <em>Panax ginseng</em> extract G115 and its constituents on organelle transport and signalling. Due to coordinated bidirectional movement of their pigmented granules (melanosomes), in response to defined chemical signals, melanophores are capable of fast colour changes and provide a great model for the study of intracellular transport. The movement is regulated by alterations in cyclic adenosine 3’:5’-monophosphate (cAMP) concentration, where a high or low level induce anterograde (dispersion) or retrograde (aggregation) transport respectively, resulting in a dark or light cell. Here we demonstrate that <em>Panax ginseng</em> and its constituents ginsenoside Rc and Rd and flavonoid quercetin induce a concentration-dependent anterograde transport of melanosomes. The effect of ginseng is shown to be independent of cAMP changes and protein kinase A activation. Upon incubation of melanophores with a combination of Rc or Rd and quercetin, a synergistic increase in anterograde movement was seen, indicating cooperation between the ginsenoside and flavonoid parts of ginseng. Protein kinase C (PKC) inhibitor Myristoylated EGF-R Fragment 651-658 decreased the anterograde movement stimulated by ginseng and ginsenoside Rc and Rd. Moreover, ginseng, but not ginsenosides or quercetin, stimulated an activation of 44/42-mitogen activated protein kinase (MAPK), previously shown to be involved in both aggregation and dispersion of melanosomes. PKC-inhibition did not affect the MAPK-activation, suggesting a role for PKC in the ginseng- and ginsenoside-induced dispersion but not as an upstream activator of MAPK.</p> / <p><em>Panax ginseng </em>är ett av de vanligaste naturläkemedlen i världen och används traditionellt för att öka kroppens uthållighet, motståndskraft och styrka. Ginseng är ett komplext ämne bestående av ett antal olika substanser, inklusive ginsenosider, flavonoider, vitaminer och enzymer, av vilka de steroidlika ginsenosiderna anses vara de mest aktiva beståndsdelarna. Flavonoider (som finns i till exempel frukt och grönsaker) och ginseng har genom forskning visat sig motverka bland annat hjärt-och kärlsjukdomar, diabetes, cancer och demens. Trots den omfattande användningen är dock mekanismen för hur ginseng verkar fortfarande oklar. I den här studien har vi använt pigmentinnehållande celler, melanoforer, från afrikansk klogroda för att undersöka effekterna av <em>Panax ginseng</em> på pigment-transport och dess maskineri. Melanoforer har förmågan att snabbt ändra färg genom samordnad förflyttning av pigmentkorn fram och tillbaka i cellen, och utgör en utmärkt modell för studier av intracellulär transport. Förflyttningen regleras av förändringar i halten av cykliskt adenosin-monofosfat (cAMP) i cellen, där en hög eller låg koncentration medför spridning av pigment över hela cellen (dispergering) eller en ansamling i mitten (aggregering), vilket resulterar i mörka respektive ljusa celler. Här visar vi att <em>Panax ginseng</em>, ginsenosiderna Rc och Rd samt flavonoiden quercetin stimulerar en dispergering av pigmentkornen. När melanoforerna inkuberades med en kombination av ginsenosid Rc eller Rd och quercetin, kunde en synergistisk ökning av dispergeringen ses, vilket tyder på en samverkan mellan ginsenosid- och flavonoid-delarna av ginseng. Ett protein som tidigare visats vara viktigt för pigmenttransporten är mitogen-aktiverat protein kinas (MAPK), och här visar vi att också melanoforer stimulerade med ginseng, men dock inte med ginsenosider eller quercetin, innehåller aktiverat MAPK. Genom att blockera enzymet protein kinas C (PKC) (känd aktivator av dispergering), minskade den ginseng- och ginsenosid-inducerade dispergeringen, medan aktiveringen av MAPK inte påverkades alls. Detta pekar på en roll för PKC i pigment-transporten men inte som en aktivator av MAPK.</p>

Panax ginseng is a traditional herb that has been used for over 2000 years to promote health and longevity. Active components of ginseng include ginsenosides, polysaccharides, flavonoids, polyacetylenes, peptides, vitamins, phenols and enzymes, of which the ginsenosides are considered to be the major bioactive constituents. Although widely used, the exact mechanisms of ginseng and its compounds remain unclear. In this thesis we use melanophores from Xenopus laevis to investigate the effects of Panax ginseng extract G115 and its constituents on organelle transport and signalling. Due to coordinated bidirectional movement of their pigmented granules (melanosomes), in response to defined chemical signals, melanophores are capable of fast colour changes and provide a great model for the study of intracellular transport. The movement is regulated by alterations in cyclic adenosine 3’:5’-monophosphate (cAMP) concentration, where a high or low level induce anterograde (dispersion) or retrograde (aggregation) transport respectively, resulting in a dark or light cell. Here we demonstrate that Panax ginseng and its constituents ginsenoside Rc and Rd and flavonoid quercetin induce a concentration-dependent anterograde transport of melanosomes. The effect of ginseng is shown to be independent of cAMP changes and protein kinase A activation. Upon incubation of melanophores with a combination of Rc or Rd and quercetin, a synergistic increase in anterograde movement was seen, indicating cooperation between the ginsenoside and flavonoid parts of ginseng. Protein kinase C (PKC) inhibitor Myristoylated EGF-R Fragment 651-658 decreased the anterograde movement stimulated by ginseng and ginsenoside Rc and Rd. Moreover, ginseng, but not ginsenosides or quercetin, stimulated an activation of 44/42-mitogen activated protein kinase (MAPK), previously shown to be involved in both aggregation and dispersion of melanosomes. PKC-inhibition did not affect the MAPK-activation, suggesting a role for PKC in the ginseng- and ginsenoside-induced dispersion but not as an upstream activator of MAPK. / Panax ginseng är ett av de vanligaste naturläkemedlen i världen och används traditionellt för att öka kroppens uthållighet, motståndskraft och styrka. Ginseng är ett komplext ämne bestående av ett antal olika substanser, inklusive ginsenosider, flavonoider, vitaminer och enzymer, av vilka de steroidlika ginsenosiderna anses vara de mest aktiva beståndsdelarna. Flavonoider (som finns i till exempel frukt och grönsaker) och ginseng har genom forskning visat sig motverka bland annat hjärt-och kärlsjukdomar, diabetes, cancer och demens. Trots den omfattande användningen är dock mekanismen för hur ginseng verkar fortfarande oklar. I den här studien har vi använt pigmentinnehållande celler, melanoforer, från afrikansk klogroda för att undersöka effekterna av Panax ginseng på pigment-transport och dess maskineri. Melanoforer har förmågan att snabbt ändra färg genom samordnad förflyttning av pigmentkorn fram och tillbaka i cellen, och utgör en utmärkt modell för studier av intracellulär transport. Förflyttningen regleras av förändringar i halten av cykliskt adenosin-monofosfat (cAMP) i cellen, där en hög eller låg koncentration medför spridning av pigment över hela cellen (dispergering) eller en ansamling i mitten (aggregering), vilket resulterar i mörka respektive ljusa celler. Här visar vi att Panax ginseng, ginsenosiderna Rc och Rd samt flavonoiden quercetin stimulerar en dispergering av pigmentkornen. När melanoforerna inkuberades med en kombination av ginsenosid Rc eller Rd och quercetin, kunde en synergistisk ökning av dispergeringen ses, vilket tyder på en samverkan mellan ginsenosid- och flavonoid-delarna av ginseng. Ett protein som tidigare visats vara viktigt för pigmenttransporten är mitogen-aktiverat protein kinas (MAPK), och här visar vi att också melanoforer stimulerade med ginseng, men dock inte med ginsenosider eller quercetin, innehåller aktiverat MAPK. Genom att blockera enzymet protein kinas C (PKC) (känd aktivator av dispergering), minskade den ginseng- och ginsenosid-inducerade dispergeringen, medan aktiveringen av MAPK inte påverkades alls. Detta pekar på en roll för PKC i pigment-transporten men inte som en aktivator av MAPK.

Ultraviolet light is damaging but fish have evolved protective mechanisms, which allows them to live in shallow water reefs, high in UV radiation. This thesis details my investigation into the physiological ecology of solar ultraviolet (UV) absorbing compounds, known as mycosporine-like amino acids found in the external epithelial mucus, and examines the supporting role potentially played by a UV-induced DNA repair mechanism in coral reef fish of the Indo-Pacific. Using reverse phase chromatography and UV spectrophotometry, I examined whether the distribution of MAA compounds across different areas of the body is correlated with differential UV exposure. Comparisons were made between the MAA content and the absorbance spectra of mucus from the dorsal, ventral, caudal and head body surface areas in five species of Scaridae (Chlorurus sordidus, Scarus schlegeli, S. niger, S. psittacus and S. globiceps) from Ningaloo Reef, Coral Bay, Western Australia. All fish analysed had at least five MAAs present, and results showed that fish had increased UV absorbance in mucus over the dorsal area, which receives the brunt of UV radiation. Little UV protection was found in mucus from the ventral area, which receives the lower level of UV radiation mostly via reflection of the sand and reef surfaces. Furthermore, UV absorbance per mg dry mucus versus standard fish length showed that there is a positive relationship in C. sordidus with increasing size. I examined whether there is a difference in the quantity of UV screening compounds found in the mucus of fish along a longitudinal geographical gradient from inshore reefs (Lizard Island, Great Barrier Reef) to the outer edge reefs to oceanic reefs (Osprey Reef). MAA absorbance increased with longitudinal distance from the mainland landmass of Australia to more oligotrophic outer reefs, where UV attenuation is reduced and the ocean is more transparent to UV wavelength. I determined that fish living on inshore, more turbid reefs where UV attenuation in shallow waters is high have lower levels of MAA protection than fish from clear oceanic reefs. Furthermore, there seems to be a direct relationship between light attenuation and exposure with the quantity of protective sunscreening found in the mucus of reef fish. It is know that UV irradiation decreases with water depth and that mucus from fish with deep habitats absorbs less UV than that of fish from shallow habitats. It is unknown however, whether this UV protection is variable within the same individuals and if so, how fast changes 11 occur. To test this, I relocated 9 ambon damselfish from a deep reef (18 m) to a shallow reef (1.5 m) to expose fish to increased levels of UV and relocated another 7 fish from a shallow to a deep reef to expose fish to decreased levels of UV. One week after relocation, all fish were returned to their original reef site to determine whether MAA levels would return to their initial levels. Fish relocated to a shallower depth were recovered and had a 60% (SD+/-2%) increase in mucus UV absorbance. Conversely, the fish relocated to a deeper depth were recovered and had a 41% (SD+/-1%) decrease mucus UV absorbance. No difference was found between UV absorbance of relocated and original fish at both depth. Six days after fish were returned to their original reef, mucus UV absorbance levels had returned to 67% +/- 4% of the original level. These results show that mucus UV absorbance is variable in individual ambon damselfish and that the sunscreen protection typical for a certain depth is reached in relocated fish within just a few days of relocation. The rate of MAA loss is higher than the accumulation of MAAs suggesting that diet is not the sole determining factor involved in the sequestration of MAAs to mucus. The cleaner fish Labroides dimidiatus performs a mutualistic service by removing ectoparasites such as gnathiid isopods as well other dead infected tissue from its clients. Cleaner fish however are also known to feed on client mucus. The benefits of eating mucus until recently were unclear. In this study, we analysed the mucus of several cleaner fish clients to determine whether mucus feeding has a nutritional advantage over gnathiids and whether cleaner fish obtain their own MAA protection through this dietary mucus ingestion. Results show that host fish that are infected with gnathiids of poor nutritional value, in contrast to those that harbour gnathiids with higher nutritional value, continuously exude mucus that has both high nutritional value and high MAA content. These findings support the conclusion that in a competitive market for cleaners some host fish are forced to offer more than parasites to cleaners. Ultraviolet light that is not filtered by UV absorbing compounds such as MAA may still lead to DNA damage such as the formation of cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts (6-4 PPs). However, coral reef fish have alternative mechanisms to overcome UV induced damage via the photolyase DNA repair mechanisms. We experimentally demonstrated for the first time that a coral reef fish species, the moon wrasse Thalassoma lunare has the ability to repair DNA damage via photoreactivation. Fish both with and without MAA protection were irradiated with UVB wavelength to induce DNA lesions. Half of the experimental fish were then exposed to photoreactivating wavelength to induce DNA repair 12 while the other fish were blocked from the repair mechanisms. Fish which had undergone DNA repair had the lowest number of lesions regardless of mucus MAA protection. When fish were blocked from photoreactivation wavelengths MAA sunscreens clearly served a photoprotective role. The amount of damage was greatest in fish which both lacked MAAs and which were also blocked from photoreactivating wavelengths. Thus for the overall UV protection of fish both the MAA sunscreens as well as the DNA repair system play a significant role in counteracting UV damage. Ultraviolet protection by MAA sunscreens is ubiquitous in marine fish. To date the same 5 MAA compounds (palythine (λmax 320 nm), asterina (λmax 330 nm), palythinol (λmax 332 nm), usujirene (λmax 357 nm) and palythene (λmax 360nm) have been identified in the mucus of several different species of reef fish from Australia. Here we report the first evidence of the presence of additional UV absorbing compounds found in the mucus of fish from Indonesia. Using UV spectroscopy the mucus of four species of fish was compared between both geographical regions. The presence of an additional peak between 294-296 nm wavelengths suggests the presence of gadusol and/or deoxygadusol, which are photoprotective compounds, thought to be the precursors of MAAs. Thus, UV protecting compounds in the mucus of fish may not be as conserved between different regions as previously assumed. Our knowledge concerning the effect of UV radiation has advanced considerably in the past decade and my research findings contribute to the better understanding of protective mechanisms of marine fish. The correlations I have found between UV attenuation/exposure, depth, and longitude of sampled individuals lead me to believe that mucus UV absorbing MAA compounds are a highly efficient adaptive defence.