Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.     

Characterization of melanin in human iridal and choroidal melanocytes from eyes with various colored irides

Variance in iris color is related to the incidence of several important ocular diseases, including uveal melanoma and age-related macular degeneration. The purposes of this study were to determine the quantity and the types of melanin in cultured human uveal melanocytes in relation to the iris color. Sixty-one cell cultures of pure uveal melanocytes were isolated from donor eyes with various iris colors. The amount of eumelanin (EM) and pheomelanin (PM) of these cells was measured by chemical degradation and microanalytical high-performance liquid chromatography (HPLC) methods. The total amount of melanin was measured by both microanalytical methods and spectrophotometry. Total melanin content, measured by HPLC and spectrophotometry, correlated well with r = 0.872 (P < 0.0001). The quantity and type of melanin in iridal and choroidal melanocytes showed no significant difference (P > 0.05). When cells became senescent, the levels of EM, PM and total melanin were significantly increased. In both growing and senescent melanocytes, the quantity and type of melanin were closely correlated to the iris color. In cells from eyes with dark-colored irides (dark brown and brown), the amount of EM, the ratio of EM/PM and total melanin were significantly greater than that from eyes with light-colored irides (hazel, green, yellow-brown and blue) (P < 0.0001). The quantity of PM in uveal melanocytes from eyes with light-colored irides was slightly greater than that from dark-colored irides, although not statistically significant (P > 0.05). The present study shows that iris color is determined by both the quantity and the type of melanin in uveal melanocytes. These results suggest a possibility that uveal melanin in eyes with dark-colored irides is eumelanic at the surface and acts as an antioxidant while that in eyes with light-colored irides exposes pheomelanic core and behaves as a pro-oxidant.     

Utilization of cyanobacterial biomass from water bloom for bioproduction of lactic acid

Cyanobacterial biomass obtained from water blooms was successfully utilized as a material for lactic acid production. The starch contained in the biomass could be converted to D- and L-lactic acid with 80–90% yield by Lactobacillus amylovorus, in a manner similar to that contained in laboratory-cultured cyanobacterial biomass. The starch was also available for L-lactic acid production with similar high yields by L. agilis and L. ruminis that specifically produce L-lactic acid. The lactic acid production from the cyanobacterial biomass did not require any supplements such as yeast extract which are essential for lactic acid production from reagent soluble starch, indicating that nutrients contained in the cyanobacterial biomass might be effectively used for the production instead of the supplements. The starch content of the fresh cyanobacterial biomass from water bloom was increased from 10 to 19 and 24% by cultivation in 1 and 5% CO₂ in air, respectively. Using such starch-rich biomass, the concentration of lactic acid produced was successfully increased without changes in the conversion yield. These results indicate that wastewater bloom cyanobacteria could be utilized for the production of a useful compound, lactic acid.     

Cyclic Oscillations in Melanin Composition within Hairs of Baboons

The wild‐type agouti‐banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of α‐melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such ‘agouti’‐banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non‐pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana‐Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.     

Characterization of a novel congenic strain of diabetic fatty (WBN/Kob-Lepr(fa)) rat

The WBN/Kob-Lepr^fa rat is a new congenic strain for the fa allele of the leptin receptor gene (Lepr). Homozygous (fa/fa) WBN/Kob-Lepr^fa rats provide a model of non-insulin-dependent diabetes with obesity. Here, we describe the characteristics of this new animal model in detail. At 7 weeks of age, both male and female obese WBN/Kob rats showed inflammatory cell infiltration of the pancreas that suggested pan-pancreatitis and an abnormal OGTT. At 3 months of age, both male and female obese WBN/Kob rats developed overt diabetes mellitus associated with severe chronic pancreatitis. In contrast, lean female WBN/Kob rats do not develop pancreatitis or diabetes. In WBN/Kob rats, this mutation might promote the onset of severe pancreatitis, leading to the rapid development of diabetes mellitus.     

Kinetic coupling of folding and prolyl isomerization of beta2-microglobulin studied by mutational analysis

β₂-Microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of β2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of β2-m, we studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V β2-ms) as well as the wild-type β2-m. W39 β2-m is a triple mutant in which both of the authentic Trp residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 β2-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V β2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic coupling of unfolding/refolding and prolyl isomerization, which has tended to be neglected in recent studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance.     

LIM kinase 1 modulates opsonized zymosan-triggered activation of macrophage-like U937 cells. Possible involvement of phosphorylation of cofilin and reorganization of actin cytoskeleton.

We have previously reported that cofilin, an actin-binding protein, plays an important role in phagocyte functions, such as respiratory burst, phagocytosis, and chemotaxis. On the other hand, it was recently found that LIM motif-containing kinase (LIMK) phosphorylates cofilin. In this work, we investigated the roles of LIMK in activated phagocytes. The results of immunostaining showed that in dormant phagocytes the endogenous LIMK1 was diffusely distributed in the cytosol of macrophage-like U937 cells, and when activated by opsonized zymosan (OZ), it was translocated to plasma membranes. Green fluorescence protein (GFP)-conjugated LIMK was expressed in the phagocytes, and the GFP-positive cells were isolated by a fluorescence-activated cell sorter. The isolated wild-type LIMK-overexpressing cells produced superoxide at a rate that was 3.2-fold higher than that of only GFP-expressing control cells, whereas the respiratory burst of dominant negative LIMK1(D460A)-expressing cells decreased to 31% of that of the control cells. Phagocytic activity monitored by using Texas Red-labeled OZ was also decreased in the D460A-expressing cells. By immunoblotting using a specific anti-phosphorylated cofilin antibody, it was revealed that in the OZ-activated wild-type LIMK1-GFP-expressing cells, the phosphorylated cofilin increased by 2.3-fold, and that in the OZ-activated D460A-GFP-expressing cells, the phosphorylated cofilin decreased to 47% of that of only GFP-expressing cells (mock control). Furthermore, in the wild-type LIMK1-expressing cells, OZ-evoked increase in filamentous actin was markedly enhanced, whereas in the dominant negative LIMK1-expressing cells, the total level of F-actin was strongly suppressed. These results suggest that LIMK1 regulates the functions of phagocytes through phosphorylation of cofilin and enhances the formation of filamentous actin.     

Glycyrrhizae Radix attenuates peroxynitrite-induced renal oxidative damage through inhibition of protein nitration

We investigated the protective effects of Glycyrrhizae Radix extract against peroxynitrite (ONOO^-)-induced oxidative stress under in vivo as well as in vitro conditions. The extract showed strong ONOO^- and nitric oxide (NO) scavenging effects under in vitro system, in particular higher activity against ONOO^-. Furthermore, elevations of plasma 3-nitrotyrosine levels, indicative of in vivo ONOO^- generation and NO production, were shown using a rat in vivo ONOO^--generation model of lipopolysaccharide injection plus ischemia-reperfusion. The administration of Glycyrrhizae Radix extract at doses of 30 and 60 mg/kg body weight/day for 30 days significantly reduced the concentrations of 3-nitrotyrosine and NO and decreased inducible NO synthase activity. In addition, the nitrated tyrosine protein level and myeloperoxidase activity in the kidney were significantly lower in rats given Glycyrrhizae Radix extract than in control rats. However, the administration of Glycyrrhizae Radix extract did not result in either significant elevation of glutathione levels or reduction of lipid peroxidation in renal mitochondria. Moreover, the in vivo ONOO^- generation system resulted in renal functional impairment, reflected by increased plasma levels of urea nitrogen and creatinine, whereas the administration of Glycyrrhizae Radix extract reduced these levels significantly, implying that the renal dysfunction induced by ONOO^- was ameliorated. The present study suggests that Glycyrrhizae Radix extract could protect the kidneys against ONOO^- through scavenging ONOO^- and/or its precursor NO, inhibiting protein nitration and improving renal dysfunction caused by ONOO^-.     

Identification of a radical formed in the reaction mixtures of oxidized phosphatidylcholine with ferrous ions using HPLC-ESR and HPLC-ESR-MS

Identification of free radicals was performed for the reaction mixtures of autoxidized 1,2-dilinoleoylphosphatidylcholine (DLPC) with ferrous ions (or DLPC hydroperoxide with ferrous ions) and of DLPC with soybean lipoxygenase using electron spin resonance (ESR), high performance liquid chromatography (HPLC)-ESR and HPLC-ESR-mass spectrometry (MS) combined use of spin trapping technique. ESR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (a^N=1.58 mT and a^Hβ=0.26 mT). Outstanding peaks with almost same retention times (autoxidized DLPC, 36.9 min; DLPC hydroperoxide, 35.0 min; DLPC with soybean lipoxygenase, 37.1 min) were observed on the elution profile of the HPLC-ESR analyses of the reaction mixtures. HPLC-ESR-MS analyses of the reaction mixtures gave two ions at m/z 266 and 179, suggesting that 4-POBN/pentyl radical adduct forms in these reaction mixtures.