Quantitative analysis of eumelanin and pheomelanin in humans,mice, and other animals: a comparative review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole-2,3,5-tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

Inhibition of gastric inhibitory polypeptide signaling prevents obesity

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.     

Inhibitory effects of U73122 and U73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line

P2X7 receptors are ATP-gated ion channels and play important roles in microglial functions in the brain. Activation of P2X7 receptors by ATP or its agonist BzATP induces Ca2+ influx from extracellular space, followed by the formation of non-selective membrane pores that is permeable to larger molecules, such as fluorescent dye. To determine whether phospholipase C (PLC) is involved in the activation of P2X7 receptors in microglial cells, U73122, a specific inhibitor of PLC, and its inactive analogue U73343 were examined on ATP and BzATP-induced channel and pore formation in an immortalized C57BL/6 mouse microglial cell line (MG6-1). ATP induced both a transient and a sustained increase in the intracellular Ca2+ concentration ([Ca2+]i) in MG6-1 cells, whereas BzATP evoked only a sustained increase. U73122, but not U73343, inhibited the transient [Ca2+]i increase involving Ca2+ release from intracellular stores through PLC activation. In contrast, both U73122 and U73343 inhibited the sustained [Ca2+]i increase either prior or after the activation of P2X7 receptor channels by ATP and BzATP. In addition, these U-compounds inhibited the influx of ethidium bromide induced by ATP and BzATP, suggesting possible PLC-independent blockage of the process of P2X7-associated channel and pore formations by U-compounds in C57BL/6 mouse microglial cells.     

Achacin induces cell death in HeLa cells through two different mechanisms

Achacin, which belongs to the L-amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H(2)O(2). In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H(2)O(2) scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z-VAD-fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H(2)O(2) through the L-amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L-amino acids by achacin.     

The role of Clock in the plasticity of circadian entrainment

The mammalian circadian clock lying in suprachiasmatic nucleus (SCN) is synchronized to about 24 h by the environmental light-dark cycle (LD). The circadian clock exhibits limits of entrainment above and below 24 h, beyond which it will not entrain. Little is known about the mechanisms regulating the limits of entrainment. In this study, we show that wild-type mice entrain to only an LD 24 h cycle, whereas Clock mutant mice can entrain to an LD 24, 28, and 32 h except for LD 20 h and LD 36 h cycle. Under an LD 28 h cycle, Clock mutant mice showed a clear rhythm in Per2 mRNA expression in the SCN and behavior. Light response was also increased. This is the first report to show that the Clock mutation makes it possible to adapt the circadian oscillator to a long period cycle and indicates that the clock gene may have an important role for the limits of entrainment of the SCN to LD cycle.     

The common deletion found in patient reexamined after 33 years and comparison with complete mtDNA sequences of maternal relatives

In 1966, a male (17 years old) was clinically examined at the National Institutes of Health (NIH) and diagnosed with Idiopathic Progressive External Ophthalmoplegia (IPEO). A muscle biopsy showing ragged-red fibers implicated mitochondrial involvement. Since the sequence of human mitochondrial DNA (mtDNA) was not determined until 1981, no genetic confirmation of the disease was possible at that time. In 1999, clinical reexamination and sequencing the entire mtDNA of the patient and living maternal relatives (mother and brother) indicated a progressive mitochondrial myopathy and the presence of the 4977 base pair (bp) deletion (the common deletion) in the patient.     

Phenotypic stability of mature dendritic cells tuned by TLR or CD40 to control the efficiency of cytotoxic T cell priming

It is generally accepted that after stimulation immature DCs turn into mature DCs, which present exogenous antigens together with their MHC class I molecules and then activate the antigen-specific CTLs. Although both TLR and CD40 stimulation appeared to provide the same effects on DC maturation, CD40-dependent CTL activation is much more potent than CTL activation through LPS stimulation. Despite their different outcomes, the factors that lead mature DCs to different functions remain largely undefined. In this study, we defined the transient maturation and subsequent deactivation of DCs by TLR stimuli, including those by LPS and CpG-ODN. In contrast, CD40 stimulation induced stable mature DCs that elicited sufficient CTL proliferation. The deactivated DCs, which we defined as "expired DCs," were phenotypically similar to immature DCs, except for their phenotype stability, MHC class I expression level and IL-10 production. Moreover, the functions of expired DCs were comparable to those of immature DCs in terms of CTL induction and tolerogenicity. These results may provide an explanation for the role of CD40 stimulation in antigen-specific CTL induction.     

Ability of a Microbial Consortium to Remove Pesticide,Carbendazim and 2,4-Dichlorophenoxyacetic Acid

A bacterial consortium capable of simultaneously degrading the fungicide, carbendazim, and the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) was obtained by enrichment of soil samples collected from paddy fields in Japan. This consortium was acclimated in a continuously fed culture with 20 M carbendazim and 2 mM 2,4-D as sole carbon sources using a glass column reactor. By denaturing gradient gel electrophoresis, we observed changes in the bacterial population following the degradation of the both pesticides. This acclimated consortium completely degraded up to 100 M carbendazim and 3 mM 2,4-D within 36 and 24 h, respectively, in batch culture, but a lag time was observed after precultivation in a rich medium. The immobilization of the consortium on a polyester support enhanced the degradation ability of this consortium compared with the use of free cells. This microbial consortium could be useful for bioremediation at sites contaminated with these pesticides.     

Ion-exchange and adsorption of Fe(III) by Sepia melanin

Sepia eumelanin is associated with many metal ions, yet little is known about its metal binding capacity and the chemical nature of the binding site(s). Herein, the natural concentrations of metal ions are presented and the ability to remove metals by exposure of the melanin granules to EDTA is quantified. The results reveal that the binding constants of melanin at pH 5.8 for Mg(II), Ca(II), Sr(II) and Cu(II) are, respectively, 5, 4, 14 and 34 times greater than the corresponding binding constants of these ions with EDTA. By exposing Sepia eumelanin to aqueous solutions of FeCl(3), the content of bound Fe(III) can be increased from a natural concentration of approximately 180 ppm to a saturation limit of approximately 80 000 ppm or 1.43 mmol/g of melanin. Similar saturation limits are found for Mg(II) and Ca(II). Exposure of Sepia melanin granules to aqueous solutions containing Ca(II) results in the stoichiometric replacement of the initially bound Mg(II), arguing that these two ions occupy the same binding site(s) in the pigment. The pH-dependent binding of Mg(II) and Ca(II) suggests coordination of these ions to carboxylic acid groups in the pigment. Mg(II) and Ca(II) can be added to a Fe(III)-saturated melanin sample without affecting the amount of Fe(III) pre-adsorbed, clearly establishing Fe(III) and Mg(II)/Ca(II) occupy different binding sites. Taking recent Raman spectroscopic data into account, the binding of Fe(III) is concluded to involve coordination to o-dihydroxyl groups. The effects of metal ion content on the surface morphology were analyzed. No significant changes were found over the full range of Fe(III) concentration studied, which is supported by the Brunauer-Emmett-Teller surface area analysis. These observations imply the existence of channels within the melanin granules that can serve to transport metal ions.     

AtPex14p maintains peroxisomal functions by determining protein targeting to three kinds of plant peroxisomes

We previously isolated an Arabidopsis: peroxisome-deficient ped2 mutant by its resistance to 2,4-dichlorophenoxybutyric acid. Here, we describe the isolation of a gene responsible for this deficiency, called the PED2 gene, by positional cloning and confirmed its identity by complementation analysis. The amino acid sequence of the predicted protein product is similar to that of human Pex14p, which is a key component of the peroxisomal protein import machinery. Therefore, we decided to call it AT:Pex14p. Analyses of the ped2 mutant revealed that AT:Pex14p controls intracellular transport of both peroxisome targeting signal (PTS)1- and PTS2-containing proteins into three different types of peroxisomes, namely glyoxysomes, leaf peroxisomes and unspecialized peroxisomes. Mutation in the PED2 gene results in reduction of enzymes in all of these functionally differentiated peroxisomes. The reduction in these enzymes induces pleiotropic defects, such as fatty acid degradation, photorespiration and the morphology of peroxisomes. These data suggest that the AT:Pex14p has a common role in maintaining physiological functions of each of these three kinds of plant peroxisomes by determining peroxisomal protein targeting.