The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

Eumelanin and Phaeomelanin Contents of Human Epidermis and Cultured Melanocytes

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeomelanin/eumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanin/eumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanin/eumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in the epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.     

FLAVOPROTEINS IN THE LEAVES OF HIGHER PLANTS CATALYZING THE OXIDATION OF L-GLUTAMATE

1. Two forms of enzyme capable of catalyzing the oxidation of L-glutamate(and L-aspartate) were isolated from the leaves of spinach andseparated from each other by column-chromatographic purificationon calcium phosphate and anion exchangers. They were distinguishedas GD₁ (L-glutamate dehydrogenase 1) and GD₂ (L-glutamate dehydrogenase2). The purification procedures and some fundamental propertiesof the partially purified enzymes were investigated.
2. It wasdiscovered that the enzymes did not require any cofactor,ie., neither dialysis nor precipitation with ammonium sulfatecaused a fall in enzyme activities and the addition of DPN andTPN to the reaction mixture did not accelerate the reactionrate
3. From the results of spectroscopic investigation GD₁ andGD₂were shown to be flavoproteins, although their prostheticgrouphas not yet been identified The activity of GD₁ was enhancedby the addition of FAD or FMN, while GD₂ was not acceleratedby these factors.
4. The characteristics of the two enzymes includingsubstrate specificity,MICHAELIS constant, optimum pH of thereaction and specificityfor electron acceptors were compared.
5. From the stoichiometric study of the oxidation of L-glutamatewith these enzymes, it was confirmed that the reaction is representedby the following equation: L-glutamate+oxidized dye+h₂o
6. Among various inhibitors tested,molecular oxygen which couldfunction as electron acceptor ofL-glutamate oxidation in thepresence of GD₁ was found to causea strong inhibition uponthe same reaction with TTC as el acceptor.The inhibition wasconfirmed to be due to hydrogen peroxideproduced as a resultof the aerobic oxidation of L-glutamate.

(Received July 25, 1962; )     

Reductive carboxylation and amination of keto acids by spinach chloroplasts

1. In spinach chloroplasts, the occurrence of malic enzyme,isocitrate dehydrogenase, glutamate dehydrogenase and alaninedehydrogenase was confirmed. 2. In the presence of ammonia, pyruvate and -ketoglutarate andpyruvate were photoreductively aminated to glutamate and alanine,respectively. 3. In the absence of ammonia, pyruvate and -ketoglutarate werephotoreductively carboxylated to malate and isocitrate, respectively. 4. Photoreductive carboxylation of pyruvate and -ketoglutaratewas suppressed by molecular oxygen. Inhibition was partly dueto oxidation of photoreduced NADP or NAD. (Received August 4, 1969; )     

Amino-acid Sequence of λ Phage Endolysin

WE wish to present a preliminary report of the amino-acid sequence of λ endolysin. This protein is a lytic enzyme¹ and its structural gene, R, maps toward the right end of λ DNA². Conditional mutants as well as frame-shift mutants (R. Thomas, personal communication) have been isolated and analysed³. Hogness et al.⁴ developed a technique to assay the gene activity of the fragmented λ DNA, which suggested that it might be possible to isolate a small segment of DNA containing the endolysin gene. Purification, immunological properties and end group analysis of λ endolysin were studied by Black and Hogness^5–7.     

PHYTOCHROME ACTION IN ORYZA SATIVA L. III. THE SEPARATION OF PHOTOPERCEPTIVE SITE AND GROWING ZONE IN COLEOPTILES, AND AUXIN TRANSPORT AS EFFECTOR SYSTEM

• 1 In 4-day-old etiolated rice seedlings, 3 mm of the coleoptile tip did mainly perceive the photostimulus to cause the phytochrome-dependent inhibition of coleoptile elongation. At this age, cell elongation occurred most in the middle portion of coleoptiles in the dark, and was reversibly controlled by a brief exposure of the tip to red and far-red light. Thus, the photoperceptive site was evidently separated from the growing zone in intact rice coleoptiles.
• 2 The red-light-induced inhibition of coleoptile elongation was nullified by the removal of tip followed by the exogenous application of IAA. The sensitivity of thus treated coleoptiles to IAA was gradually lost during intervening darkness between the irradiation and the decapitation, and a 50% loss was obtained at ca. 6th hour at 26°C.
• 3 Polar auxin transport from coleoptile tips was remarkably prevented at the period between, at least, 2nd and 4th hour after red irradiation, and it recovered to the level of dark control by the 6th hour. Far-red light given immediately after red irradiation reversed the yield of diffusible auxin up to that of far-red control.

     

Prothoracicotropic Hormone Bioassay: Pupal-Adult Bombyx Assay

Blockage of adult development by brain removal and its resumption by application of the prothoracicotropic hormone (PTTH) were studied using pupae of a racial hybrid J-122 × C-115 of Bombyx mori . A log-linear dose-response relationship was obrained after injection of a PTTH solution. The Bombyx -unit of PTTH has been defined from this dose-response curve.     

A redescription of Cythere japonica Hanai, 1959 (Podocopida: Ostracoda)

Cythere japonica was proposed by Hanai (1959) as a new species of the genus Cythere , from the Pleistocene Sawane Formation in Sado Island, Niigata Prefecture, Japan. The characters of carapace were already known, but the appendages have not been previously described. The authors formerly considered that this species should be separate from the genus Cythere sensu stricto because it has twice as many sieve-type pore systems as typical Cythere species, and a markedly higher carapace. The existence of living Cythere japonica in the tidal zone of north-west Japan is confirmed, and its taxonomic position re-examined on the basis of its appendages and the ontogeny of pore systems. The appendages, except for the copulatory organ, are almost identical with those of other Cythere species, and their pore systems share the same pattern in and before the A-4 moult stage. On the basis of these features this species should be retained in the genus Cythere. Phylogenetic relationships are considered on the basis of the ontogeny of pore systems.
The abdominal segments of podocopid Ostracoda, which have always been regarded as difficult to observe because of their fusion, are shown clearly by the SEM.     

Additive apportioning of lepidopteran and coleopteran species diversity across spatial and temporal scales in a cool–temperate deciduous forest in Japan

Abstract.  1. Forest entomofauna retain high diversity, and examining beta diversity, or species turnover, among assemblages in a forest community is vital to elucidate the source of this diversity.
2. Under the DIVERSITAS in Western Pacific and Asia–International Biodiversity Observation Year (DIWPA–IBOY) project for simultaneously documenting biodiversity throughout the Western Pacific and Asian Region, 892 lepidopteran species (51 742 specimens) and 355 coleopteran species (11 633 specimens) were collected in 2001 by light traps in a cool–temperate forest in northern Japan.
3. This study evaluated the beta diversity of lepidopteran and coleopteran communities by ecological categories (i.e. trap location, forest strata, sampling days, and months), and assessed the habitat preferences of lepidopteran and coleopteran species.
4.  anova -like additive apportioning models were used to quantify the beta diversity among the categories. The models simultaneously provide assessments of whether species distributions are biased in favour of particular habitats.
5. Significantly high beta diversity was observed among months for both Lepidoptera and Coleoptera. The category of months corresponded fairly well to the number of specialist species detected in the category, although a remarkably large number of significant specialists in Coleoptera were observed on strata.
6. The high beta diversity and number of specialist species among strata in both communities indicate that stratification between canopy and ground, and seasonal variation, played major roles in species composition and the rich entomofauna in the forest. Highly mobile adults were influenced by the vertical spatial scale, as previously suggested for larvae.     

Insect Melanogenesis. II. Inability of Manduca Phenoloxidase to Act on 5, 6-Dihydroxyindole-2-Carboxylic Acid1

Eumelanins in animals are biosynthesized by the combined action of tyrosinase, 3, 4-dihydroxyphenylalanine (DOPA)chrome isomerase, and other factors. Two kinds of eumelanins were characterized from mammalian systems; these are 5,6-dihydroxyindole (DHI)-melanin and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin. In insects, melanin biosynthesis is initiated by phenoloxidase and supported by DOPAchrome isomerase (decarboxylating). Based on the facts that DOPA is a poor substrate for insect phenoloxidases and DHI is the sole product of insect DOPAchrome isomerase reaction, it is proposed that insects lack DHICA-melanin. Accordingly, the phenoloxidase isolated from the hemolymph of Manduca sexta failed to oxidize DHICA. Control experiments reveal that mushroom tyrosinase, as well as laccase, which is a contaminant in the commercial preparations of mushroom tyrosinase, are capable of oxidizing DHICA. Neither the whole hemolymph nor the cuticular extracts of M. sexta possessed any detectable oxidase activity towards this substrate. Thus, insects do not seem to produce DHICA-eumelanin. A useful staining procedure to localize DHICA oxidase activity on gels is also presented.