Structure and function of the conidiospore pigments of Penicillium cyclopium.

The cell wall of mature, green condiospores of Penicillium cyclopium Westling contains at least two pigments: A green chromoprotein which is extractable by means of formic acid or liquified phenol and a black insoluble pigment. Both fractions after long term treatment with boiling conc. HCl leave black amorphous residues which, due to their chemical and physico-chemical properties, belong to the group of melanins. The chemical structure of these melanins is still unidentified. No degradation products typical for indol-type or catechol-type melanins have so far been detected. During spore maturation parallel to an increase of pigmentation (determined by remission), the melanin residue left after acid hydrolysis of spores increases. The mature, dark green spores of the wild type strain contain about 40% melanin, the yellow-green spores of the mutant aux-glu 1 about 36%. The unpigmented spores of mutant res-eth 1 possess a melanin content of only about 5%. This value is nearly the same as that found in hyphae, which in all strains are yellowish-brown. The heavily pigmented condia of the wild type strain are about 100-times less sensitive to UV-radiation compared with the unpigmented spores of the mutant res-eth 1. The reduced sensitivity indicates that, as with other microorganisms, the conidia pigments of P. cyclopium are protective components of the spores.     

The patchwork mouse phenotype: implication for melanocyte replacement in the hair follicle.

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

Mutant gene expression in mouse aggregation chimeras. 8. The effect of the white gene on coat pigmentation

Aggregation of mouse embryos produced 11 chimaeras Miwh/+C/C----+/+c/c and 8 chimaeras +/+C/C----+/+c/c (control). Chimaerism was detected by mosaicism of coat retinal pigment epithelium and by electrophoretic pattern of glucose phosphate isomerase. All chimaeras showed a common pattern of pigmented and unpigmented hair regions that alternated as stripes of different length and width and extended from spine in lateral-ventral direction. However, white coat color predominated in Miwh/+C/C----+/+c/c chimaeras due to a higher proportion of unpigmented zones as well as to weakening of hair color in pigmented areas. Besides, distal regions of limbs were always unpigmented in Miwh/+C/C----+/+c/c chimaeras and completely or partially pigmented in +/+C/C----+/+c/c chimaeras. Pigmented hair regions are often located on the ventral trunk surface where the Miwh/+ heterozygotes usually had an unpigmented spot. The examination of hairs, taken from the same regions of gray coloration, revealed the presence of pigmented, unpigmented and mosaic hairs. The proportion of unpigmented hairs was much higher in Miwh/+C/C----+/+c/c chimaeras than in +/+C/C----+/+c/c chimaeras. The data obtained indicate that a single Miwh gene dose reduced proliferative activity of melanoblasts which resulted in weakening of coat pigmentation.     

The Patchwork Mouse Phenotype: Implication for Melanocyte Replacement in the Hair Follicle

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

Trade-Offs between Predation Risk and Growth Benefits in the Copepod Eurytemora affinis with Contrasting Pigmentation

Intraspecific variation in body pigmentation is an ecologically and evolutionary important trait; however, the pigmentation related trade-offs in marine zooplankton are poorly understood. We tested the effects of intrapopulation phenotypic variation in the pigmentation of the copepod Eurytemora affinis on predation risk, foraging, growth, metabolic activity and antioxidant capacity. Using pigmented and unpigmented specimens, we compared (1) predation and selectivity by the invertebrate predator Cercopagis pengoi, (2) feeding activity of the copepods measured as grazing rate in experiments and gut fluorescence in situ, (3) metabolic activity assayed as RNA:DNA ratio in both experimental and field-collected copepods, (4) reproductive output estimated as egg ratio in the population, and (5) total antioxidant capacity. Moreover, mitochondrial DNA (mtDNA) COI gene variation was analysed. The pigmented individuals were at higher predation risk as evidenced by significantly higher predation rate by C. pengoi on pigmented individuals and positive selection by the predator fed pigmented and unpigmented copepods in a mixture. However, the antioxidant capacity, RNA:DNA and egg ratio values were significantly higher in the pigmented copepods, whereas neither feeding rate nor gut fluorescence differed between the pigmented and unpigmented copepods. The phenotypic variation in pigmentation was not associated with any specific mtDNA genotype. Together, these results support the metabolic stimulation hypothesis to explain variation in E. affinis pigmentation, which translates into beneficial increase in growth via enhanced metabolism and antioxidant protective capacity, together with disadvantageous increase in predation risk. We also suggest an alternative mechanism for the metabolic stimulation via elevated antioxidant levels as a primary means of increasing metabolism without the increase in heat absorbance. The observed trade-offs are relevant to evolutionary mechanisms underlying plasticity and adaptation and have the capacity to modify strength of complex trophic interactions.     

In vitro clonal analysis of quail cardiac neural crest development

The developmental potentials of cardiac neural crest cells were investigated by in vitro clonal analysis. Five morphologically distinct types of clones were observed: (1) "pigmented" clones contained melanocytes only; (2) "mixed" clones consisted of pigmented and unpigmented cells; (3) "unpigmented dense" clones consisted of flattened, closely aligned unpigmented cells; (4) "unpigmented loose" clones consisted of a few loosely arranged, flattened cells; and (5) "unpigmented large" clones included a large number of small, stellate cells that were highly proliferative. The binding patterns of antibodies against lineage-specific markers showed that cells in the different clones expressed characteristic phenotypes. The following phenotypes were expressed in addition to pigment cells: smooth muscle cells, connective tissue cells, chondrocytes, and cells in the sensory neuron lineage. Mixed clones expressed all five phenotypes. Unpigmented dense clones contained smooth muscle cells, connective tissue cells, chondrocytes, and sensory neurons. Unpigmented loose clones exclusively consisted of smooth muscle cells, whereas unpigmented large clones contained chondrocytes and sensory neuron precursors. Based on these results, the following conclusions can be drawn: (1) Pigmented and unpigmented loose clones are most likely formed by precursors that are committed to the melanogenic and myogenic cell lineages, respectively. (2) Mixed and unpigmented dense clones are derived from pluripotent cells with the capacity to give rise to four or five phenotypes. (3) Unpigmented large clones originate from progenitor cells that appear to have a partially restricted developmental potential, that is, these cells are capable of generating two phenotypes in clonal cultures. Thus, the data indicate that the early migratory cardiac neural crest is a heterogeneous population of cells, consisting of pluripotent cells, cells with a partially restricted developmental potential, and cells committed to a particular cell lineage.     

Mitochondrial biogenesis during fungal spore germination: respiration and cytochrome c oxidase in Neurospora crassa.

The germination of conidiospores of wild-type Neurospora crassa was found to be dependent upon the function of the cytochrome-mediated electron transport pathway. The cyanide-insensitive alternate oxidase did not contribute significantly to the respiration of these germinating spores. The dormant spores contained all of the cytochrome components and a catalytically active cytochrome c oxidase required for the activity of the standard respiratory pathway, and these preserved components were responsible for the accelerating rates of oxygen uptake which began immediately upon suspension of the spores in an incubation medium. Mitochondria of the dormant spores contained all of the subunit peptides of the functional cytochrome c oxidase; nevertheless, de novo synthesis of these subunits began at low rates in the first stages of germination. Reactivation of the respiratory system of germinating N. crassa spores seems not to be dependent initially upon the function of either the mitochondrial or cytoplasmic protein-synthesizing systems. The respiratory activity of spores of three mutant cytochrome c oxidase-deficient strains of N. crassa also was found to depend upon the function of the cytochrome electron transport pathway; the dormant and germinating spores of these strains contained a catalytically active cytochrome c oxidase. Cytochrome c oxidase may be present in the dormant and germinating spores of these strains as the result of a developmental-phase-specific synthesis of and requirement for the enzyme.     

Fonsecaea monophora色素毒力性研究

目的探讨黑色素是否为Fonsecaea monophora的一个重要毒力因子。方法从Fonsecaea monophora的分生孢子突变株(CBS122845)传代接种产生白色突变株(CBS 125149)。透射电子显微镜(TEM)下观察到黑色素是位于分生孢子细胞壁表面上的电子致密颗粒。通过碱-酸法提取来自两个不同菌株的细胞壁色素颗粒。建立不同菌株或色素颗粒与活化巨噬细胞(RAW264.7)共培养体系,通过实时荧光相对定量PCR检测i-NOS基因的表达,格里斯法检测一氧化氮(NO)的表达结果,ELISA检测IL-12、TNF-α、IL-10的表达结果。结果色素型分生孢子和其色素颗粒能够降低巨噬细胞诱导型一氧化氮合酶(I-NOS)基因的表达和抑制一氧化氮的合成(P<0.05)。提高Th2细胞因子表达,同时抑制Th1细胞因子表达(P<0.05)。结论黑色素可能是Fonsecaea monophora逃避巨噬细胞对其氧化应激的重要机制。同时黑色素下调Th1免疫应答,可能利于真菌的持续感染。     

Cell Clustering and Pleiotropy in White-Variegated Eyes and Malpighian Tubes of DROSOPHILA HYDEI

The type of variegation of eyes of white-mottled mutants of D. hydei, either small-spotted or large-spotted, depends on the specific chromosome rearrangement involved. This distinction between mutants, though handsome, is not absolute because very seldomly small-spotted types do show a larger pigment aggregate and some "large-spotted" flies have no large spots at all, but only minor spots. Because of a pleiotropic action of the white gene, we could study the variegation of Malpighian tubules. The quasi-linear array of Malpighian cells enables a thorough statistical analysis. The problem was in how far the variegation of the tubes that is correlated with the variegation of eyes (in as far as numbers of pigmented and unpigmented cells are concerned) is also connected with a cell-lineage type of determination. Statistics now show that in spite of temperature-induced great variation in the percentage of pigmented cells, all types studied show a nearly random distribution of pigmented and unpigmented cells in the Malpighian tubules. This implies that the cell-lineage type of determination is not only largely mutant-specific but also organ-specific, i.e., limited to eyes. A basic gradient, however, as characteristic for eyes, was also found in Malpighian tubes.     

Melanomas display increased cytoprotection to hypericin-mediated cytotoxicity through the induction of autophagy

Photodynamic therapy (PDT) as a regime for melanoma is of limited success due to factors such as the efficacy of the photosensitizer used, penetration depth and the presence of pigment. We characterised a pigmented and an unpigmented melanoma cell line with respect to their phenotypes. Cell viability was assessed after exposure to hypericin, a UVA-activated photosensitizer. Exposure to 3 μM activated hypericin induced a cytoprotective (autophagic) response from both cell lines. However, the pigmented cells accumulated a large amount of glycogen in their cytoplasm. We hypothesise that the treatment induces an initial cytoprotective response through autophagy, but with increased stress results in a different mode of cell death in pigmented melanoma cells from unpigmented cells. These results indicate that hypericin-PDT could be an adjuvant therapy for melanoma.     

Tumor-Promoting Phorbol Esters Promote Melanogenesis and Prevent Expression of the Adrenergic Phenotype in Quail Neural Crest Cells

Tumor-promoting phorbol esters were used to manipulate the in vitro development of neural crest cells. When plated at clonal density in secondary culture, quail neural crest cells from the trunk region gave rise to three types of colonies, pigmented, unpigmented, and mixed. Pigmented colonies consisted exclusively of melanocytes; up to 50% of the unpigmented and mixed colonies contained adrenergic nerve cells which could be identified by a catecholamine-specific histofluorescence method. Addition of potent tumor promoters to the culture medium shortened the doubling time of neural crest cells and altered their morphologic appearance. It also delayed the onset of pigmentation, prevented the expression of the adrenergic phenotype, reduced the number of unpigmented and mixed colonies, and increased the number of pigmented colonies, most likely by directing progenitor cells preferentially to the melanogenic pathway. There was a clear correlation between the ability of phorbol esters to promote skin tumors in mice and their ability to interfere with the in vitro development of quail neural crest cells. The potent promoters 12–0–tetradecanoyl phorbol 13–acetate (TPA) and phorbol 12,13–didecanoate (PDD) were most effective, phorbol 12,13–diacetate (PDA) was considerably less effective, the nonpromoting analogues 4–0–methyl 12–0–tetradecanoyl phorbol 13–acetate (4–0–Me-TPA) and 4α-phorbol 12,13–didecanoate (4α-PDD) and the parent alcohol phorbol (PHR) had little or no effect.     

higher-order phylogeny of plasmodial slime molds (Myxogastria) based on elongation factor 1-A and small subunit rRNA gene sequences

The Myxogastria are common soil microorganisms with a life cycle comprised of a plasmodial trophic stage and large fruiting bodies generally visible with the unaided eye. Until now, their classification has been based exclusively on a combination of morphological, ultrastructural, and developmental characters. Our study is the first attempt to examine phylogenetic relationships among these taxa using molecular data. Partial small-subunit ribosomal RNA and/or elongation factor 1-alpha gene sequences were obtained from eleven, mostly field-collected species representing the five orders of Myxogastria. Nineteen sequences were obtained and subjected to phylogenetic analysis together with 10 sequences available from GenBank. Separate and combined analyses of the two data sets support the division of Myxogastria into three distinct groups. The most basal clade consists of the Echinosteliales, an order considered to have affinities with Protostelia. The three species examined possess unpigmented or slightly pigmented spores. The second group consists of Liceales and Trichiales, taxa characterized by the presence of clear, but pigmented, spores. The third group consists of the two remaining orders, Physarales and Stemonitales, both possessing dark spores. This suggests that spore pigmentation is an evolutionarily conservative character in myxogastrians, and that the simple morphology of echinostelids is not a derived feature.     

Genetic relationships between Nereimyra punctata and N. woodsholea (Hesionidae, Polychaeta)

We present a COI-based parsimony analysis of the relationships between the shallow water, pigmented hesionid polychaete Nereimyra punctata , and a deep-water, unpigmented form with sympatric distribution in Norway and Sweden. Apart from the pigmentation differences, the two forms exhibit no observed morphological differences. The terminals are represented by four specimens each of the two forms from the Trondheimsfjord in Norway, and four each of the two forms from northern Bohuslän in Sweden, plus members of the two hesionids Heteropodarke and Ophiodromus as outgroups. In addition, the analysis includes a topotype of the morphologically similar and unpigmented Nereimyra woodsholea from the Middle Atlantic Bight off the US east coast. The equally weighted matrix includes 132 informative characters. All most-parsimonious trees unequivocally indicate that specimens belonging to the same form (pigmented or unpigmented) from different areas are cladistically closer related to each other than different forms from the same areas. Nereimyra woodsholea is nested within the unpigmented deeper group of the Norwegian and Swedish specimens, thus indicating that this name should be applied to the deep-water form in Norway and Sweden.     

Antioxidant enzymatic systems in pigment tissue of amphibia

Superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities in pigmented and unpigmented liver tissues of frog and albino rat, respectively, were studied. Our results show that pigmented tissue is lacking in manganese superoxide dismutase activity and that the main enzymatic activity utilized in the cytosol by pigmented cells to reduce the hydrogen peroxide to water is represented by catalase; on the contrary, for the same reaction, the cells of albino rat liver primarily utilize the glutathione peroxidase activity. Both a low glutathione peroxidase activity and a low glutathione reductase activity were found in pigmented tissue of frog liver when compared with unpigmented tissue of rat liver. In light of our results, we also report a hypothetical interrelationship between melanin and reduced glutathione: We believe that in pigmented cells the melanin could act as a reducing physiological agent replacing the glutathione in the reduction of hydrogen peroxide. This reducing action of melanin could cause a diminished need for GSH and therefore could provoke the low glutathione peroxidase and reductase activities in pigmented tissue.     

Surface hydrophobicity of Aspergillus nidulans conidiospores and its role in pellet formation

Formation of pellets by Aspergillus nidulans is primarily due to agglomeration of the fungal conidiospores. Although agglomeration of conidiospores has been known for a long time, its mechanism has not been clearly elucidated. To study the influence of the fungal conidiospore wall hydrophobicity on conidiospore agglomeration, pellet formation of an A. nidulans wild type and strains deleted in the conidiospore-wall-associated hydrophobins DewA and RodA was compared at different pH values. From contact angle measurements, RodA was found to be more important for the surface hydrophobicity than DewA. The absence of either hydrophobin led to a decrease in the relative amount of biomass present as pellets at all pH values as well as a decrease in the average size of the pellets. For all strains, an increasing alkalinity of the medium resulted in an increased pellet formation. Together with measurements of electrophoretic mobility, it is concluded that both the electrical charge and hydrophobicity of the conidiospores affects the pellet formation but that the conidiospore agglomeration process cannot be ascribed to these factors alone.     

Premature Graying as a Consequence of Compromised Antioxidant Activity in Hair Bulb Melanocytes and Their Precursors

Intricate coordinated mechanisms that govern the synchrony of hair growth and melanin synthesis remain largely unclear. These two events can be uncoupled in prematurely gray hair, probably due to oxidative insults that lead to the death of oxidative stress-sensitive melanocytes. In this study, we examined the gene expression profiles of middle (bulge) and lower (hair bulb) segments that had been micro-dissected from unpigmented and from normally pigmented hair follicles from the same donors using quantitative real-time RT-PCR (qPCR) arrays. We found a significant down-regulation of melanogenesis-related genes (TYR, TYRP1, MITF, PAX3, POMC) in unpigmented hair bulbs and of marker genes typical for melanocyte precursor cells (PAX3, SOX10, DCT) in unpigmented mid-segments compared with their pigmented analogues. qPCR, western blotting and spin trapping assays revealed that catalase protein expression and hydroxyl radical scavenging activities are strongly repressed in unpigmented hair follicles. These data provide the first clear evidence that compromised antioxidant activity in gray hair follicles simultaneously affects mature hair bulb melanocytes and their immature precursor cells in the bulge region.     

DNA relatedness among species of Enterobacter and Serratia.

Species of Enterobacter and Serratia were examined for deoxyribonucleic acid relatedness to Klebsielleae, to atypical erwiniae, and to other members of Enterobacteriaceae. Deoxyribonucleic acid hybridization and then hydroxyapatite chromatography was the technique used to assess relatedness. Strains of Enterobacter cloacae formed two separate hybridization groups that correlate with the presence or absence of yellow pigment. Pigmented E. cloacae were 75-100% related, but they were only 40-50% related to unpigmented strains. Conversely, unpigmented strains were 70% or more related but were only 40-50% related to the pigmented strains. Both pigmented and unpigmented E. cloacae were 40-45% related to Enterobacter aerogenes and klebsiellae, and 20-30% related to Serratia species and Enterobacter hafniae. Atypical erwiniae were highly related to E. cloacae. Serratia marcescens strains formed one closely related group. Serratia liquefaciens strains formed a single, more disperse, relatedness group, as did isolates of Serratia rubidaea. These species were related throughout a substantial portion of their genomes. A group of lysine-positive "Citrobacter-like" strains were 40-50% related to Serratia species. Only four E. hafniae strains were tested. Two of these were highly related, while the other two were only 50% related to the reference strain. Enterobacter hafniae was only 15-20% related to other Enterobacteriaceae.     

Ion currents and membrane domains in the cleaving Xenopus egg

We used an extracellular vibrating probe to measure ion currents through the cleaving Xenopus laevis egg. Measurements indicate sharp membrane heterogeneities. Current leaves the first cleavage furrow after new, unpigmented membrane is inserted. This outward current may be carried by K+ efflux. No direct involvement of the Na+,K+-ATPase in the generation of this outward current is detected at first cleavage. Inward current enters the old, pigmented membrane; however, it does not enter uniformly. The inward current is largest at the old membrane bordering the new membrane. This suggests a heterogeneous ion channel distribution within the old membrane. Experiments suggest that the inward current may be carried by Na+ influx, Ca2+ influx, and Cl- efflux. No steady currents were detected during grey crescent formation, the surface contraction waves preceding cleavage, or with groove formation at the beginning of cleavage.     

Effects of Beauveria bassiana on embryos of the inland silverside fish (Menidia beryllina).

A chemical toxicity and teratogenicity test was adapted to assess potential adverse effects of a microbial pest control agent on a nontarget fish. Developing embryos of the inland silverside, Menidia beryllina, were exposed to conidiospores of the insect-pathogenic fungus Beauveria bassiana. Embryo rupture and death were observed. Embryo rupture did not always result in death, nor was death always associated with embryo rupture. Adherence of spores to the chorion, followed by germination and penetration by the germ tube, probably caused the embryos to rupture. Statistically significant (P less than or equal to 0.05) responses were observed in tests in which conidiospore concentrations were greater than or equal to 8.3 x 10(4) or less than or equal to 1.5 x 10(6)/ml. Conidiospores treated with a dispersant (biological detergent) showed significantly less binding (P less than or equal to 0.01) to embryos than did untreated spores. Both detergent-treated and heat-killed spores failed to cause significant adverse effects.     

Effects of Beauveria bassiana on embryos of the inland silverside fish (Menidia beryllina).

A chemical toxicity and teratogenicity test was adapted to assess potential adverse effects of a microbial pest control agent on a nontarget fish. Developing embryos of the inland silverside, Menidia beryllina, were exposed to conidiospores of the insect-pathogenic fungus Beauveria bassiana. Embryo rupture and death were observed. Embryo rupture did not always result in death, nor was death always associated with embryo rupture. Adherence of spores to the chorion, followed by germination and penetration by the germ tube, probably caused the embryos to rupture. Statistically significant (P less than or equal to 0.05) responses were observed in tests in which conidiospore concentrations were greater than or equal to 8.3 x 10(4) or less than or equal to 1.5 x 10(6)/ml. Conidiospores treated with a dispersant (biological detergent) showed significantly less binding (P less than or equal to 0.01) to embryos than did untreated spores. Both detergent-treated and heat-killed spores failed to cause significant adverse effects.