Increased level of p27 subunit of proteasomes and its co-localization with tyrosinase in amelanotic melanoma cells indicate its direct role in the regulation of melanin biosynthesis

Proteasomes have been shown to be involved in the regulation of melanin biosynthesis in melanoma cells. Here we report on the correlation between proteasome subunits and Tyrosinase (Tyr) activity in different cell phenotypes, and thereby regulation of melanin biosynthesis in B16F10 mouse melanoma cells. Our results indicated that the quantity of proteasome subunit p27 is higher and that of the enzyme Tyr and its activity are lower in amelanotic melanoma cells, while the reverse is true in melanotic melanoma cells. Proteasome subunit p27, compared to another subunit p31, shows increased co-localization with Tyr and Tyrosinase related protein 1 (Trp1) in amelanotic cells to a greater extent than that in melanotic cells. On exposure to cycloheximide, increased Tyr degradation was seen in amelanotic cells, as indicated by increased co-localization of p27 and Tyr. Further, exposure of amelanotic melanoma cells with proteasome-specific inhibitor MG132 resulted in an increased Tyr activity, increased levels of Tyr and Trp1, leading to increased melanin synthesis. These results therefore suggest that proteasomes, particularly p27 subunit, are directly involved in the regulation of melanin biosynthesis in mouse melanoma cells.     

Phylogeography of the cosmopolitan marine parasite Kudoa thyrsites (Myxozoa: Myxosporea)

Kudoa thyrsites (Myxozoa: Multivalvulida) is a cosmopolitan marine parasite of fishes associated with post-mortem tissue degradation. Financial losses incurred as a result of these infections are of concern to commercial fisheries. There is conflicting evidence whether K. thyrsites represents a cryptic species complex. Myxospore morphology is very similar for K. thyrsites across its range, but preliminary genetic analyses show some differences. Kudoa thyrsites and the morphologically similar Kudoa histolytica were examined from hosts in British Columbia, Canada, Oregon, USA, Chile, England, South Africa, Australia, and Japan. We compared myxospore morphology and DNA sequences of heat shock protein 70 and the small subunit, large subunit, and internal transcribed spacer 1 of the ribosomal DNA. There was some morphological variation between regional representatives, inconsistent with genetic analyses. Phylogenetically, major separations correlated to four broad geographic regions: Japan, Australia, eastern Pacific, and eastern Atlantic. Within these regions there was little additional genetic structure. These data are evidence for regional subdivision of K. thyrsites suggesting global transplantation of fishes has yet to homogenize these distinctions. Within regions, parasite gene flow appears to be high between host species, suggesting little host specificity and minimal cryptic speciation. Our data also indicate that K. histolytica is not a valid species, as it was morphologically and genetically indistinguishable from K. thyrsites.     

Assessing Risk of Unintended Antigen Occurrence in Food: A Case Instance for Maize-Expressed LT-B

Plant-based antigen production represents an innovative strategy for low cost vaccine production and delivery. Successfully advancing plant-made antigen production in open field systems requires understanding of confinement integrity and consequences of inadvertent occurrence in the food supply. The food safety implications of confinement loss and inadvertent antigen occurrence in the food supply can be effectively addressed using quantitative exposure assessment along with knowledge of properties of specific antigens. We report here a food safety risk assessment for the maize-expressed heat-labile enterotoxin subunit B of Escherichia coli (LT-B). In addition to dietary exposure assessment, food safety considerations for maize-expressed LT-B included assessment of allergenic potential, levels and sites of transgenic protein expression, history of use, post-translational glycosylation, protein processing and digestive stability, mammalian functionality and toxicity, and compositional characteristics of the transformed plant. As shown for LT-B, inadvertent occurrence in the food supply of a plant-produced antigen constitutes a minimal human health concern principally because of limited exposure potential.     

Molecular cloning and sequence analysis of the β-1,3-glucan synthase catalytic subunit gene from a medicinal fungus, Cordyceps militaris

The entomopathogenic fungus Cordyceps militaris belongs to vegetable wasps and plant worms and is used as herbal medicine, but β-1,3-glucan biosynthesis has been poorly studied in C. militaris. The fungal FKS1 gene encodes an integral membrane protein that is the catalytic subunit of β-1,3-glucan synthase. Here, we isolated cDNA clones encoding a full-length open reading frame of C. militaris FKS1. Cordyceps militaris Fks1 protein is a 1981 amino acid protein that shows significant similarity with other fungal Fks proteins. This study is the first report of molecular cloning of the β-1,3-glucan synthase catalytic subunit gene from vegetable wasps and plant worms.     

A Daphnia Parasite (Caullerya mesnili) Constitutes a New Member of the Ichthyosporea,a Group of Protists near the Animal–Fungi Divergence

ABSTRACT. Caullerya mesnili is a protozoan endoparasite in the gut epithelium of Daphnia, which causes regular epidemics in lakes throughout Europe. Its classification has remained unchanged for over a century, leaving it placed with the Haplosporidia, despite speculation that this position is incorrect. The difficulty in classifying C. mesnili stems from its few known morphological and ecological characteristics, as well as a lack of genetic markers. Here we sequenced the nuclear small subunit (SSU) and internal transcribed spacer rDNA regions of C. mesnili samples from 10 locations. Based on sequence similarities, we suggest the re‐classification of C. mesnili to the Ichthyosporea, a class of protists near the animal–fungi divergence. We report average intragenomic variation of 0.75% and 2.27% in the SSU and internal transcribed spacer regions, respectively. From electron micrographs and light microscopy of histological sections we determined that C. mesnili spores grow within the intestinal epithelium where they establish themselves intercellularly. In addition, we confirmed previous accounts regarding the high virulence of this parasite. Caullerya mesnili reduces host lifespan, the number of clutches, and the total number of offspring. This high selection pressure placed on hosts supports the importance of C. mesnili as a model parasite for the study of host–parasite biology in permanent lakes.     

Phylogenetic Relationships of Orders Within the Class Colpodea (Phylum Ciliophora) Inferred from Small Subunit rRNA Gene Sequences

Molecular analyses have been used recently to refine our knowledge of phylogenetic relationships within the ciliated protozoa (phylum Ciliophora). A current Hennigian phylogeny of the orders in the class Colpodea, based on light and electron microscopic analyses, makes three important assumptions with regard to apomorphic character states, namely, (1) that the kreyellid silver line evolved early in colpodean phylogeny, separating bryometopids, such as Bryometopus, from all other colpodeans; (2) that the macro–micronuclear complex is an autapomorphy of the cyrtolophosidids, such as Platyophrya; and (3) that merotelokinetal stomatogenesis is an apomorphic character of colpodids, such as Colpoda, Bresslaua, and Pseudoplatyophrya. These predictions of relationships within the class Colpodea were investigated by determining the complete small subunit rRNA gene sequences for the colpodid Bresslaua vorax, the grossglockneriid Pseudoplatyophrya nana, and the cyrtolophosidid Platyophrya vorax and a partial sequence for the bryometopid Bryometopus sphagni. These sequences were combined with the previously published complete SSrRNA sequences for the colpodid Colpoda inflata and the bursariomorphid Bursaria truncatella. The affiliations were assessed using both distance matrix and maximum-parsimony analyses. The tree topologies for the class Colpodea were identical in all analyses, with bootstrap support for bifurcations always exceeding 60%. The results suggest the following. (1) Since the clade including Bryometopus and its sister taxon, Bursaria, is never basal, the kreyellid silver-line system evolved later in colpodean phylogeny and does not separate bryometopids from all other colpodeans. (2) Since Platyophrya is always the sister taxon to the other five genera, there is a fundamental phylogenetic significance for its macro–micronuclear complex. (3) Since the colpodids, Colpoda, Bresslaua, and Pseudoplatyophrya, always group in one clade, merotelokinetal stomatogenesis appears to be a derived character state. Received: 30 June 1998 / Accepted: 3 December 1998     

The Role of the Supernumerary Subunit of Rhodobactersphaeroides Cytochrome bc 1 Complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group,is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc ₁ complex.This subunit is involved in Q binding and the structural integrity of the complex. When thecytochrome bc ₁ complex is photoaffinity labeled with [³H]azido-Q derivative, radioactivity isfound in subunits IV and I (cytochrome b), indicating that these two subunits are responsiblefor Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R.sphaeroides chromosome, the resulting strain (RSIV) requires a period of adaptation beforethe start of photosynthetic growth. The cytochrome bc ₁ complex in adapted RSIVchromatophores is labile to detergent treatment (60–75% inactivation), and shows a four-fold increasein the K _m for Q₂H₂. The first two changes indicate a structural role of subunit IV; the thirdchange supports its Q-binding function. Tryptophan-79 is important for structural andQ-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GSTfusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunitIV is functionally active as it can restore the bc ₁ complex activity from the three-subunit corecomplex to the same level as that of wild-type or complement complex. Three regions in thesubunit IV sequence, residues 86–109, 77–85, and 41–55, are essential for interaction withthe core complex because deleting one of these regions yields a subunit completely or partiallyunable to restore cytochrome bc ₁ from the core complex.     

Dissecting subdomains involved in multiple functions of the CK2β subunit

We have characterized several subdomains of the subunit of protein kinase CK2. The N-terminal half of the protein exhibits a pseudo-substrate segment in tandem with a polyamine binding domain responsible for the activation of the kinase by these polybasic compounds. Study of the chemical features of this polyamine binding site showed that polyamine analogs exhibiting the highest affinity for CK2 are the best CK2 activators. Mutational analysis disclosed that glutamic residues lying in the polyacidic region of the CK2 subunit are involved in the interaction with polyamine molecules and allowed the delineation of an autonomous binding domain. Furthermore, this regulatory domain was shown to mediate the association of CK2 with plasma membrane.The C-terminal domain of the CK2 subunit plays a role in the oligomerization of the kinase since it was observed that a truncated form of this subunit lacking its 33-last amino acids was incompetent for the assembly of polymeric forms of CK2. Altogether, our results support the notion that the subunit of CK2 is a modular protein made by the association of interdependent domains that are involved in its multiple functions.