Identification of a unicellular,non-pigmented alga that mediates growth inhibition in anuran tadpoles: a new species of the genus Prototheca (Chlorophyceae: Chlorococcales)

A non-pigmented, unicellular alga isolated from the faeces of British anuran tadpoles and which is associated with growth inhibition in these tadpoles, was described and identified using cytological, ultrastructural, nutrient assimilation and immunological studies. The alga possessed all the distinctive morphological features of the genus Prototheca, it grew weakly on Prototheca Isolation Medium (PIM), it required thiamine for continued growth and replication, and it could assimilate the five major substrates used to speciate the protothecans. All of these characteristics, together with previous nucleic acid hybridisation studies, indicated that the microorganism belonged to the genus Prototheca. There are currently five species recognised as valid (Pore, 1985 & 1986): Prototheca zopfii Kruger, 1884, P. wickerhamii Tubaki & Soneda, 1959, P. moriformis Kruger, 1884, P. stagnora Cooke, 1968 and P. ulmea Pore, 1986.The immunology showed that the new species was related to two of the protothecans, but overall it showed that the alga was antigenically distinct from the other protothecans tested in the immunoassay. This, together with its inability to grow strongly on PIM, its ability to assimilate a wide rage of carbon substrates and its ability to mediate growth inhibition in anuran tadpoles, indicated a new species of Prototheca. We therefore propose the name Prototheca richardsi sp. n.     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Thermal denaturation of the DNA-ethidium complex. Redistribution of the intercalated dye during melting.

The temperature dependence of the circular dichroism of the DNA-ethidium bromide complex at elevated temperatures provides evidence that the optical activity of the complex near 307 nm originates from interactions between intercalated dye molecules while the optical activity near 515 nm results from singly intercalated ethidium bromide molecules. The behavior of the circular dichroism of the complex at elevated temperatures also explains the higher ellipticities near 307 nm which characterize complexes formed between ethidium bromide and denaturated DNA. Finally the circular dichroism data indicate that the melting of the complex takes place in a stepwise manner with some DNA regions, probably AT-rich regions, dissociating first. The implications of these findings regarding the inhibiting effect of ethidium bromide on the function of DNA polymerase are examined.     

Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system

The skin commensal and opportunistic pathogen Staphylococcus epidermidis is the leading cause of nosocomial and biofilm-associated infections. Little is known about the mechanisms by which S. epidermidis protects itself against the innate human immune system during colonization and infection. We used scanning electron microscopy to demonstrate that the exopolysaccharide intercellular adhesin (PIA) resides in fibrous strands on the bacterial cell surface, and that lack of PIA production results in complete loss of the extracellular matrix material that has been suggested to mediate immune evasion. Phagocytosis and killing by human polymorphonuclear leucocytes was significantly increased in a mutant strain lacking PIA production compared with the wild-type strain. The mutant strain was also significantly more susceptible to killing by major antibacterial peptides of human skin, cationic human beta-defensin 3 and LL-37, and anionic dermcidin. PIA represents the first defined factor of the staphylococcal biofilm matrix that protects against major components of human innate host defence.     

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus

Supernumerary mini-chromosomes–a unique type of genomic structural variation–have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.     

NFκB regulates expression of Polo-like kinase 4

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulated NFκB activity in cancer can lead to increased genomic instability and uncontrolled proliferation.     

C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.     

Cadmium decontamination and reversal potential of teratological forms of the diatom Planothidium frequentissimum (Bacillariophyceae) after experimental contamination

While the induction of teratology by cadmium (Cd) on diatoms is already known, reversal kinetics are not well documented. This study aims to understand the viability of diatoms exhibiting teratological frustules and their reproduction capacities within a Cd‐impacted population to predict their return to normal diatom forms. We worked on a frequently encountered species in French hydrosystems: Planothidium frequentissimum (Lange‐Bertalot) Round & L. Bukhtiyarova. First, a 21‐d contamination phase highlighted increasing inductionof different teratological types in response to two levels of Cd contamination: 20 and 100 μg · L^?1. The deformity counting indicated that Cd firstly generated striae and mixed teratologies, then affected the central area and the valves. Second, a 28‐d decontamination phase demonstrated the Cd depuration capacity of Planothidium frequentissimum. Cd half‐lives appeared relatively low, ~6 d for the 100 μg · L^?1 condition. Moreover, the decontamination phase showed a decrease in teratology abundances, but a still incomplete recovery after 28 d. Deformations of the striae appeared to be the most sustainable phenotype since they were still significantly higher than in reference cultures at the end of the decontamination phase for both Cd cultures.