Inference of population structure and patterns of gene flow in canine heartworm (Dirofilaria immitis)

Understanding the genetic variation within a parasitic species is crucial to implementing successful control programs and preventing the dispersal of drug resistance alleles. We examined the population genetics and structure of canine heartworm (Dirofilaria immitis) by developing a panel of 11 polymorphic microsatellite loci for this abundant parasite. In total, 192 individual nematodes were opportunistically sampled from 9 geographic regions in the United States and Mexico and genotyped. Population genetic analyses indicate the presence of 4 genetic clusters. The canine heartworm samples used in this study were characterized by low heterozygosity, with eastern and central North America experiencing high levels of reciprocal gene flow. Geographic barriers impede the movement of vectors and infected hosts west of the Rocky Mountains and south of the Central Mexican Plateau. This, combined with corridors of contiguous habitat, could influence the spread of drug resistance alleles.     

Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8(+) T cells in B6.Ly49h(-/-) mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H(+) NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h(-/-) mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H(+) NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H(+) NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.     

The effect of stimulus type and background noise on hearing abilities of the round goby Neogobius melanostomus

The auditory abilities of the round goby Neogobius melanostomus were quantified using auditory evoked potential recordings, using tone bursts and conspecific call stimuli. Fish were tested over a range of sizes to assess effects of growth on hearing ability. Tests were also run with and without background noise to assess the potential effects of masking in a natural setting. Neogobius melanostomus detected tone bursts from 100 to 600 Hz with no clear best frequency in the pressure domain but were most sensitive to 100 Hz tone stimuli when examined in terms of particle acceleration. Responses to a portion of the N. melanostomus call occurred at a significantly lower threshold than responses to pure tone stimulation. There was no effect of size on N. melanostomus hearing ability, perhaps due to growth of the otolith keeping pace with growth of the auditory epithelium. Neogobius melanostomus were masked by both ambient noise and white noise, but not until sound pressure levels were relatively high, having a 5-10 dB threshold shift at noise levels of 150 dB re 1 μPa and higher but not at lower noise levels.     

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.     

Endocytosis of Candida albicans by vascular endothelial cells is associated with tyrosine phosphorylation of specific host cell proteins

Candida albicans escapes from the bloodstream by invading the endothelial cell lining of the vasculature. In vitro, C. albicans invades endothelial cells by inducing its own endocytosis. We examined whether this process is regulated by the tyrosine phosphorylation of endothelial cell proteins. We found that endocytosis of wild-type C. albicans was accompanied by the tyrosine phosphorylation of two endothelial cell proteins with molecular masses of 80 and 82 kDa. The phosphorylation of these proteins was closely associated with the endocytosis of C. albicans because these proteins were phosphorylated in response to the endocytosis of both live and killed organisms, but they were not phosphorylated in endothelial cells infected with a poorly endocytosed strain of C. albicans. The tyrosine kinase inhibitors genistein and tyrphostin 47 blocked the phosphorylation of the two endothelial cell proteins and significantly reduced endocytosis of C. albicans. Therefore, C. albicans probably induces its own endocytosis by stimulating the tyrosine phosphorylation of two endothelial cell proteins.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

A signal peptide secretion screen in<Emphasis Type="Italic"> Fucus distichus</Emphasis> embryos reveals expression of glucanase,EGF domain-containing,and LRR receptor kinase-like polypeptides during asymmetric cell growth

Zygotes of the brown alga Fucus distichus (L.) Powell develop polarity prior to the first embryonic cell division and retain a pattern of asymmetric growth during early embryogenesis. In order to identify F. distichus polypeptides secreted during asymmetric cell growth, we used a functional assay in Saccharomyces cerevisiae to screen a cDNA library generated from asymmetrically growing Fucus embryos for sequences encoding polypeptides that function as signal peptides for secretion. We isolated and sequenced 222 plasmids containing Fucus cDNAs encoding signal peptide activity. The cDNA inserts from these plasmids were translated in silico into 244 potential polypeptide sequences, 169 of which are predicted to contain signal peptides. BlastP analysis of the Fucus sequences revealed similarity between many Fucus proteins and cell surface proteins that function in development in other eukaryotes, including epidermal growth factor (EGF)-like repeat-containing proteins, plant leucine-rich repeat (LRR)-receptor kinases, and algal -1, 3-exoglucanase. However, most of the isolated Fucus polypeptides lack similarity to known proteins. The isolation of cDNAs encoding secreted Fucus proteins provides an important step toward characterizing cell surface proteins important for asymmetric organization and growth in fucoid embryos.Abbreviations ECM extracellular matrix - EGF epidermal growth factor - ER endoplasmic reticulum - LRR leucine-rich repeat - SST signal sequence trap - WAK wall-associated kinase     

Genetic analysis of peptidoglycan biosynthesis in mycobacteria: characterization of a ddlA mutant of Mycobacterium smegmatis

A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation in ddlA, the gene encoding D-alanine:D-alanine ligase. Enzymatic assays using recombinant proteins and D-cycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.     

Membrane recycling occurs during asymmetric tip growth and cell plate formation inFucus distichus zygotes

Summary Zygotes of the brown algaFucus distichus undergo a series of intracellular changes resulting in the establishment of a polar growth axis prior to the first embryonic cell division. In order to examine the dynamics of membrane recycling which occur in the zygote during polar growth of the rhizoid, we probed living Fucus zygotes with the vital stain FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylammo)phenyl)hexatrienyl)pyridinium dibromide. In newly fertilized, spherical zygotes, FM4-64 staining is symmetric and predominantly in the perinuclear region which is rich in endoplasmic reticulum, Golgi, and vacuolar membranes. As rhizoid or tip growth is initiated, this population of stained membranes becomes asymmetrically redistributed, concentrating at the rhizoid tip and extending centrally to the perinuclear region. This asymmetric localization is maintained in the zygote throughout polar growth of the rhizoid and during karyokinesis. Subsequently, FM4-64 staining also begins to accumulate in a central location between the daughter nuclei. As cytokinesis proceeds, this region of stain expands laterally from this central location, perpendicular to the plane of polar rhizoid outgrowth. The staining pattern thus delineates the formation of a cell plate, similar spatially to the accumulation of nascent plate membranes of higher plants. Treatment of Fucus zygotes with brefeldin-A inhibits both asymmetric growth of the rhizoid and formation of a new cell plate. These data suggest that inF. distichus FM4-64 is labeling a Golgi-derived membrane fraction that appears to be recycling between the site of tip growth, perinuclear region, and new cell plate.Abbreviations AF after fertilization - ASW artificial seawater - BFA brefeldin A - ER endoplasmic reticulum - FM4-64 N-(3-triethylam-moniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide