Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Nuclear DNA identification of migrating bull trout captured at the Puget Sound Energy diversion dam on the White River,Washington State

Bull trout (Salvelinus confluentus) is a char listed as threatened under the United States Endangered Species Act throughout its range in the coterminous United States. Substantial morphological similarities between bull trout and Dolly Varden (S. malma) make field identification difficult. This has resulted in an incomplete understanding of their distribution and abundance in Washington State where these two species occur sympatrically. We used three diagnostic nuclear loci to determine the species of char collected at a trap on the White River in southern Puget Sound (Washington State, USA). Each of the 104 samples revealed the expected bull trout genotype at all three loci. This work presents three principle results: (i) the presence of a migratory bull trout population in southern Puget Sound; (ii) no evidence of migratory Dolly Varden over 3 years; and (iii) no evidence of hybridization was detected. These results also demonstrate how molecular markers can provide information essential to the conservation and management of these species.     

Regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded RNA and viral infection

In this study the regulation of macrophage expression of cyclooxygenase-2 (COX-2) in response to dsRNA and virus infection was examined. Treatment of RAW 264.7 macrophages with dsRNA results in COX-2 mRNA accumulation and protein expression and the production of PGE(2). Similar to dsRNA, encephalomyocarditis virus (EMCV) infection of RAW 264.7 cells stimulates COX-2 expression and PGE(2) accumulation. The dsRNA-dependent protein kinase (PKR), which has been shown to participate in the regulation of gene expression in response to dsRNA and virus infection, does not appear to participate in the regulation of COX-2 expression by macrophages. Expression of dominant negative mutants of PKR in RAW 264.7 cells fails to attenuate dsRNA- and EMCV-induced COX-2 expression or PGE(2) production. Furthermore, dsRNA and EMCV stimulate COX-2 expression and PGE(2) accumulation to similar levels in macrophages isolated from wild-type and PKR-deficient mice. Recently, a novel PKR-independent role for the calcium-independent phospholipase A(2) (iPLA(2)) in the regulation of inducible NO synthase expression by macrophages in response to virus infection has been identified. The selective iPLA(2) suicide substrate inhibitor bromoenol lactone prevents dsRNA- and EMCV-stimulated inducible NO synthase expression; however, bromoenol lactone does not attenuate dsRNA- or EMCV-induced COX-2 expression by macrophages. In contrast, inhibition of NF-kappaB activation prevents dsRNA-stimulated COX-2 expression and PGE(2) accumulation by macrophages. These findings indicate that virus infection and treatment with dsRNA stimulate COX-2 expression by a mechanism that requires the activation of NF-kappaB and that is independent of PKR or iPLA(2) activation.     

A catalytically inactive form of protein kinase C-associated kinase/receptor interacting protein 4, a protein kinase C beta-associated kinase that mediates NF-kappa B activation,interferes with early B cell development

Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) beta-associated kinase that links PKC to NF-kappaB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCbeta, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.     

TCR signal transduction in antigen-specific memory CD8 T cells

Memory T cells are more responsive to Ag than naive cells. To determine whether memory T cells also have more efficient TCR signaling, we compared naive, effector, and memory CD8 T cells of the same antigenic specificity. Surprisingly, initial CD3 signaling events are indistinguishable. However, memory T cells have more extensive lipid rafts with higher phosphoprotein content before TCR engagement. Upon activation in vivo, they more efficiently induce phosphorylation of-LAT (linker for activation of T cells), ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38. Thus, memory CD8 T cells do not increase their TCR sensitivity, but are better poised to augment downstream signals. We propose that this regulatory mechanism might increase signal transduction in memory T cells, while limiting TCR cross-reactivity and autoimmunity.     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Subfamilies of CR1 non-LTR retrotransposons have different 5'UTR sequences but are otherwise conserved

CR1 elements and CR1-related (CR1-like) elements are a novel family of non-LTR retrotransposons that are found in all vertebrates (reptilia, amphibia, fish, and mammals), whereas more distantly related elements are found in several invertebrate species. CR1 elements have several features that distinguish them from other non-LTR retrotransposons. Most notably, their 3' termini lack a polyadenylic acid (poly A) tail and instead contain 2-4 copies of a unique 8 bp repeat. CR1 elements are present at approximately 100,000 copies in the chicken genome. The vast majority of these elements are severely 5' truncated and mutated; however, six subfamilies (CR1-A through CR1-F) are resolved by sequence comparisons. One of these subfamilies (i.e. CR1-B) previously was analyzed in detail. In the present study, we identified several full-length elements from the CR1-F subfamily. Although regions within the open reading frames and 3' untranslated regions of CR1-F and CR1-B elements are well conserved, their respective 5' untranslated regions are unrelated. Thus, our results suggest that new CR1 subfamilies form when elements with intact open reading frames acquire new 5' UTRs, which could, in principle, function as promoters.     

Extremely low levels of genetic polymorphism in endosymbionts (Buchnera) of aphids (Pemphigus)

Molecular evolutionary studies have suggested that vertically transmitted endosymbionts are subject to accumulation of deleterious mutations through genetic drift. Predictions of this hypothesis for patterns of intraspecific polymorphism were borne out in the single relevant study available, on the symbiont Buchnera aphidicola of Uroleucon ambrosiae. In order to examine the generality of this result, we surveyed DNA sequence variation in Buchnera of the distantly related aphid, Pemphigus obesinymphae. In contrast to Uroleucon species, Pemphigus species have complex life cycles with few dispersal stages. Despite these differences, P. obesinymphae showed patterns of variation at two Buchnera loci and one mitochondrial locus that were remarkably similar to those reported previously for Buchnera of U. ambrosiae. In the western US, Buchnera was nearly monomorphic, and in the eastern US, synonymous divergence ranged from 0.08 to 0.16%. Most polymorphisms involved rare alleles, consistent with a recent range of ancestral polymorphism, probably due to demographic fluctuations in aphid populations. These results support the generality of small effective population size in Buchnera and their aphid hosts.