Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.     

Cardiac glycogen accumulation after dexamethasone is regulated by AMPK

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (Dex)-treated animals, glycogen accumulation was enhanced. We examined the influence of 5'-AMP-activated protein kinase (AMPK) on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. After Dex, cardiac tissue had a limited contribution toward the development of whole body insulin resistance. Measurement of glucose transporter 4 (GLUT4) at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated with increased GLUT4 mRNA. Both total and phosphorylated AMPK increased after Dex. Immunoprecipitation of Akt substrate of 160 kDa (AS160) followed by Western blot analysis demonstrated no change in Akt phosphorylation at Ser(473) and Thr(308) in Dex-treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr(172), which correlated well with AS160 phosphorylation. In Dex-treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas glycogen synthase kinase-3-beta phosphorylation was augmented. Our data suggest that AMPK-mediated glucose entry combined with the activation of glycogen synthase and a reduction in glucose oxidation (Qi et al., Diabetes 53: 1790-1797, 2004) act together to promote glycogen storage. Should these effects persist chronically in the heart, they may explain the increased morbidity and mortality observed with long-term excesses in endogenous or exogenous glucocorticoids.     

Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339-345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd(2+) (current with 100 microM Cd(2+)/control current at -140 mV = 67.6 +/- 2.9%, 109.3 +/- 3.1%, and 103.8 +/- 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at -140 mV (to 43.7 +/- 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: approximately 6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339-345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.     

Selection through male function favors smaller floral display size in the common morning glory Ipomoea purpurea (Convolvulaceae)

In self-compatible, hermaphroditic plants, display size-the number of flowers open on a plant at one time-is believed to be influenced by trade-offs between increasing geitonogamous selfing and decreasing per-flower pollen export as display size increases. Experimental results presented here indicate that selection through male function favors smaller display sizes in Ipomoea purpurea. In small arrays, plant display size was manipulated experimentally, and female selfing rate, male outcross success, and total male fitness were estimated using genetic markers and likelihood and regression analyses. As would be expected if larger displays experience greater geitonogamy, selfing rate increased with display size. However, the per-flower amount of pollen exported to other plants decreased with display size. The magnitude of this effect is more than sufficient to offset the increase in selfing rate, resulting in reduced per-flower total male fitness with increasing display size. The low values of inbreeding depression previously reported for this species would enhance this effect.     

Predictable patterns of disruptive selection in stickleback in postglacial lakes

Disruptive selection is often assumed to be relatively rare, because it is dynamically unstable and hence should be transient. However, frequency-dependent interactions such as intraspecific competition may stabilize fitness minima and make disruptive selection more common. Such selection helps explain the maintenance of genetic variation and may even contribute to sympatric speciation. There is thus great interest in determining when and where disruptive selection is most likely. Here, we show that there is a general trend toward weak disruptive selection on trophic morphology in three-spine stickleback (Gasterosteus aculeatus) in 14 lakes on Vancouver Island. Selection is inferred from the observation that, within a lake, fish with intermediate gill raker morphology exhibited slower growth than phenotypically extreme individuals. Such selection has previously been shown to arise from intraspecific competition for alternate resources. However, not all environments are equally conducive to disruptive selection, which was strongest in intermediate-sized lakes where both littoral and pelagic prey are roughly balanced. Also, consistent with theory, we find that sexual dimorphism in trophic traits tends to mitigate disruptive selection. These results suggest that it may be possible to anticipate the kinds of environments and populations most likely to experience disruptive selection.     

Localized decrease of beta-catenin contributes to the differentiation of human embryonic stem cells

Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies.     

Ecological genomics of mutualism decline in nitrogen-fixing bacteria

Anthropogenic changes can influence mutualism evolution; however, the genomic regions underpinning mutualism that are most affected by environmental change are generally unknown, even in well-studied model mutualisms like the interaction between legumes and their nitrogen (N)-fixing rhizobia. Such genomic information can shed light on the agents and targets of selection maintaining cooperation in nature. We recently demonstrated that N-fertilization has caused an evolutionary decline in mutualistic partner quality in the rhizobia that form symbiosis with clover. Here, population genomic analyses of N-fertilized versus control rhizobium populations indicate that evolutionary differentiation at a key symbiosis gene region on the symbiotic plasmid (pSym) contributes to partner quality decline. Moreover, patterns of genetic variation at selected loci were consistent with recent positive selection within N-fertilized environments, suggesting that N-rich environments might select for less beneficial rhizobia. By studying the molecular population genomics of a natural bacterial population within a long-term ecological field experiment, we find that: (i) the N environment is indeed a potent selective force mediating mutualism evolution in this symbiosis, (ii) natural variation in rhizobium partner quality is mediated in part by key symbiosis genes on the symbiotic plasmid, and (iii) differentiation at selected genes occurred in the context of otherwise recombining genomes, resembling eukaryotic models of adaptation.     

Successive evaluation of multiple parameters in individual cells: the SEMPIC photometric system.

An automated microphotometric system has been designed for the successive assessment of a maximum number of parameters from individual cells. The hardware and software elements are described as well as the various photometric functions that can be performed. The aim is to obtain a multiparameter analysis of morphology, cytochemistry, cell surface characteristics, and metabolic activity including cell cycle characteristics for any set of cells selected from a sample. The combination of DNA synthesis rate determination in a really quantitative approach with the analysis of features of nuclear chromatin texture opens up a new field for associating morphology with functional properties. Examples of application are presented to demonstrate the applicability of the system in a heterogeneous cell population of a very limited sample size. For this purpose, human peripheral blood cells grown in diffusion chambers in preirradiated mice were investigated. It is shown that, depending on their functions, different cell types may have quite different nuclear-to-cell area ratios. Further, a crude morphological parameter, such as the degree of basophilia of Giemsa-stained cells, may be associated with the rate of DNA synthesis, thus bearing information on the replicative activity of a cell. Cell surface properties related to the leukemia-associated cALL antigen are investigated in a human-derived cell line and correlated with cell-cycle characteristics. It is concluded that different antigenic sites rather than cell-cycle dependent differences of antigen density account for the outcome of a bimodal distribution.