Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates

The Candida albicans ALS (agglutinin-like sequence) gene family encodes eight cell-surface glycoproteins, some of which function in adhesion to host surfaces. ALS genes have a central tandem repeat-encoding domain comprised entirely of head-to-tail copies of a conserved 108-bp sequence. The number of copies of the tandemly repeated sequence varies between C. albicans strains and often between alleles within the same strain. Because ALS alleles can encode different-sized proteins that may have different functional characteristics, defining the range of allelic variability is important. Genomic DNA from C. albicans strains representing the major genetic clades was PCR amplified to determine the number of tandemly repeated sequence copies within the ALS5 and ALS6 central domain. ALS5 alleles had 2-10 tandem repeat sequence copies (mean=4.82 copies) while ALS6 alleles had 2-8 copies (mean=4.00 copies). Despite this variability, tandem repeat copy number was stable in C. albicans strains passaged for 3000 generations. Prevalent alleles and allelic distributions varied among the clades for ALS5 and ALS6. Overall, ALS6 exhibited less variability than ALS5. ALS5 deletions can occur naturally in C. albicans via direct repeats flanking the ALS5 locus. Deletion of both ALS5 alleles was associated particularly with clades III and SA. ALS5 exhibited allelic polymorphisms in the coding region 5' of the tandem repeats; some alleles resembled ALS1, suggesting recombination between these contiguous loci. Natural deletion of ALS5 and the sequence variation within its coding region suggest relaxed selective pressure on this locus, and that Als5p function may be dispensable in C. albicans or redundant within the Als family.     

Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.     

Long-term evaluation of a selective retrograde coronary venous perfusion model in pigs (Sus scrofa domestica)

The lack of suitable target vessels remains a challenge for aortocoronary bypass grafting in end-stage coronary heart disease. This study aimed to investigate the arterialization of cardiac veins as an alternative myocardial revascularization strategy in an experimental long-term model in pigs. Selective retrograde perfusion of a coronary vein (aorta to coronary vein bypass, retrobypass) before ligation of the ramus interventricularis paraconalis (equivalent to the left anterior descending artery in humans) was performed in 20 German Landrace pigs (Sus scrofa domestica). Retroperfusion of the left anterior descending vein was performed in 10 pigs (RP+) but not in the other 10 (RP-), and the vena cordis magna was ligated (L+) in 5 pigs in each of these groups but left open (L-) in the remaining animals. Hemodynamic performance (for example, cardiac output) was significantly better in the group that underwent selective retroperfusion with proximal ligation of vena cordis magna (RP+L+; 4.1 L/min) compared with the other groups (RP+L-, 2.5 L/min; RP-L+, 2.2 L/min; RP-L-, 1.9 L/min). Long-term survival was significantly better in RP+L+ pigs (112±16 d) than in all other groups. Histologic follow-up studies showed significantly less necrosis in the RP+L+ group compared with all other groups. Venous retroperfusion is an effective technique to achieve long-term survival after acute occlusion of the left anterior descending artery in a pig model. In this model, proximal ligation of vena cordis magna is essential.     

Vegetative characteristics of three low-lying Florida coastal rivers in relation to flow, light, salinity and nutrients

The Chassahowitzka, Homosassa and Crystal rivers along the central Gulf coast of Florida were studied from 1998 to 2000 to identify factors controlling the abundance and distribution of submersed aquatic vegetation (SAV). Each of these three low-lying coastal rivers are spring-fed and exhibit low to moderate absolute flow rates (flows in either direction because of tidal influences, 0.06–0.46ms^–1) with only 14 of the stations sampled for SAV having flow rates in excess of 0.25ms^–1. At those stations where flow rates exceeded 0.25ms–1, the substrate was generally comprised of exposed limestone outcroppings and did not provide a favorable habitat for either submersed macrophytes or macroalgae. The remaining sampling stations, where flow rates were less than 0.25ms–1, had suitable substrates (e.g. mud, mud/sand, and sand) for the colonization and subsequent growth of SAV. Light availability and salinity were determined to be major factors affecting the distribution and abundance of SAV. Sampling stations, where the percent of incident light at the surface reaching the substrate was less than 10, had little or no SAV biomass. Low SAV biomass was also linked to sites where annual average salinities exceeded 3.5. Nutrient loads and nutrient concentrations accounted for little variance in SAV biomass after accounting for flow and related substrate type, light and salinity. These latter factors control the distribution and abundance of SAV in these three Florida coastal rivers.     

Rapid self-assembly of alpha-synuclein observed by in situ atomic force microscopy

Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.     

A comparison between aquatic birds of lakes and coastal rivers in Florida

Aquatic birds were counted on five Gulf coast Florida rivers to determine if these river systems supported densities, biomass and species richness similar to those found on Florida lakes. Forty-two species were identified and for the species that were found on both Florida streams and lakes similar densities and biomass were encountered. As with Florida lakes, stream bird abundance and species richness were higher in winter months than in summer months, a consequence of migratory bird populations. Total bird abundance, biomass per unit of phosphorus, and species richness per unit of area were similar to data collected on Florida lakes. Thus, Florida rivers are capable of supplying sufficient resources to maintain bird densities, biomass and species richness values similar to lakes of equal size and nutrient concentrations and are therefore important habitats for aquatic bird populations. An examination of individual habitat characteristics indicates that water depth was inversely correlated and submersed aquatic vegetation was positively correlated with bird density, biomass and species richness within the river systems. While both habitat characteristics are important they are also inversely related making it difficult to separate the individual significance of each characteristic.     

Histidine button engineered into cardiac troponin I protects the ischemic and failing heart

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.     

Spontaneous Autoimmunity in the Absence of IL-2 Is Driven by Uncontrolled Dendritic Cells

BALB/c IL-2-deficient (IL-2-KO) mice develop systemic autoimmunity, dying within 3 to 5 wk from complications of autoimmune hemolytic anemia. Disease in these mice is Th1 mediated, and IFN-γ production is required for early autoimmunity. In this study, we show that dendritic cells (DCs) are required for optimal IFN-γ production by T cells in the IL-2-KO mouse. Disease is marked by DC accumulation, activation, and elevated production of Th1-inducing cytokines. IL-2-KO DCs induce heightened proliferation and cytokine production by naive T cells compared with wild-type DCs. The depletion of either conventional or plasmacytoid DCs significantly prolongs the survival of IL-2-KO mice, demonstrating that DCs contribute to the progression of autoimmunity. Elimination of Th1-inducing cytokine signals (type 1 IFN and IL-12) reduces RBC-specific Ab production and augments survival, indicating that cytokines derived from both plasmacytoid DCs and conventional DCs contribute to disease severity. DC activation likely precedes T cell activation because DCs are functionally activated even in an environment lacking overt T cell activation. These data indicate that both conventional and plasmacytoid DCs are critical regulators in the development of this systemic Ab-mediated autoimmune disease, in large part through the production of IL-12 and type 1 IFNs.     

Selective inhibition of leukotriene receptor BLT-2 reduces vascular oxidative stress and improves endothelial function in ApoE?/? mice

Atherosclerosis is a chronic inflammatory disease and represents the main cause of death in the industrialized world. Metabolites of the arachidonic acid derived from the 5-lipoxygenase pathway are known as leukotrienes that mediate various inflammatory processes during atherogenesis. Leukotriene B4 elicits the overexpression of several proinflammatory proteins, promotes chemotaxis and foam cell formation via BLT receptors. Currently, little is known about the implications of the BLT2 receptor in atherogenesis. Here, we tested whether selective inhibition of this receptor influences the progression of atherosclerosis in mice. Apolipoprotein-E deficient mice were fed a high-fat, cholesterol-rich diet to create atherosclerotic conditions (each group n?=?9). Simultaneously, mice received the pharmacologic BLT2 inhibition (Ly) by intraperitoneal injection every second day 5?mg/kg bw or vehicle. After 8 weeks, mice were killed and experiments were performed. Vascular superoxide release was diminished in mice treated with Ly compared with the control group (68?±?15 vs 131?±?20 RLU, P?=?0.01), as measured by L012 assay. Next, endothelial function was assessed by organ chamber experiments. Endothelial-dependent relaxation was improved in mice treated with the BLT2 receptor antagonist. To determine whether selective inhibition of the BLT2 receptor affects the atherosclerotic plaque growth, immunohistochemical stainings of the aortic root were performed. Oil red O staining revealed no significant differences between both groups (36?±?3% vs 38?±?3%). Monocyte infiltration into the vessel wall was analyzed using Moma-2 staining. No significant differences were observed between both groups (31?±?3% vs 34?±?2%). Selective inhibition of the BLT2 receptor in mice reduces the release of vascular reactive oxygen species and improves endothelial function in mice. Further experiments are necessary in order to obtain tissue-specific and mechanistical insights.     

A monoclonal antibody specific for Candida albicans Als4 demonstrates overlapping localization of Als family proteins on the fungal cell surface and highlights differences between Als localization in vitro and in vivo

The Candida albicans agglutinin-like sequence (ALS) family encodes large cell surface glycoproteins that function in adhesion of the fungus to host and abiotic surfaces. Monoclonal antibodies (mAbs) specific for each Als protein were developed to study Als localization on the C. albicans surface. An anti-Als4 mAb demonstrated that Als4 covers the surface of yeast cells, with a greater abundance of Als4 on cells grown at 30 °C compared to 37 °C. On germ tubes, Als4 is localized in a restricted area proximal to the mother yeast. Immunolabeling with several anti-Als mAbs showed overlapping localization of Als1 and Als4 on yeast cells and Als1, Als3 and Als4 on germ tubes. Overlapping localization of Als proteins was also observed on yeast and hyphae recovered from mouse models of disseminated and oral candidiasis. Differences between Als localization in vivo and in vitro suggested changes in regulation of Als production in the host compared to the culture flask. Characterization with the anti-Als mAbs reveals the simultaneous presence and differences in relative abundance of Als proteins, creating an accurate image of Als representation and localization that can be used to guide conclusions regarding individual and collective Als protein function.