Structural and evolutionary analysis of an orangutan foamy virus

The full-length proviral genome of a foamy virus infecting a Bornean orangutan was amplified, and its sequence was analyzed. Although the genome showed a clear resemblance to other published foamy virus genomes from apes and monkeys, phylogenetic analysis revealed that simian foamy virus SFVora was evolutionarily equidistant from foamy viruses from other hominoids and from those from Old World monkeys. This finding suggests an independent evolution within its host over a long period of time.     

Influence of different combinations of bacteria and yeasts in voice prosthesis biofilms on air flow resistance

Laryngectomized patients use silicone rubber voice prostheses to rehabilitate their voice. However, biofilm formation limits the lifetime of voice prostheses. The presence of particular combinations of bacterial and yeast strains in voice prosthesis biofilms has been suggested to be crucial for causing valve failure. In order to identify combinations of bacterial and yeast strains causative to failure of voice prostheses, the effects of various combinations of bacterial and yeast strains on air flow resistances of Groningen button voice prostheses were determined. Biofilms were grown on Groningen button voice prostheses by inoculating so-called artificial throats with various combinations of clinically relevant bacterial and yeast strains. After 3 days, all throats were perfused three times daily with 250 ml phosphate buffered saline and at the end of each day the artificial throats were filled with growth medium for half an hour. After 7 days, the air flow resistances of the prostheses were measured. These air flow resistances were expressed relative to the air flow resistances of the same prostheses prior to biofilm formation. This study shows that biofilms causing strong increases in air flow resistance (26 to 28 cm water.s/l) comprised combinations of microorganisms, involving Candida tropicalis, Staphylococcus aureus and Rothia dentocariosa. This revised version was published online in June 2006 with corrections to the Cover Date.     

Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood

A combination of techniques was used to localise the O-acetyl substituents in xylo-oligosaccharides, which are present in hydrolysates of hydrothermally treated Eucalyptus wood. Reversed-phase (RP)-high performance liquid chromatography (HPLC) coupled on-line to both a mass spectrometer and an evaporating light scattering (ELS) detector provided data about the order of elution of the various O-acetylated oligomers. The retention of the oligomers on the column depended on the number and position of the O-acetyl substituents within the xylo-oligosaccharides. One dimensional (1D)- and two dimensional (2D)-(1)H NMR spectroscopy was used to study the structural features of several xylotetramers separated by RP-HPLC, each having one O-acetyl substituent. O-Acetyl migration was proven to have occurred in these xylo-oligosaccharides. Mainly O-acetyl migration within the same xylosyl residue was observed. RP-HPLC-NMR was performed in order to study the structural features of the acetylated oligomers 'on-line' avoiding O-acetyl migration. Finally, the precise location of the 2-O- or 3-O-acetyl substituent in 6 xylotetramers and 4 xylotrimers separated by RP-HPLC was determined.     

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins

The coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen.     

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

The cell membrane and the struggle for life of lactic acid bacteria

The major life-threatening event for lactic acid bacteria (LAB) in their natural environment is the depletion of their energy sources and LAB can survive such conditions only for a short period of time. During periods of starvation LAB can exploit optimally the potential energy sources in their environment usually by applying proton motive force generating membrane transport systems. These systems include in addition to the proton translocating F_oF₁-ATPase: a respiratory chain when hemin is present in the medium, electrogenic solute uptake and excretion systems, electrogenic lactate/proton symport and precursor/ product exchange systems. Most of these metabolic energy-generating systems offer as additional bonus the prevention of a lethal decrease of the internal and external pH. LAB have limited biosynthetic capacities and rely heavily on the presence of essential components such as sources of amino acids in their environment. The uptake of amino acids requires a major fraction of the available metabolic energy of LAB. The metabolic energy cost of amino acid uptake can be reduced drastically by accumulating oligopeptides instead of the individual amino acids and by proton motive force-generating efflux of excessively accumulated amino acids. Other life-threatening conditions that LAB encounter in their environment are rapid changes in the osmolality and the exposure to cytotoxic compounds, including antibiotics. LAB respond to osmotic upshock or downshock by accumulating or releasing rapidly osmolytes such as glycine-betaine. The life-threatening presence of cytotoxic compounds, including antibiotics, is effectively counteracted by powerful drug extruding multidrug resistance systems. The number and variety of defense mechanisms in LAB is surprisingly high. Most defense mechanisms operate in the cytoplasmic membrane to control the internal environment and the energetic status of LAB. Annotation of the functions of the genes in the genomes of LAB will undoubtely reveal additional defense mechanisms.     

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4oC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.     

Multiple mechanisms compensate to enhance tumor-protective CD8(+) T cell response in the long-term despite poor CD8(+) T cell priming initially: comparison between an acute versus a chronic intracellular bacterium expressing a model antigen

We evaluated CD8(+) T cell responses against the dominant CTL epitope, OVA(257-264), expressed by an acute (Listeria monocytogenes (LM) OVA) vs a chronic pathogen (Mycobacterium bovis bacillus Calmette-Guérin (BCG) OVA) to reveal the influence on CD8(+) T cell memory and consequent protection against a challenge with OVA-expressing tumor cells. Infection with lower doses of both pathogens resulted in stronger bacterial growth but weaker T cell memory indicating that memory correlates with pathogen dose but not with bacterial expansion. The CD8(+) T cell response induced by LM-OVA was helper T cell-independent and was characterized by a rapid effector response followed by a rapid, but massive, attrition. In contrast, BCG-OVA induced a delayed and weak response that was compensated for by a longer effector phase and reduced attrition. This response was partly dependent on CD4(+) T cells. CD8(+) T cell response induced by BCG-OVA, but not LM-OVA, was highly dependent on pathogen persistence to compensate for the weak initial CD8(+) T cell priming. Despite a stronger initial T cell response with LM-OVA, BCG-OVA provided more effective tumor (B16OVA) control at both local and distal sites due to the induction of a persistently activated acquired, and a more potent innate, immunity.     

Ca(2+) -independent vesicular catecholamine release in PC12 cells by nanomolar concentrations of Pb(2+)

Effects of Pb(2+) on vesicular catecholamine release in intact and ionomycin-permeabilized PC12 cells were investigated using carbon fibre microelectrode amperometry. Changes in intracellular Pb(2+) and Ca(2+) were measured from indo-1 fluorescence by confocal laser scanning microscopy. Depolarization of intact cells and superfusion of permeabilized cells with saline containing > or = 100 microm Ca(2+) rapidly evokes quantal catecholamine release. Superfusion with up to 10 microm Pb(2+) -containing saline evokes release of similar catecholamine quanta after a concentration-dependent delay. Thresholds to induce exocytosis within 30 min of exposure are between 1 and 10 microm Pb(2+) in intact cells and between 10 and 30 nm Pb(2+) in permeabilized cells. Additional inhibition of exocytosis occurs in permeabilized cells exposed to 10 microm Pb(2+). Using membrane-impermeable and -permeable chelators it is demonstrated that intracellular Ca(2+) is not required for Pb(2+) -induced exocytosis. In indo- 1-loaded cells Pb(2+) reduces the fluorescence intensity after a concentration-dependent delay, whereas the fluorescence ratio, indicating intracellular Ca(2+) concentration, remains unchanged. The delay to detect an increase in free intracellular Pb(2+) (> or = 30 nm) is much longer than the delay to Pb(2+) -induced exocytosis, indicating that cytoplasmic components buffer Pb(2+) with high affinity. It is concluded that Pb(2+) acts as a high-affinity substitute for Ca(2+) to trigger essential steps leading to vesicular catecholamine release, which occurs when only approximately 20% of the intracellular high-affinity binding capacity ( approximately 2 attomol/cell) is saturated with Pb(2+).