Enhanced biological phosphorus removal in a sequencing batch reactor using propionate as the sole carbon source

An enhanced biological phosphorus removal (EBPR) system was developed in a sequencing batch reactor (SBR) using propionate as the sole carbon source. The microbial community was followed using fluorescence in situ hybridization (FISH) techniques and Candidatus 'Accumulibacter phosphatis' were quantified from the start up of the reactor until steady state. A series of SBR cycle studies was performed when 55% of the SBR biomass was Accumulibacter, a confirmed polyphosphate accumulating organism (PAO) and when Candidatus 'Competibacter phosphatis,' a confirmed glycogen-accumulating organism (GAO), was essentially undetectable. These experiments evaluated two different carbon sources (propionate and acetate), and in every case, two different P-release rates were detected. The highest rate took place while there was volatile fatty acid (VFA) in the mixed liquor, and after the VFA was depleted a second P-release rate was observed. This second rate was very similar to the one detected in experiments performed without added VFA. A kinetic and stoichiometric model developed as a modification of Activated Sludge Model 2 (ASM2) including glycogen economy, was fitted to the experimental profiles. The validation and calibration of this model was carried out with the cycle study experiments performed using both VFAs. The effect of pH from 6.5 to 8.0 on anaerobic P-release and VFA-uptake and aerobic P-uptake was also studied using propionate. The optimal overall working pH was around 7.5. This is the first study of the microbial community involved in EBPR developed with propionate as a sole carbon source along with detailed process performance investigations of the propionate-utilizing PAOs. (C) 2004 Wiley Periodicals, Inc.

Discovery, structure and biological activities of the cyclotides

The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaccae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.

Structure-function studies of the plant cyclotides: The role of a circular protein backbone

The traditional idea of proteins as linear chains of amino acids is being challenged with the discovery of miniproteins that contain a circular backbone. The cyclotide family is the largest group of circular proteins and is characterized by an amide-circularized protein backbone and six conserved cysteine residues. These conserved cysteines are paired to form a knotted network of disulfide bonds. The combination of the circular backbone and a cystine knot, known as the cyclic cystine knot (CCK) motif, confers exceptional stability upon the cyclotides. This review discusses the role of the circular backbone based on studies of both the oxidative folding of kalata B1, the prototypical cyclotide, and a comparison of the structure and activity of kalata B1 and its acyclic permutants.

Studies on the cryopreservation of common wombat (Vombatus ursinus) spermatozoa

In an attempt to develop a gamete-recovery protocol for the northern hairy nosed wombat (Lasiorhinus krefftii), spermatozoa were removed from the cauda epididymides of four common wombats (Vombatus ursinus) and cryopreserved following a variety of prefreeze storage conditions. Spermatozoa stored for 72 h at 4 degrees C within the testicle before cryopreservation tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability (% motile P< 0.01; rate of movement P< 0.01; % live P< 0.01) than sperm recovered on the day of post-mortem, stored in a test tube for 72 h at 4 degrees C and then frozen. The effect of post-thaw dilution with Tris citrate fructose (TCF) diluent on the survival of epididymal common wombat spermatozoa was also investigated. Motility (P< 0.05), rate of sperm movement (P< 0.01) and the percentage of live spermatozoa (P< 0.05) were all significantly greater when spermatozoa were thawed and diluted immediately in TCF than when thawed without dilution. The present study also reports, for the first time, a successful pellet method of freezing wombat spermatozoa on dry ice; volumes of 0.25 and 0.5mL resulted in higher post- thaw survival compared with 0.1-mL pellets.

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: Investigations of the cyclotide kalata B1 in biological fluids

A rapid method has been developed for the quantification of the prototypic cyclotide kalata B I in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity (R-2 > 0.99) in the concentration range 0.05-10 mu g/mL with a limit of detection of 0.05 mu g/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological samples without the need for extensive extraction procedures. (c) 2005 Elsevier B.V. All rights reserved.

Lessons from tomographic studies of the mammalian Golgi

Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure -function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space. (c) 2005 Elsevier B.V. All rights reserved.

Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance

In this study we have demonstrated the interactions of kalata B1 and its naturally occurring analogue kalata B6 with five model lipid membranes and have analyzed the binding kinetics using surface plasmon resonance. Two kalata peptides showed a higher affinity for the phosphatidylethanolamine-containing membranes, indicating that the peptides would bind selectively to bacterial membranes. Also we have optimized the procedure for the immobilization of five liposome mixtures and have shown that the procedure provides reproducible levels of immobilized liposomes and could be used to screen the selective binding of putative antimicrobial peptides to model mammalian or microbial phospholipid membranes. (C) 2004 Elsevier Inc. All rights reserved.

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha- D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (K-d = 0.15 muM) than mannose (K-d = 2.3 muM). Exploration of the binding affinities of alpha-D-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.

Pulsed EPR studies of a bacterial sulfite-oxidizing enzyme with pH-invariant hyperfine interactions from exchangeable protons

Variable-frequency pulsed electron paramagnetic resonance studies of the molybdenum(V) center of sulfite dehydrogenase (SDH) clearly show couplings from nearby exchangeable protons that are assigned to a (MoOHn)-O-v group. The hyperfine parameters for these exchangeable protons of SDH are the same at both low and high pH and similar to those for the high-pH forms of sulfite oxidases (SOs) from eukaryotes. The SDH proton parameters are distinctly different from the low-pH forms of chicken and human so.

Interpretation of the temperature dependence of rate constants in biosensor studies

A comparison is made between Arrhenius and transition-state analyses of the temperature dependence of rate constants reported in four published biosensor studies. Although the Eyring transition-state theory seemingly affords a more definitive solution to the problem of characterizing the activation energetics, the analysis is equivocal because of inherent assumptions about reaction mechanism and the magnitude of the transmission coefficient. In view of those uncertainties it is suggested that a preferable course of action entails reversion to the empirical Arrhenius analysis with regard to the energy of activation and a preexponential factor. The former is essentially equivalent to the enthalpy of activation, whereas the magnitude of the latter indicates directly the extent of disparity between the frequency of product formation and the universal frequency factor (temperature multiplied by the ratio of the Boltzmann and Planck constants) and hence the likelihood of a more complicated kinetic mechanism than that encompassed by the Eyring transition-state theory. (C) 2004 Elsevier Inc. All rights reserved.

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis. (c) 2005 Wiley Periodicals, Inc.

NMR studies of exchange between intra- and extracellular glutathione in human erythrocytes

Glutathione is the main source of intracellular antioxidant protection in the human erythrocyte and its redox status has frequently been used as a measure of oxidative stress. Extracellular glutathione has been shown to enhance intracellular reduced glutathione levels in some cell types. However, there are conflicting reports in the literature and it remains unclear as to whether erythrocytes can utilise extracellular glutathione to enhance the intracellular free glutathione pool. We have resolved this issue using a C-13-NMR approach. The novel use of L-gamma-glutamyl-L-cysteinyl-[2-C-13] glycine allowed the intra- and extracellular glutathione pools to be distinguished unequivocally, enabling the direct and non-invasive observation over time of the glutathione redox status in both compartments. The intracellular glutathione redox status was measured using H-1 spin-echo NMR, while C-13[H-1-decoupled] NMR experiments were used to measure the extracellular status. Extracellular glutathione was not oxidised in the incubations, and did not affect the intracellular glutathione redox status. Extracellular glutathione also did not affect erythrocyte glucose metabolism, as measured from the lactate-to-pyruvate ratio. The results reported here refute the previously attractive hypothesis that, in glucose-starved erythrocytes, extracellular GSH can increase intracellular GSH concentrations by releasing bound glutathione from mixed disulfides with membrane proteins.

Committee report: Guidelines for human startle eyeblink electromyographic studies

The human startle response is a sensitive, noninvasive measure of central nervous system activity that is Currently used in a wide variety of research and clinical settings. In this article, we raise methodological issues and present recommendations for optimal methods of startle blink electromyographic (EMG) response elicitation, recording, quantification, and reporting. It is hoped that this report Will foster more methodological validity and reliability in research using the startle response, Lis well Lis increase the detail with which relevant methodology is reported in publications using this measure.

Applicability of experience from laboratory reactors with biological phosphorus removal in full-scale plants

Enhanced biological phosphorus removal (EBPR) has been used at many wastewater treatment plants all over the world for many years. In this study a full-scale sludge with good EBPR was tested with P-release batch tests and combined FISH/MAR (fluorescence in situ hybridisation and microautoradiography). Proposed models of PAOs and GAOs (polyphosphate- and glycogen-accumulating organisms) and microbial methods suggested from studies of laboratory reactors were found to be applicable also on sludge from full-scale plants. Dependency of pH and the uptake of both acetate and propionate were studied and used for calculations for verifying the models and results from microbial methods. All rates found from the batch tests with acetate were higher than in the batch tests with propionate, which was explained by the finding that only those parts of the bacterial community that were able to take up acetate anaerobically were able to take up propionate anaerobically.