Diffusional properties of methanogenic granular sludge: 1H NMR characterization

The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22 degrees C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H(2)O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H(2)O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55 degrees C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10 degrees C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90 degrees C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl(2) did not affect the H(2)O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.     

Outcome study: periareolar mammaplasty patients' perspective

Residual scars on occasion compromise patient satisfaction with breast reduction procedures. Periareolar breast reduction was proposed to minimize the scarring produced by the operation. This technique was criticized predominantly for producing a breast with low projection, for recurrence of ptosis after surgery, and for widening of the periareolar scar. The purpose of this study was to evaluate patient satisfaction from this technique. In the first branch of the study, 11 patients who had periareolar or circumareolar breast reduction were compared with 13 patients who had vertical scar reduction mammaplasty. The groups were matched for patient's age, size of reduction, and follow-up period. The patients were contacted by phone and requested to comment on four aspects of their operation: breast size, symmetry, quality of scars, and appearance and position of the nipple-areola complex. The replies were converted to numerical values and analyzed statistically. A panel of three women, prospective patients for breast reduction, were presented with standardized five-view before-and-after photographs and requested to score the results using the same criteria. Their responses were analyzed in the same manner. Both phases of the study produced similar results. Breast reduction done through a periareolar scar scored higher than a vertical scar technique. The operation did not differ with respect to shape, symmetry, or nipple-areola shape but did with respect to dissatisfaction with scars, which affected the overall result. This experimental model of assessing results of cosmetic operations is proposed as a tool to assess the patient's rather than the surgeon's perspective of a surgical technique, and it could find application in assessing other cosmetic operations.     

Long-term adaptation of methanol-fed thermophilic (55 °C) sulfate-reducing reactors to NaCl

A laboratory-scale upflow anaerobic sludge bed (UASB) reactor was operated during 273 days at increasing NaCl concentrations (0.5–12.5 g NaCl l^–1) to assess whether the stepwise addition of the salt NaCl results in the acclimation of that sludge. The 6.5-l thermophilic (55 °C), sulfidogenic [a chemical oxygen demand (COD) to SO₄^2– ratio of 0.5] UASB reactor operated at an organic loading rate of 5 g COD l^–1 day^–1, a hydraulic retention time of 10 h and was fed with methanol as the sole electron donor. The results show that the adaptation of the thermophilic, sulfidogenic methanol-degrading biomass to a high osmolarity environment is unlikely to occur. Sulfide was the main mineralization product from methanol degradation, regardless of the NaCl concentration added to the influent. However, sulfide production in the reactor steadily decreased after the addition of 7.5 g NaCl l^–1, whereas acetate production was stimulated at that influent NaCl concentration. Batch tests performed with sludge harvested from the UASB reactor when operating at different influent salinities confirmed that acetate is the main metabolic product at NaCl concentrations higher than 12.5 g l^–1. The apparent order of NaCl toxicity towards the different trophic groups was found to be: sulfate-reducing bacteria > methane-producing archaea > acetogenic bacteria.     

Biological reduction of nitric oxide in aqueous Fe(II)EDTA solutions

The reduction of nitric oxide (NO) in aqueous solutions of Fe(II)EDTA is one of the core processes in BioDeNOx, an integrated physicochemical and biological technique for NO(x)() removal from industrial flue gases. NO reduction in aqueous solutions of Fe(II)EDTA (20-25 mM, pH 7.2 +/- 0.2) was investigated in batch experiments at 55 degrees C. Reduction of NO to N(2) was found to be biologically catalyzed with nitrous oxide (N(2)O) as an intermediate. Various sludges from full-scale denitrifying and anaerobic reactors were capable to catalyze NO reduction under thermophilic conditions. The NO reduction rate was not affected by the presence of ethanol or acetate. EDTA-chelated Fe(II) was found to be a suitable electron donor for the biological reduction of nitric oxide to N(2), with the concomitant formation of Fe(III)EDTA. In the presence of ethanol, EDTA-chelated Fe(III) was reduced to Fe(II)EDTA. This study strongly indicates that redox cycling of FeEDTA plays an important role in the biological denitrification process within the BioDeNOx concept.     

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.     

ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro

Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg(2+), whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.     

Structure of apo-phosphatidylinositol transfer protein alpha provides insight into membrane association

Phosphatidylinositol transfer protein alpha (PITP alpha) is a ubiquitous and highly conserved protein in multicellular eukaryotes that catalyzes the exchange of phospholipids between membranes in vitro and participates in cellular phospholipid metabolism, signal transduction and vesicular trafficking in vivo. Here we report the three-dimensional crystal structure of a phospholipid-free mouse PITP alpha at 2.0 A resolution. The structure reveals an open conformation characterized by a channel running through the protein. The channel is created by opening the phospholipid-binding cavity on one side by displacement of the C-terminal region and a hydrophobic lipid exchange loop, and on the other side by flattening of the central beta-sheet. The relaxed conformation is stabilized at the proposed membrane association site by hydrophobic interactions with a crystallographically related molecule, creating an intimate dimer. The observed open conformer is consistent with a membrane-bound state of PITP and suggests a mechanism for membrane anchoring and the presentation of phosphatidylinositol to kinases and phospholipases after its extraction from the membrane. Coordinates have been deposited in the Protein Data Bank (accession No. 1KCM).     

Enrichment of homoacetogens converting H2/CO2 into acids and ethanol and simultaneous methane production

An anaerobic granular sludge was enriched to utilize H₂/CO₂ in a continuous gas-fed up-flow anaerobic sludge reactor by applying operating conditions expected to produce acetic acid, butyric acid, and ethanol. Three stages of fermentation were found: Stage I with acetic acid accumulation with the highest concentration of 35 mM along with a pH decrease from initial 6 to 4.5. In Stage II, H₂/CO₂ was replaced by 100% H₂ to induce solventogenesis, whereas butyric acid was produced with the highest concentration of 2.5 mM. At stage III with 10 µM tungsten (W) addition, iso-valeric acid, valeric acid, and caproic acid were produced at pH 4.5–5.0. In the batch tests inoculated with the enriched sludge taken from the bioreactor (day 70), however, methane production occurred at pH 6. Exogenous 15 mM acetate addition enhanced both the H₂ and CO₂ consumption rate compared to exogenous 10, 30, and 45 mM acetate by the enriched sludge. Exogenous acetate was failed to be converted to ethanol using H₂ as electron donor by the enriched acetogens.     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

Hyphal and mycelial interactions betweenAgaricus bisporus andScytalidium thermophilum on agar media

The interaction betweenAgaricus bisporus andScytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy.A. bisporus combatively replacesS. thermophilum in culture on agar media. The antagonistic effect ofA. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration ofS. thermophilum protoplasm, resulting in a total loss of viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 mm·d^–1 following contact withS. thermophilum mycelium.