The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.     

Flexible Community Structure Correlates with Stable Community Function in Methanogenic Bioreactor Communities Perturbed by Glucose

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050–4057, 2000).     

Anaerobic Granular Sludge Bioreactor Technology

Anaerobic digestion is a mature wastewater treatment technology, with worldwide application. The predominantly applied bioreactor designs, such as the upflow anaerobic sludge blanket and expanded granular sludge bed, are based on the spontaneous formation of granular sludge. Despite the exploitation of granular reactors at full-scale for more than two decades, the mechanisms of granulation are not completely understood and numerous theories have been put forward to describe the process from a biological, ecological and engineering point of view. New technological opportunities are emerging for anaerobic digestion, aided by an improved understanding of microbiological and environmental factors affecting the formation and activity of anaerobic granular sludge.     

DNA asymmetric strand bias affects the amino acid composition of mitochondrial proteins.

Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Compositional Bias May Affect Both DNA-Based and Protein-Based Phylogenetic Reconstructions

It is now well-established that compositional bias in DNA sequences can adversely affect phylogenetic analysis based on those sequences. Phylogenetic analyses based on protein sequences are generally considered to be more reliable than those derived from the corresponding DNA sequences because it is believed that the use of encoded protein sequences circumvents the problems caused by nucleotide compositional biases in the DNA sequences. There exists, however, a correlation between AT/GC bias at the nucleotide level and content of AT- and GC-rich codons and their corresponding amino acids. Consequently, protein sequences can also be affected secondarily by nucleotide compositional bias. Here, we report that DNA bias not only may affect phylogenetic analysis based on DNA sequences, but also drives a protein bias which may affect analyses based on protein sequences. We present a striking example where common phylogenetic tools fail to recover the correct tree from complete animal mitochondrial protein-coding sequences. The data set is very extensive, containing several thousand sites per sequence, and the incorrect phylogenetic trees are statistically very well supported. Additionally, neither the use of the LogDet/paralinear transform nor removal of positions in the protein alignment with AT- or GC-rich codons allowed recovery of the correct tree. Two taxa with a large compositional bias continually group together in these analyses, despite a lack of close biological relatedness. We conclude that even protein-based phylogenetic trees may be misleading, and we advise caution in phylogenetic reconstruction using protein sequences, especially those that are compositionally biased. Received: 19 February 1998 / Accepted: 28 August 1998     

Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.     

Development of a size-dependent aerosol deposition model utilising human airway epithelial cells for evaluating aerosol drug delivery

Aerosol delivery to the airways of the human respiratory tract, followed by absorption, constitutes an alternative route of administration for compounds unsuitable for delivery by conventional oral and parenteral routes. The target for aerosol drug delivery is the airways epithelium, i.e. tracheal, bronchial, bronchiolar and alveolar cells, which become the site of drug deposition. These epithelial layers also serve as a barrier to the penetration of inhaled material. An in vitro model for aerosol deposition and transport across epithelia in the human airways may be a good predictor of in vivo disposition. The present preliminary studies begin an investigation that blends the dynamics of aerosol delivery and the basis of an in vitro simulated lung model to evaluate the transport properties of a series of molecular weight marker compounds across human-derived bronchiolar epithelial cell monolayers. An Andersen viable cascade impactor was used as a delivery apparatus for the deposition of size-segregated particles onto monolayers of small airway epithelial cells and Calu-3 cells. It was shown that these cell layers can withstand placement in the impactor, and that permeability can be tested subsequent to removal from the impactor.     

The influence of formulation and spacer device on the in vitro performance of solution chlorofluorocarbon-free propellant-driven metered dose inhalers

The purpose of this study was to evaluate the hypothesis that spacer devices have limited effect on the in vitro fine particle dose emitted from solution metered dose inhalers containing different proportions of HFA134a [1,1,1,2-tetrafluoroethane] propellant. Two solution formulations (80% and 97.5% wt/wt HFA134a) were tested across the actuator alone, actuator plus Aerochamber, and Ace holding chamber. Particle size distributions were determined using laser diffraction (LD) and cascade impaction (CI). Multimodal particle size distributions were identified using LD. CI analyses were characterized by a major mode located at ≈0.5 μm. The fine particle dose emitted from the inhaler spacer combinations containing 97.5% HFA134a was independent of the device setup used. Fine particle doses were influenced by spacer setup in 80% HFA134a formulations, indicating different plume dynamics of low vapor pressure formulations. Sampling inlet deposition was approximately O when spacer devices were used with either formulation. When spacers were not used, sampling inlet deposition was increased significantly. However, inlet deposition with the 97.5% HFA134a formulation was significantly less than that of the 80% HFA134a formulation (≈25% of emitted dose compared with 69% respectively). Thus, high propellant concentration formulations appear to have more robust in vitro performance. This is particularly important given the preponderance of poor patient compliance that is associated with spacer use. High propellant concentrations had the advantage of fine particle doses that were independent of the device setup and significantly lowered sampling inlet deposition when no spacer was used.