Biogenic nanosized iron oxides obtained from cultivation of iron bacteria from the genus <Emphasis Type="Italic">Leptothrix</Emphasis>

A detailed investigation of nanostructured iron oxides/(oxy)hydroxides gathered after cultivation of bacteria from the genus Leptothrix as iron (II) oxidizers is presented. A specific type of medium is selected for the cultivation of the bacteria. Results for sediment powder and bio-film on glass substrate samples from the same media are discussed. XRD, Raman spectroscopy, SEM, and TEM images and PPMS measurements are used to prove the exact composition of the biogenic products and to interpret the oxidation process. Analysis of the data collected shows that around 80 % of the iron (II) from the growth medium has been transformed into iron (III) in the form of different (oxy)hydroxides, with the rest found to be in a mixed 2,5 valence in magnetite. Our investigation shows that the bio-film sample has a phase content different from that of the powdered biomass and that lepidocrocite (γ-FeOOH) is the predominant and the initial biogenic phase in both samples. Magnetite nanoparticles are a secondary product in the bio-film, part of which possesses a defective quasi-maghemite surface layer. In the powdered biomass, the oxidation steps are not fully completed. The initial products are non-stoichiometric and due to the mixed ferric and ferrous ions present, they develop into: (i) lepidocrocite (γ-FeOOH) as a basic sediment, (ii) magnetite (Fe₃O₄) and (iii) goethite (α-FeOOH) in small quantities. The average size of all iron-bearing particles is found to be below 30 nm. The magnetic measurements performed show a superparamagnetic behavior of the material at room temperature.     

Sorption of Fe (Hydr)Oxides to the Surface of Shewanella putrefaciens: Cell-Bound Fine-Grained Minerals Are Not Always Formed De Novo

Shewanella putrefaciens, a gram-negative, facultative anaerobe, is active in the cycling of iron through its interaction with Fe (hydr)oxides in natural environments. Fine-grained Fe precipitates that are attached to the outer membranes of many gram-negative bacteria have most often been attributed to precipitation and growth of the mineral at the cell surface. Our study of the sorption of nonbiogenic Fe (hydr)oxides revealed, however, that large quantities of nanometer-scale ferrihydrite (hydrous ferric oxide), goethite (α-FeOOH), and hematite (α-Fe₂O₃) adhered to the cell surface. Attempts to separate suspensions of cells and minerals with an 80% glycerin cushion proved that the sorbed minerals were tightly attached to the bacteria. The interaction between minerals and cells resulted in the formation of mineral-cell aggregates, which increased biomass density and provided better sedimentation of mineral Fe compared to suspensions of minerals alone. Transmission electron microscopy observations of cells prepared by whole-mount, conventional embedding, and freeze-substitution methods confirmed the close association between cells and minerals and suggested that in some instances, the mineral crystals had even penetrated the outer membrane and peptidoglycan layers. Given the abundance of these mineral types in natural environments, the data suggest that not all naturally occurring cell surface-associated minerals are necessarily formed de novo on the cell wall.     

Iron transformations induced by an acid-tolerant Desulfosporosinus species

The mineralogical transformations of Fe phases induced by an acid-tolerant, Fe(III)- and sulfate-reducing bacterium, Desulfosporosinus sp. strain GBSRB4.2 were evaluated under geochemical conditions associated with acid mine drainage-impacted systems (i.e., low pH and high Fe concentrations). X-ray powder diffractometry coupled with magnetic analysis by first-order reversal curve diagrams were used to evaluate mineral phases produced by GBSRB4.2 in media containing different ratios of Fe(II) and Fe(III). In medium containing Fe predominately in the +II oxidation state, ferrimagnetic, single-domain greigite (Fe₃S₄) was formed, but the addition of Fe(III) inhibited greigite formation. In media that contained abundant Fe(III) [as schwertmannite; Fe₈O₈(OH)₆SO₄ · nH₂O], the activities of strain GBSRB4.2 enhanced the transformation of schwertmannite to goethite (α-FeOOH), due to the increased pH and Fe(II) concentrations that resulted from the activities of GBSRB4.2.     

Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins

The β₂ subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe₂^III-Tyr^•) that is essential for nucleotide reduction. The β₂ subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β′). Although only β is capable of iron binding and Tyr^• formation, cells lacking β′ are either dead or exhibit extremely low Tyr^• levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β′ is required for iron loading and Tyr^• formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr^•-reducing agent hydroxyurea. Transient induction of β′ in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr^• levels in β. Tyr^• can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β′ and is inhibited by adding an iron chelator prior to, but not after, β′ supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr^• levels and ββ′ activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr^• cofactor in RNR.     

M?ssbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions

The Mo–Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in ⁵⁷Fe-enriched medium. The Mössbauer spectrum of the Mo–Fe protein, as isolated in the presence of Na₂S₂O₄, showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with δ=0.65mm/s and ΔE=3.05mm/s at 4.2°K, corresponded to two iron atoms/molecule of protein and it is interpreted as being due to a high-spin ferrous, spin-coupled pair of iron atoms. The iron atoms of species M4 may be involved in the quaternary structure of the protein. Species M5, with δ=0.61mm/s and ΔE=0.83mm/s at 77°K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe₄S₄ or Fe₂S₂ low-spin ferrous iron clusters. Species M6, with δ=0.37mm/s and ΔE=0.71mm/s at 77°K, also corresponded to eight iron atoms/molecule of protein and, at 4.2°K, became a broad shallow absorption, characteristic of magnetic hyperfine interaction. Oxidation of the Mo–Fe protein with the redox dye Lauth''s Violet did not affect the activity of the protein but changed species M4, M5 and M6 into the species M1 (δ=0.37mm/s, ΔE=0.75mm/s at 77°K, broad magnetic component at 4.2°K) and M2 (δ=0.35mm/s, ΔE=0.9mm/s at 4.2°K). In the presence of the Fe protein, Na₂S₂O₄, ATP and Mg²⁺, the M6 component of the Mo–Fe protein was replaced by species M7 with δ=0.46mm/s, ΔE=1.04mm/s at 4.2°K. The change in Mössbauer parameters associated with the M6 → M7 transformation was very similar to the change observed on reduction of the high-potential Fe protein from Chromatium vinosum. In contrast, Na₂S₂O₄-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had δ=0.50mm/s, ΔE=0.9mm/s at 195°K, and at 4.2°K broadened in a manner characteristic of a magnetic hyperfine interaction, associated with half-integral spin, equally distributed over all four atoms of the Fe protein. The Mössbauer spectra of the Mo–Fe and the Fe protein under argon were unaffected by the reducible substrates N₂ and C₂H₂ and the inhibitor CO in the presence of ATP, Mg²⁺ and Na₂S₂O₄. A number of Mössbauer spectral species associated with inactivated Mo–Fe and Fe proteins are described and discussed.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

Increased Cytokine and Nitric Oxide Levels in Serum of Dogs Experimentally Infected with Rangelia vitalii

This study aimed to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and nitrite/nitrate (NO_x) in serum of dogs experimentally infected with Rangelia vitalii. Twelve female mongrel dogs were divided into 2 groups; group A (uninfected controls) composed by healthy dogs (n=5) and group B consisting of dogs inoculated with R. vitalii (n=7). Animals were monitored by blood smear examinations, which showed intraerythrocytic forms of the parasite on day 5 post-infection (PI). Blood samples were collected through the jugular vein on days 0, 10, and 20 PI to determine the serum levels of IFN-γ, TNF-α, IL-1, IL-6, and NO_x. Cytokines were assessed by ELISA quantitative sandwich technique, and NO_x was measured by the modified Griess method. Cytokine levels (IFN-γ, TNF-α, IL-1, and IL-6) were increased (P<0.01) in serum of infected animals. Serum levels of NO_x were also increased on days 10 PI (P<0.01) and 20 PI (P<0.05) in infected animals. Therefore, the infection with R. vitalii causes an increase in proinflammatory cytokines and nitric oxide content. These alterations may be associated with host immune protection against the parasite.     

Alzheimer's disease drug candidates stabilize A-β protein native structure by interacting with the hydrophobic core

Deposition of amyloid fibrils, consisting primarily of Aβ₄₀ and Aβ₄₂ peptides, in the extracellular space in the brain is a major characteristic of Alzheimer''s disease (AD). We recently developed new (to our knowledge) drug candidates for AD that inhibit the fibril formation of Aβ peptides and eliminate their neurotoxicity. We performed all-atom molecular-dynamics simulations on the Aβ₄₂ monomer at its α-helical conformation and a pentamer fibril fragment of Aβ₄₂ peptide with or without LRL and fluorene series compounds to investigate the mechanism of inhibition. The results show that the active drug candidates, LRL22 (EC₅₀ = 0.734 μM) and K162 (EC₅₀ = 0.080 μM), stabilize hydrophobic core I of Aβ₄₂ peptide (residues 17–21) to its α-helical conformation by interacting specifically in this region. The nonactive drug candidates, LRL27 (EC₅₀ > 10 μM) and K182 (EC₅₀ > 5 μM), have little to no similar effect. This explains the different behavior of the drug candidates in experiments. Of more importance, this phenomenon indicates that hydrophobic core I of the Aβ₄₂ peptide plays a major mechanistic role in the formation of amyloid fibrils, and paves the way for the development of new drugs against AD.     

Crystal structure of H2O2-dependent cytochrome P450SPalpha with its bound fatty acid substrate: insight into the regioselective hydroxylation of fatty acids at the alpha position

Cytochrome P450_SPα (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H₂O₂-dependent P450. P450_SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450_SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the C_α of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450_SPα. Mutations at the active site and the F–G loop of P450_SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450_BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450_SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450_BSβ.     

M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase

The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222₁ with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K_d of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD⁺ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria.     

The amino-terminus of nitric oxide sensitive guanylyl cyclase α₁ does not affect dimerization but influences subcellular localization

Background

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β₁-subunit. A splice variant (C-α₁) of the α₁-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61–128 of the α₁-subunit are mandatory for quantitative heterodimerization implying that the C-α₁-splice variant should lose its capacity to dimerize quantitatively.

Methodology/Principal Findings

In the current study we demonstrate preserved quantitative dimerization of the C-α₁-splice by co-purification with the β₁-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β₁-subunit and the α₁-subunit or the C-α₁ variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α₁/β₁ and C-α₁/β₁ than the negative control. In addition we show that lack of the amino-terminus in the α₁ splice variant directs it to a more oxidized subcellular compartment.

Conclusions/Significance

We conclude that the amino-terminus of the α₁-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.     

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single α-helical globular domain, microplusin consists of five α-helices: α1 (residues Gly-9 to Arg-21), α2 (residues Glu-27 to Asn-40), α3 (residues Arg-44 to Thr-54), α4 (residues Leu-57 to Tyr-64), and α5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.     

Longer leukocyte telomere length is associated with smaller hippocampal volume among non-demented APOE ε3/ε3 subjects

Telomere length shortens with cellular division, and leukocyte telomere length is used as a marker for systemic telomere length. The hippocampus hosts adult neurogenesis and is an important structure for episodic memory, and carriers of the apolipoprotein E ε4 allele exhibit higher hippocampal atrophy rates and differing telomere dynamics compared with non-carriers. The authors investigated whether leukocyte telomere length was associated with hippocampal volume in 57 cognitively intact subjects (29 ε3/ε3 carriers; 28 ε4 carriers) aged 49–79 yr. Leukocyte telomere length correlated inversely with left (r_s = −0.465; p = 0.011), right (r_s = −0.414; p = 0.025), and total hippocampus volume (r_s = −0.519; p = 0.004) among APOE ε3/ε3 carriers, but not among ε4 carriers. However, the ε4 carriers fit with the general correlation pattern exhibited by the ε3/ε3 carriers, as ε4 carriers on average had longer telomeres and smaller hippocampi compared with ε3/ε3 carriers. The relationship observed can be interpreted as long telomeres representing a history of relatively low cellular proliferation, reflected in smaller hippocampal volumes. The results support the potential of leukocyte telomere length being used as a biomarker for tapping functional and structural processes of the aging brain.     

Association between IgM anti-herpes simplex virus and plasma amyloid-beta levels

Objective

Herpes simplex virus (HSV) reactivation has been identified as a possible risk factor for Alzheimer''s disease (AD) and plasma amyloid-beta (Aβ) levels might be considered as possible biomarkers of the risk of AD. The aim of our study was to investigate the association between anti-HSV antibodies and plasma Aβ levels.

Methods

The study sample consisted of 1222 subjects (73.9 y in mean) from the Three-City cohort. IgM and IgG anti-HSV antibodies were quantified using an ELISA kit, and plasma levels of Aβ_1–40 and Aβ_1–42 were measured using an xMAP-based assay technology. Cross-sectional analyses of the associations between anti-HSV antibodies and plasma Aβ levels were performed by multi-linear regression.

Results

After adjustment for study center, age, sex, education, and apolipoprotein E-e4 polymorphism, plasma Aβ_1–42 and Aβ_1–40 levels were specifically inversely associated with anti-HSV IgM levels (β = −20.7, P = 0.001 and β = −92.4, P = 0.007, respectively). In a sub-sample with information on CLU- and CR1-linked SNPs genotyping (n = 754), additional adjustment for CR1 or CLU markers did not modify these associations (adjustment for CR1 rs6656401, β = −25.6, P = 0.002 for Aβ_1–42 and β = −132.7, P = 0.002 for Aβ_1–40; adjustment for CLU rs2279590, β = −25.6, P = 0.002 for Aβ_1–42 and β = −134.8, P = 0.002 for Aβ_1–40). No association between the plasma Aβ_1–42-to-Aβ_1–40 ratio and anti-HSV IgM or IgG were evidenced.

Conclusion

High anti-HSV IgM levels, markers of HSV reactivation, are associated with lower plasma Aβ_1–40 and Aβ_1–42 levels, which suggest a possible involvement of the virus in the alterations of the APP processing and potentially in the pathogenesis of AD in human.     

Mineral Products of Pyrrhotite Oxidation by Thiobacillus ferrooxidans

The biological leaching of pyrrhotite (Fe_1-xS) by Thiobacillus ferrooxidans was studied to characterize the oxidation process and to identify the mineral weathering products. The process was biphasic in that an initial phase of acid consumption and decrease in redox potential was followed by an acid-producing phase and an increase in redox potential. Elemental S was one of the first products of pyrrhotite degradation detected by X-ray diffraction. Pyrrhotite oxidation also yielded K-jarosite [KFe₃(SO₄)₂(OH)₆], goethite (α-FeOOH), and schwertmannite [Fe₈O₈(OH)₆SO₄] as solid-phase products. Pyrrhotite was mostly depleted after 14 days, whereas impurities in the form of pyrite (cubic FeS₂) and marcasite (orthorhombic FeS₂) accumulated in the leach residue.     

Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean Room Environments

In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4°C to 65°C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 10⁶ cells/m². However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (α- and β-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.     

Relationships between Structural Dynamics and Functional Kinetics in Oligomeric Membrane Receptors

Recent efforts to broaden understanding of the molecular mechanisms of membrane receptors in signal transduction make use of rate-equilibrium free-energy relationships (REFERs), previously applied to chemical reactions, enzyme kinetics, and protein folding. For oligomeric membrane receptors, we distinguish between a), the Leffler parameter α_L, to characterize the global transition state for the interconversion between conformations; and b), the Fersht parameter, ϕ_F, to assign the degree of progression of individual residue positions at the transition state. For both α_L and ϕ_F, insights are achieved by using harmonic energy profiles to reflect the dynamic nature of proteins, as illustrated with single-channel results reported for normal and mutant nicotinic receptors. We also describe new applications of α_L based on published results. For large-conductance calcium-activated potassium channels, data are satisfactorily fit with an α_L value of 0.65, in accord with REFERs. In contrast, results reported for the flip conformational state of glycine and nicotinic receptors are in disaccord with REFERs, since they yield α_L values outside the usual limits of 0–1. Concerning published ϕ_F values underlying the conformational wave hypothesis for nicotinic receptors, we note that interpretations may be complicated by variations in the width of harmonic energy profiles.     

Inhibition of Lung Fluid Clearance and Epithelial Na+ Channels by Chlorine, Hypochlorous Acid, and Chloramines

We investigated the mechanisms by which chlorine (Cl₂) and its reactive byproducts inhibit Na⁺-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na⁺ channels (ENaC) by measuring AFC in mice exposed to Cl₂ (0–500 ppm for 30 min) and Na⁺ and amiloride-sensitive currents (I_Na and I_amil, respectively) across Xenopus oocytes expressing human α-, β-, and γ-ENaC incubated with HOCl (1–2000 μm). Both Cl₂ and HOCl-derived products decreased AFC in mice and whole cell and single channel I_Na in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I_Na in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of I_Na by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the γ-ENaC subunit restored the ability of HOCl to inhibit I_Na. Finally, I_Na of oocytes expressing wild type α- and γ-ENaC and a mutant form of βENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage.     

Nematicidal and Propagation Activities of Thyme Red and White Oil Compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

The toxic and propagation effects on Bursaphelenchus xylophilus of 28 Thymus vulgaris red oil and white oil compounds were examined using direct contact and cotton ball bioassays. Results were compared with those of the trunk-injection nematicides emmamectin benzoate, levamisol hydrochloride and morantel tartrate. In direct contact bioassays, geraniol (LC₅₀, 0.47 mg/ml) was the most toxic compound, followed by thymol (1.08 mg/ml), carvacrol (1.23 mg/ml) and terpinen-4-ol (2.61 mg/ml). In cotton ball tests with 20 inactive compounds at 2 mg/cotton ball, p-cymene significantly inhibited propagation (propagation ratio [PR] 8), compared with the castor oil-ethanol-treated control (PR 56). Propagation stimulation was observed with (–)-caryophyllene oxide, (+)-ledene, (+)- and (–)-limonene, linalool oxide, β-myrcene, (–)-α-phellandrene, (+)-α-pinene and γ-terpinene (PR 63–100). The other 10 compounds exhibited low to moderate levels of propagation inhibition (PR 36–56). At 0.1 μg/cotton ball, emmamectin benzoate and morantel tartrate exhibited complete suppression of propagation, whereas a very low level of propagation inhibition was obtained from levamisol hydrochloride (PR 6). In conclusion, propagation-stimulating compounds can exist in plants in addition to nematicidal compounds, and careful use of plant preparations containing high quantities of these compounds is mandatory.