Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1----3 to the asparagine-linked N-acetylglucosamine residue

The ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.     

Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals

In this study reactive oxygen species (ROS) generated in the respiratory chain were measured and the quantitative relationship between inhibition of the respiratory chain complexes and ROS formation was investigated in isolated nerve terminals. We addressed to what extent complex I, III and IV,respectively, should be inhibited to cause ROS generation. For inhibition of complex I, III and IV, rotenone, antimycin and cyanide were used, respectively, and ROS formation was followed by measuring the activity of aconitase enzyme. ROS formation was not detected until complex III was inhibited by up to 71 +/- 4%, above that threshold inhibition, decrease in aconitase activity indicated an enhanced ROS generation. Similarly, threshold inhibition of complex IV caused an accelerated ROS production. By contrast, inactivation of complex I to a small extent (16 +/- 2%) resulted in a significant increase in ROS formation, and no clear threshold inhibition could be determined. However, the magnitude of ROS generated at complex I when it is completely inhibited is smaller than that observed when complex III or complex IV was fully inactivated. Our findings may add a novel aspect to the pathology of Parkinson's disease, showing that a moderate level of complex I inhibition characteristic in Parkinson's disease leads to significant ROS formation. The amount of ROS generated by complex I inhibition is sufficient to inhibit in situ the activity of endogenous aconitase.     

Transglutaminase 2 Contributes to Apoptosis Induction in Jurkat T Cells by Modulating Ca2+ Homeostasis via Cross-Linking RAP1GDS1

Background

Transglutaminase 2 (TG2) is a protein cross-linking enzyme known to be associated with the in vivo apoptosis program of T cells. However, its role in the T cell apoptosis program was not investigated yet.

Results

Here we report that timed overexpression of both the wild type (wt) and the cross-linking mutant of TG2 induced apoptosis in Jurkat T cells, the wt being more effective. Part of TG2 colocalised with mitochondria. WtTG2-induced apoptosis was characterized by enhanced mitochondrial Ca²⁺ uptake. Ca²⁺-activated wtTG2 cross-linked RAP1, GTP-GDP dissociation stimulator 1, an unusual guanine exchange factor acting on various small GTPases, to induce a yet uncharacterized signaling pathway that was able to promote the Ca²⁺ release from the endoplasmic reticulum via both Ins₃P and ryanodine sensitive receptors leading to a consequently enhanced mitochondrial Ca²⁺uptake.

Conclusions

Our data indicate that TG2 might act as a Ca²⁺ sensor to amplify endoplasmic reticulum-derived Ca²⁺ signals to enhance mitochondria Ca²⁺ uptake. Since enhanced mitochondrial Ca²⁺ levels were previously shown to sensitize mitochondria for various apoptotic signals, our data demonstrate a novel mechanism through which TG2 can contribute to the induction of apoptosis in certain cell types. Since, as compared to knock out cells, physiological levels of TG2 affected Ca²⁺ signals in mouse embryonic fibroblasts similar to Jurkat cells, our data might indicate a more general role of TG2 in the regulation of mitochondrial Ca²⁺ homeostasis.     

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity

DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function. Removal of the CTD tail enables GyrA to introduce writhe into DNA in the absence of GyrB, an activity exhibited by other GyrA orthologs, but not by wild-type E. coli GyrA. Strikingly, a "tail-less" gyrase holoenzyme is markedly impaired for DNA supercoiling capacity, but displays normal ATPase function. Our findings reveal that the E. coli GyrA tail regulates DNA wrapping by the CTD to increase the coupling efficiency between ATP turnover and supercoiling, demonstrating that CTD functions can be fine-tuned to control gyrase activity in a highly sophisticated manner.     

Structure and subunit composition of GABA(A) receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. If all GABA(A) receptor subunits could randomly combine with each other, an extremely large number of GABA(A) receptor subtypes with distinct subunit composition and arrangement would be formed. Depending on their subunit composition, these receptors would exhibit distinct pharmacological and electrophysiological properties. Recent evidence, however, indicates that not all subunits can assemble efficiently with each other and form functional homo- or hetero-oligomeric receptors. In addition, the efficiency of formation of hetero-oligomeric assembly intermediates determines the subunit stoichiometry and subunit arrangement for each receptor and thus further reduces the possible heterogeneity of GABA(A) receptors in the brain. Studies investigating the subunit composition of native GABA(A) receptors support this conclusion, but also indicate that receptors composed of one, two, three, four, or five different subunits might exist in the brain. Using a recently established immunodepletion technique, the subunit composition and quantitative importance of native GABA(A) receptor subtypes can be determined. This information, together with studies on the regional, cellular and subcellular distribution of these receptor subtypes, will be the basis for a rational development of drugs that specifically affect the GABAergic system.     

Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψ_m) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψ_m and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψ_m was detected via safranin fluorescence and a TPP⁺ electrode, the rate of H₂O₂ formation was measured by Amplex UltraRed, the intramitochondrial pH (pH_in) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K⁺/H⁺ exchanger, nigericin lowered pH_in and ΔpH, followed by a compensatory increase in Δψ_m that led to an augmented H₂O₂ production. Valinomycin, a K⁺ ionophore, at low [K⁺] increased ΔpH and pH_in, decreased Δψ_m, which resulted in a decline in H₂O₂ formation. It was concluded that Δψ_m is dominant over ?pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H₂O₂ release and a decreased ?pH. This phenomenon reveals that from the pH component not ?pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψ_m might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.     

Molecular phylogeny of fungus gnats (Diptera: Mycetophilidae) revisited: position of Manotinae,Metanepsiini, and other enigmatic taxa as inferred from multigene analysis

The phylogeny of selected genera from four subfamilies of fungus gnats (Diptera: Mycetophilidae) – Manotinae, Leiinae, Sciophilinae and Gnoristinae (including Metanepsiini) – is reconstructed based on the combined analysis of five mitochondrial (12S, 16S, COI, COII, cytB) and two nuclear (28S, ITS2) gene markers. Results of the different analyses all support Manotinae as a monophyletic group, with Leiinae as the sister group. Allactoneura DeMeijere is nested in the monophyletic and strongly supported clade of Leiinae. The tribe Metanepsiini is revealed as paraphyletic and the genera Metanepsia Edwards and Chalastonepsia Søli do not appear to be closely related. The genera Docosia Winnertz, Ectrepesthoneura Enderlein, Novakia Strobl and Syntemna Winnertz were placed with a group of genera included traditionally in the Gnoristinae. The monophyly of Dziedzickia Johannsen and Phthinia Winnertz is not supported. The genera of Sciophilinae (excluding Paratinia Mik but including Eudicrana Loew) form a monophyletic group in the Bayesian model.