Evidence for Involvement of Gut-Associated Denitrifying Bacteria in Emission of Nitrous Oxide (N2O) by Earthworms Obtained from Garden and Forest Soils

Earthworms (Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) obtained from nitrous oxide (N₂O)-emitting garden soils emitted 0.14 to 0.87 nmol of N₂O h⁻¹ g (fresh weight)⁻¹ under in vivo conditions. L. rubellus obtained from N₂O-emitting forest soil also emitted N₂O, which confirmed previous observations (G. R. Karsten and H. L. Drake, Appl. Environ. Microbiol. 63:1878–1882, 1997). In contrast, commercially obtained Lumbricus terrestris did not emit N₂O; however, such worms emitted N₂O when they were fed (i.e., preincubated in) garden soils. A. caliginosa, L. rubellus, and O. lacteum substantially increased the rates of N₂O emission of garden soil columns and microcosms. Extrapolation of the data to in situ conditions indicated that N₂O emission by earthworms accounted for approximately 33% of the N₂O emitted by garden soils. In vivo emission of N₂O by earthworms obtained from both garden and forest soils was greatly stimulated when worms were moistened with sterile solutions of nitrate or nitrite; in contrast, ammonium did not stimulate in vivo emission of N₂O. In the presence of nitrate, acetylene increased the N₂O emission rates of earthworms; in contrast, in the presence of nitrite, acetylene had little or no effect on emission of N₂O. In vivo emission of N₂O decreased by 80% when earthworms were preincubated in soil supplemented with streptomycin and tetracycline. On a fresh weight basis, the rates of N₂O emission of dissected earthworm gut sections were substantially higher than the rates of N₂O emission of dissected worms lacking gut sections, indicating that N₂O production occurred in the gut rather than on the worm surface. In contrast to living earthworms and gut sections that produced N₂O under oxic conditions (i.e., in the presence of air), fresh casts (feces) from N₂O-emitting earthworms produced N₂O only under anoxic conditions. Collectively, these results indicate that gut-associated denitrifying bacteria are responsible for the in vivo emission of N₂O by earthworms and contribute to the N₂O that is emitted from certain terrestrial ecosystems.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

The c13 Ring from a Thermoalkaliphilic ATP Synthase Reveals an Extended Diameter Due to a Special Structural Region

We have structurally characterized the c-ring from the thermoalkaliphilic Bacillus sp. strain TA2.A1 F₁F_o-ATP synthase. Atomic force microscopy imaging and cryo-electron microscopy analyses confirm previous mass spectrometric data indicating that this c-ring contains 13 c-subunits. The cryo-electron microscopy map obtained from two-dimensional crystals shows less closely packed helices in the inner ring compared to those of Na⁺-binding c₁₁ rings. The inner ring of α-helices in c₁₁ rings harbors a conserved GxGxGxGxG motif, with glycines located at the interface between c-subunits, which is responsible for the close packing of these helices. This glycine motif is altered in the c₁₃ ring of Bacillus sp. strain TA2.A1 to AxGxSxGxS, leading to a change in c-c subunit contacts and thereby enlarging the c-ring diameter to host a greater number of c-subunits. An altered glycine motif is a typical feature of c-subunit sequences in alkaliphilic Bacillus species. We propose that enlarged c-rings in proton-dependent F-ATP synthases may represent an adaptation to facilitate ATP synthesis at low overall proton-motive force, as occurs in bacteria that grow at alkaline pH.     

Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils

Crucial steps in geochemical cycles are in many cases performed by more than one group of microorganisms, but the significance of this functional redundancy with respect to ecosystem functioning is poorly understood. Ammonia-oxidizing archaea (AOA) and their bacterial counterparts (AOB) are a perfect system to address this question: although performing the same transformation step, they belong to well-separated phylogenetic groups. Using pig manure amended with different concentrations of sulfadiazine (SDZ), an antibiotic that is frequently used in veterinary medicine, it was possible to affect AOB and AOA to different degrees. Addition of manure stimulated growth of AOB in both soils and, interestingly, also growth of AOA was considerably stimulated in one of the soils. The antibiotic treatments decreased the manure effect notably on AOB, whereas AOA were affected to a lower extent. Model calculations concerning the respective proportions of AOA and AOB in ammonia oxidation indicate a substantial contribution of AOA in one of the soils that further increased under the influence of SDZ, hence indicating functional redundancy between AOA and AOB.     

Allosteric activation mechanism of the alpha1beta2gamma2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha1beta2gamma2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta2Y157S; increased the EC(50)) and the conserved M2 leucine (beta2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.     

Impaired striatal Akt signaling disrupts dopamine homeostasis and increases feeding

Background

The prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address “food-abuse” disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT). Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake.

Methodology/Principal Findings

We show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH)-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH)-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia.

Conclusions/Significance

Acquired disruption of brain insulin action may confer risk for and/or underlie “food-abuse” disorders and the recalcitrance of obesity. This molecular model, thus, explains how even short-term exposure to “the fast food lifestyle” creates a cycle of disordered eating that cements pathological changes in DA signaling leading to weight gain and obesity.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.