Mechanism of anaerobic degradation of triethanolamine by a homoacetogenic bacterium

Triethanolamine (TEA) is converted into acetate and ammonia by a strictly anaerobic, gram-positive Acetobacterium strain LuTria3. Fermentation experiments with resting cell suspensions and specifically deuterated substrates indicate that in the acetate molecule the carboxylate and the methyl groups correspond to the alcoholic function and to its adjacent methylene group, respectively, of the 2-hydroxyethyl unit of TEA. A 1,2 shift of a hydrogen (deuterium) atom from -CH2-O- to =N-CH2- without exchange with the medium was observed. This fact gives evidence that a radical mechanism occurs involving the enzyme and/or coenzyme molecule as a hydrogen carrier. Such a biodegradation appears analogous to the conversion of 2-phenoxyethanol into acetate mediated by another strain of the anaerobic homoacetogenic bacterium Acetobacterium.     

De Novo Synthesis and Degradation of Lx and V Cycle Pigments during Shade and Sun Acclimation in Avocado Leaves

The photoprotective role of the universal violaxanthin cycle that interconverts violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) is well established, but functions of the analogous conversions of lutein-5,6-epoxide (Lx) and lutein (L) in the selectively occurring Lx cycle are still unclear. We investigated carotenoid pools in Lx-rich leaves of avocado (Persea americana) during sun or shade acclimation at different developmental stages. During sun exposure of mature shade leaves, an unusual decrease in L preceded the deepoxidation of Lx to L and of V to A+Z. In addition to deepoxidation, de novo synthesis increased the L and A+Z pools. Epoxidation of L was exceptionally slow, requiring about 40 d in the shade to restore the Lx pool, and residual A+Z usually persisted overnight. In young shade leaves, the Lx cycle was reversed initially, with Lx accumulating in the sun and declining in the shade. De novo synthesis of xanthophylls did not affect α- and β-carotene pools on the first day, but during long-term acclimation α-carotene pools changed noticeably. Nonetheless, the total change in α- and β-branch carotenoid pools was equal. We discuss the implications for regulation of metabolic flux through the α- and β-branches of carotenoid biosynthesis and potential roles for L in photoprotection and Lx in energy transfer to photosystem II and explore physiological roles of both xanthophyll cycles as determinants of photosystem II efficiency.It has long been recognized that photosynthesis in plants must resolve two conflicting requirements, the need to ramp up maximum light-harvesting efficiency in dim light and to wind back to lower efficiency when light is in excess, in order to maintain high rates of growth and productivity in varying light environments (Björkman, 1981; Pearcy, 1990). A wealth of research has established that plants adjust through an array of morphological and molecular events that confer photoprotection, mitigate and repair photoinactivation of PSII, and facilitate acclimation of the photosynthetic apparatus over different time scales in response to variable light regimes in wild plants, crops, and algae (Osmond et al., 1999; Demmig-Adams et al., 2006). In the context of the light reactions, low light acclimation optimizes light harvesting and energy transfer to the photosystems, particularly PSII, via enlarged functional antennae, accumulation of accessory light-harvesting pigments, and down-regulation of unnecessary competing photoprotective processes. High light acclimation involves increased photoprotection and photorepair, downsized antennae, fewer photosystems, and sometimes changes in the PSI to PSII stoichiometry (Osmond et al., 1999; Förster et al., 2005). Along with their function in energy transfer to the photosynthetic reaction centers as accessory pigments to chlorophylls, the xanthophyll pigments violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) play a central role in these transformations of the photosynthetic apparatus, especially in thermal energy dissipation and detoxification of reactive oxygen species.Two xanthophyll cycles are now known in terrestrial plants, the lutein epoxide cycle (Lx cycle) based on interconversions of lutein-5,6-epoxide (Lx) and lutein (L) synthesized from α-carotene (α-C), and the violaxanthin cycle (V cycle) based on the interconversions of V and A+Z synthesized from β-carotene (β-C; García-Plazaola et al., 2007). Presumably, both cycles are catalyzed by the same enzymes, violaxanthin epoxidase (VDE) for deepoxidation in high light and zeaxanthin epoxidase (ZE) for the reverse reactions in low light or darkness (Latowski et al., 2004). Rediscovery of the Lx cycle in the parasitic angiosperm Cuscuta reflexa (Bungard et al., 1999) has led to growing interest in differing manifestations of this cycle in terrestrial plants and its relationships to the apparently universal V cycle (Demmig-Adams, 1998). A complete Lx cycle seems to function on a daily basis in both C. reflexa and the mistletoe Amyema miquelii (Matsubara et al., 2001), even though Lx conversion to L is sometimes slower than V to A+Z and dark recovery of Lx is usually slower than that of V. Intriguingly, in shade leaves of Inga sapindoides, high concentrations of Lx were seemingly irreversibly converted to L on exposure to strong light, in marked contrast to the co-occurring, fully reversible V cycle (Matsubara et al., 2005). Similar responses have been found in other woody plants with long-lived leaves in deeply shaded canopies, including Mediterranean oaks (Quercus spp.; García-Plazaola et al., 2003), sweet bay laurel (Laurus nobilis), and avocado (Persea americana; Esteban et al., 2007, 2008). This response type is known as a truncated Lx cycle (García-Plazaola et al., 2007).The functions attributed to the Lx cycle were initially based on structural analogies between Lx and A and between L and Z (Bungard et al., 1999; Pogson and Rissler, 2000; Matsubara et al., 2001). With increased evidence for the possible role of L in photoprotection (Pogson et al., 1996, 1998; Lokstein et al., 2002; Dall''Osto et al., 2006), additional functional analogies emerged. Furthermore, recent in vitro reconstitution studies with light-harvesting complex proteins and purified pigments also support a spatial overlap of the cycles, as some pigment-binding sites can be occupied by either α- or β-xanthophylls (Matsubara et al., 2007). An attractive hypothesis is that photoconversion of Lx to L might sustain or enhance photoprotection associated with the V cycle (Demmig-Adams and Adams, 1992; Niyogi, 2000). In support of this view, it has been demonstrated in leaves of Quercus rubra and in leaflets of Inga marginata that increasing amounts of photoconverted L, which persist even when A and Z are epoxidized to V, were associated with faster engagement and higher levels of nonphotochemical quenching (NPQ) of chlorophyll fluorescence (García-Plazaola et al., 2003; Matsubara et al., 2008). Furthermore, evidence from mammalian eye research as well as from plants suggests that L also acts as a highly efficient reactive oxygen species scavenger (Kim et al., 2006; Johnson et al., 2007).Broader issues, such as the roles of short-term dynamics of the two cycles in relation to long-term processes of shade and sun acclimation and in relation to leaf development and age, are poorly understood. Nonfruiting shoots of avocado trees constitute a very suitable model system in which to address these issues. Long-lived leaves of shade-grown avocado contain some of the highest levels of Lx thus far recorded (Esteban et al., 2007; García-Plazaola et al., 2007) and have two to four flushes of leaf initiation per year that exhibit a form of delayed greening in which leaf expansion precedes increases in stomatal conductance, chlorophyll content, and CO₂ assimilation. Expanding leaves remain sinks for up to 1 month until they reach about 70% to 80% of full expansion (Schaffer et al., 1991), and stomata do not become fully functional until leaves attain 90% of full expansion (Scholefield and Kriedemann, 1979). However, shoots also retain old leaves through several flushes, and leaves from the previous season contribute significantly to total plant carbon gain (Liu et al., 2002), with photosynthesis rates up to 50% of those in new, fully expanded leaves (Heath et al., 2005). These properties offer an array of opportunities for new research into the concurrent operation of the two xanthophyll cycles.Since there have been very few studies of these complex responses, we carried out a series of short- and long-term light treatments that are likely to reflect what leaves may experience in natural environments, with the aim to gain further insight into the physiological relevance of the Lx and V cycles under those circumstances. Four types of acclimation experiments were undertaken in this study. First, short-term acclimation from shade to sun addressed fast responses to a drastic increase in the light environment, simulating a prolonged sun fleck in shaded mature leaves or exposure to a bright sunny day in young leaves that had emerged during a prolonged overcast (shaded) growth period. These experiments revealed an unexpected loss of L prior to deepoxidation of Lx and V and a reverse Lx cycle in young leaves. Second, long-term acclimation of sun leaves to prolonged shade simulated normal processes of shading by further growth of outer canopy leaves. These treatments established the very slow accumulation of Lx in avocado leaves. Third, sequential sun exposures of mature leaves over several days, followed by continuous shade, were applied to simulate successive prolonged sun flecks, mimicking stochastic canopy disturbance during severe weather events, which confirmed many responses in the above experiments, particularly the very slow epoxidation of L to Lx in prolonged shade. Fourth, long-term acclimation of young and mature leaves to sun was examined. These experiments simulated sudden changes to canopy architecture as experienced during pruning and extended our understanding of the comparative rates and magnitude of Lx and V cycle engagement. We discuss the short-and long-term kinetics of both cycles in avocado leaves of different ages during acclimation, with particular attention to the stoichiometric relationships between xanthophyll and carotenoid pools and changing PSII efficiency.     

Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen–thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2 ± 5.4%, motility following 30 min incubation at 37 °C: 58.5 ± 6.0% and following 3 h incubation: 21.7 ± 7.6%, intact acrosome: 57.1 ± 5.2%, normal morphology: 52.0 ± 5.8% and viability: 67.3 ± 6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.     

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.HIV-1³ is an enveloped retrovirus, which acquires its lipid envelope by budding from the plasma membrane of the infected host cell. Several reports have shown that the viral membrane is enriched in sphingomyelin (SM), including the unusual sphingolipid dihydrosphingomyelin (DHSM) and collectively referred to as sphingomyelins (SMs), glycosphingolipids, cholesterol (CHOL), saturated phosphoglycerolipids and phosphoinositides (1–4). Moreover the CHOL/phospholipid and protein/lipid ratios of the HIV-1 membrane are high, corresponding to a highly ordered membrane, and are presumed to be different from the overall host cell plasma membrane. Accordingly, the HIV-1 envelope has been considered to be a large raft-like membrane microdomain (3). This is in line with previous reports describing enrichment of raft markers in the HIV-1 membrane and its sensitivity to CHOL-depleting agents (5–9). Furthermore, HIV-1 glycoproteins have been suggested to localize within membrane rafts due to palmitoylation of two cysteines (10), and the main structural Gag protein has been shown to rapidly relocalize to detergent-resistant membranes after initial membrane binding (6).Membrane microdomains are dynamic assemblies resulting from the lateral interaction of lipids and proteins. Two phases coexist in the plasma membrane: the liquid-ordered phase (l_o), mainly composed of CHOL and sphingolipids (SPLs), and the liquid disordered phase (l_d), mainly composed of glycerophospholipids (11–13). In the activated state, l_o microdomains can coalesce and serve as platforms for membrane trafficking, signaling, and virus budding (14, 15). The first method to biochemically enrich membrane rafts was the purification of detergent-resistant membranes, based on their resistance to extraction with non-ionic detergent at 4 °C (16). However, this and other methods based on antibody or cholera toxin binding may lead to artificial aggregation of membrane microdomains and thus do not necessarily represent their native state (17, 18). For these reasons and because the association and dissociation of membrane microdomains appears to occur on a rapid time-scale and the raft size is too small to be optically resolved, the raft concept remains controversial. However, the determination of the HIV-1 lipidome, a native membrane purified without any detergent, has provided strong evidence for the existence of these microdomains (3).Fluorescent lipid analogs that partition preferentially into a specialized lipid phase could be an attractive tool to study membrane microdomains. However, partitioning of such dyes mainly depends on the local chemical environment and not on the phase state of the membrane (19–21). In contrast, Laurdan (6-dodecanoyl-2-dimethylaminonapthalene) is a lipophilic dye that binds to membranes independent of their phase state but reports the phase state by a change in its fluorescence emission (20). Laurdan exhibits a blue shift in its emission spectrum with increasing membrane condensation. This is caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. Thus, excitation of membrane bound Laurdan leads to two emission maxima representing differences in membrane lateral structure. Quantification of membrane order is achieved by computing the Generalized Polarization (GP) value, which is defined as normalized intensity ratio of the two emission channels. GP values range from +1 (most condensed) to −1 (most fluid). They are not biased by probe concentration, membrane ruffles, and surface modifications, such as lipoprotein binding. Furthermore, there is no preferential interaction with a specific lipid, fatty acid, or head group (20, 21). GP value correspondence to different lipid phases was estimated using liposomes with a composition similar to that of cellular membranes (22, 23). Using an equimolar mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine, CHOL, and SM as an l_o membrane, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as an l_d and solid ordered (s_o) phase, GP values below +0.25 were shown to correspond to the l_d phase, GP values between +0.25 and +0.5 to the l_o phase, and GP values above +0.5 to the s_o phase (22, 23).Laurdan has been extensively used to characterize domain formation and lateral lipid segregation in model membranes composed of different phospholipid mixtures or lipids extracted from cellular membranes (19, 22–25). It has also been used to study the membrane structure in living cells. Gaus and coworkers observed l_o domains enriched on membrane protrusions (filopodia), adhesion points, and cell-cell contacts (26). They also used Laurdan to address the physical properties of the plasma membrane around the T-cell receptor in activated T cells, observing larger and more stably ordered membrane domains at sites of T-cell activation (27). Quantitative determination of cellular plasma membrane order by fluorescence spectroscopy is complicated due to the rapid internalization and redistribution of the probes to other cellular membranes, making it difficult to interpret the fluorescence measurements over the whole cell. This problem is not encountered in purified virus particles, because they contain only a single membrane. We therefore developed an assay to study viral membrane lateral structure by fluorescence spectroscopy. For this purpose, isogenic HIV-1 particles were produced in two different cell lines, and their GP profiles were determined. In parallel, the lipid constituents were quantified by mass spectrometry. The viral membrane displayed a l_o structure in both cases, but this was more prominent for the virus derived from the T-cell line MT-4 compared with virus derived from 293T cells. The validity of this result was supported by comparing the lipidome of the two viruses, which revealed a significantly higher SMs/phosphatidylcholine (PC) ratio for the MT-4-derived virus. Accordingly, treatment with SM-binding toxins inactivated MT-4-derived virus more efficiently than 293T-derived virus, whereas both viruses exhibited similar infectivities before treatment. The reported approach allows rapid determination of differences in viral membrane order, permitting screening for compounds that perturb l_o domains, which may act as antivirals of a novel type.     

A Changing Gastric Environment Leads to Adaptation of Lipopolysaccharide Variants in Helicobacter pylori Populations during Colonization

The human gastric pathogen Helicobacter pylori colonizes the stomachs of half of the human population, and causes development of peptic ulcer disease and gastric adenocarcinoma. H. pylori-associated chronic atrophic gastritis (ChAG) with loss of the acid-producing parietal cells, is correlated with an increased risk for development of gastric adenocarinoma. The majority of H. pylori isolates produce lipopolysaccharides (LPS) decorated with human-related Lewis epitopes, which have been shown to phase-vary in response to different environmental conditions. We have characterized the adaptations of H. pylori LPS and Lewis antigen expression to varying gastric conditions; in H. pylori isolates from mice with low or high gastric pH, respectively; in 482 clinical isolates from healthy individuals and from individuals with ChAG obtained at two time points with a four-year interval between endoscopies; and finally in isolates grown at different pH in vitro. Here we show that the gastric environment can contribute to a switch in Lewis phenotype in the two experimental mouse models. The clinical isolates from different human individuals showed that intra-individual isolates varied in Lewis antigen expression although the LPS diversity was relatively stable within each individual over time. Moreover, the isolates demonstrated considerable diversity in the levels of glycosylation and in the sizes of fucosylated O-antigen chains both within and between individuals. Thus our data suggest that different LPS variants exist in the colonizing H. pylori population, which can adapt to changes in the gastric environment and provide a means to regulate the inflammatory response of the host during disease progression.     

Intestinal Microbiota Regulate Xenobiotic Metabolism in the Liver

Background

The liver is the central organ for xenobiotic metabolism (XM) and is regulated by nuclear receptors such as CAR and PXR, which control the metabolism of drugs. Here we report that gut microbiota influences liver gene expression and alters xenobiotic metabolism in animals exposed to barbiturates.

Principal findings

By comparing hepatic gene expression on microarrays from germfree (GF) and conventionally-raised mice (SPF), we identified a cluster of 112 differentially expressed target genes predominantly connected to xenobiotic metabolism and pathways inhibiting RXR function. These findings were functionally validated by exposing GF and SPF mice to pentobarbital which confirmed that xenobiotic metabolism in GF mice is significantly more efficient (shorter time of anesthesia) when compared to the SPF group.

Conclusion

Our data demonstrate that gut microbiota modulates hepatic gene expression and function by altering its xenobiotic response to drugs without direct contact with the liver.     

Global analysis of alternative splicing regulation by insulin and wingless signaling in Drosophila cells

Background  

Despite the prevalence and biological relevance of both signaling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signaling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis using splicing-sensitive microarrays of changes in alternative splicing induced by activation of two distinct signaling pathways, insulin and wingless, in Drosophila cells in culture.     

Phase relationship between skin temperature and sleep-wake rhythms in women with vascular dysregulation and controls under real-life conditions

The aim of the study was to investigate whether women with primary vascular dysregulation (VD; main symptoms of thermal discomfort with cold extremities) and difficulties initiating sleep (DIS) exhibit a disturbed phase of entrainment (Ψ) under everyday life conditions. The authors predicted a phase delay of the distal-proximal skin temperature gradient and salivary melatonin rhythms with respect to the sleep-wake cycle in women with VD and DIS (WVD) compared to controls (CON), similar to that found in their previous constant-routine laboratory data. A total of 41 young healthy women, 20 with WVD and 21 matched CON without VD and normal sleep onset latency (SOL), were investigated under ambulatory conditions (following their habitual bedtimes) during 7 days of continuous recording of skin temperatures, sleep-wake cycles monitored by actimetry and sleep-wake diaries, and single evening saliva collections for determining the circadian marker of dim light melatonin onset (DLMO). Compared to CON, WVD showed increased distal vasoconstriction at midday and in the evening, as indicated by lower distal (DIST; hands and feet) and foot-calf skin temperatures, and distal-proximal skin temperature gradients (p相似文献