Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

The melibiose permease of Salmonella typhimurium (MelB_St) catalyzes the stoichiometric symport of galactopyranoside with a cation (H⁺, Li⁺, or Na⁺) and is a prototype for Na⁺-coupled major facilitator superfamily (MFS) transporters presenting from bacteria to mammals. X-ray crystal structures of MelB_St have revealed the molecular recognition mechanism for sugar binding; however, understanding of the cation site and symport mechanism is still vague. To further investigate the transport mechanism and conformational dynamics of MelB_St, we generated a complete single-Cys library containing 476 unique mutants by placing a Cys at each position on a functional Cys-less background. Surprisingly, 105 mutants (22%) exhibit poor transport activities (<15% of Cys-less transport), although the expression levels of most mutants were comparable to that of the control. The affected positions are distributed throughout the protein. Helices I and X and transmembrane residues Asp and Tyr are most affected by cysteine replacement, while helix IX, the cytoplasmic middle-loop, and C-terminal tail are least affected. Single-Cys replacements at the major sugar-binding positions (K18, D19, D124, W128, R149, and W342) or at positions important for cation binding (D55, N58, D59, and T121) abolished the Na⁺-coupled active transport, as expected. We mapped 50 loss-of-function mutants outside of these substrate-binding sites that suffered from defects in protein expression/stability or conformational dynamics. This complete Cys-scanning mutagenesis study indicates that MelB_St is highly susceptible to single-Cys mutations, and this library will be a useful tool for further structural and functional studies to gain insights into the cation-coupled symport mechanism for Na⁺-coupled MFS transporters.     

Climatic effects on the breeding phenology and reproductive success of an arctic-nesting goose species

Climate warming is pronounced in the Arctic and migratory birds are expected to be among the most affected species. We examined the effects of local and regional climatic variations on the breeding phenology and reproductive success of greater snow geese ( Chen caerulescens atlantica ), a migratory species nesting in the Canadian Arctic. We used a long-term dataset based on the monitoring of 5447 nests and the measurements of 19 234 goslings over 16 years (1989–2004) on Bylot Island. About 50% of variation in the reproductive phenology of individuals was explained by spring climatic factors. High mean temperatures and, to a lesser extent, low snow cover in spring were associated with an increase in nest density and early egg-laying and hatching dates. High temperature in spring and high early summer rainfall were positively related to nesting success. These effects may result from a reduction in egg predation rate when the density of nesting geese is high and when increased water availability allows females to stay close to their nest during incubation recesses. Summer brood loss and production of young at the end of the summer increased when values of the summer Arctic Oscillation (AO) index were either very positive (low temperatures) or very negative (high temperatures), indicating that these components of the breeding success were most influenced by the regional summer climate. Gosling mass and size near fledging were reduced in years with high spring temperatures. This effect is likely due to a reduced availability of high quality food in years with early spring, either due to food depletion resulting from high brood density or a mismatch between hatching date of goslings and the timing of the peak of plant quality. Our analysis suggests that climate warming should advance the reproductive phenology of geese, but that high spring temperatures and extreme values of the summer AO index may decrease their reproductive success up to fledging.     

Requirement for the second coding exon of Tat in the optimal replication of macrophage-tropic HIV-1

HIV-1 Tat is essential for virus replication and is a potent transactivator of viral gene expression. Evidence suggests that Tat also influences virus infectivity and cytopathicity. Here, we find that the second coding exon of Tat contributes a novel function for the replication/infectivity of macrophage-tropic HIV-1. We show that macrophage-tropic HIV-1 which expresses the full-length two-exon form of Tat replicates better in monocyte-derived macrophages (MDM) than an otherwise isogenic virus which expresses only the one-exon form of Tat. Similarly, two-exon Tat expressing HIV-1 also replicates better than one-exon Tat expressing HIV-1 in two different models of human cells/tissue reconstituted SCID mice.     

Mortality in adult intensive care patients with severe systemic inflammatory response syndromes is strongly associated with the hypo-immune TNF −238A polymorphism

The systemic inflammatory response syndrome (SIRS) is associated with activation of innate immunity. We studied the association between mortality and measures of disease severity in the intensive care unit (ICU) and functional polymorphisms in genes coding for Toll-like receptor 4 (TLR4), macrophage migratory inhibitory factor (MIF), tumour necrosis factor (TNF) and lymphotoxin-alpha (LTA). Two hundred thirty-three patients with severe SIRS were recruited from one general adult ICU in a tertiary centre in the UK. DNA from patients underwent genotyping by 5′ nuclease assay. Genotype was compared to phenotype. Primary outcome was mortality in ICU. Minor allele frequencies were TLR4 +896G 7%, MIF 173C 16%, TNF ?238A 10% and LTA +252G 34%. The frequency of the hypoimmune minor allele TNF ?238A was significantly higher in patients who died in ICU compared to those who survived (p?=?0.0063) as was the frequency of the two haplotypes LTA +252G, TNF ?1031T, TNF ?308G, TNF ?238A and LTA +252G, TNF?1031T, TNF?308A and TNF?238A (p?=?0.0120 and 0.0098, respectively). These findings re-enforce the view that a balanced inflammatory/anti-inflammatory response is the most important determinant of outcome in sepsis. Genotypes that either favour inflammation or its counter-regulatory anti-inflammatory response are likely to influence mortality and morbidity.     

Exceptional carbon uptake in European forests during the warm spring of 2007: a data–model analysis

Temperate and boreal forests undergo drastic functional changes in the springtime, shifting within a few weeks from net carbon (C) sources to net C sinks. Most of these changes are mediated by temperature. The autumn 2006–winter 2007 record warm period was followed by an exceptionally warm spring in Europe, making spring 2007 a good candidate for advances in the onset of the photosynthetically active period. An analysis of a decade of eddy covariance data from six European forests stands, which encompass a wide range of functional types (broadleaf evergreen, broadleaf deciduous, needleleaf evergreen) and a wide latitudinal band (from 44° to 62°N), revealed exceptional fluxes during spring 2007. Gross primary productivity (GPP) of spring 2007 was the maximum recorded in the decade examined for all sites but a Mediterranean evergreen forest (with a +40 to +130 gC m^?2 anomaly compared with the decadal mean over the January–May period). Total ecosystem respiration (TER) was also promoted during spring 2007, though less anomalous than GPP (with a +17 to +93 gC m^?2 anomaly over 5 months), leading to higher net uptake than the long‐term mean at all sites (+12 to +79 gC m^?2 anomaly over 5 months). A correlative analysis relating springtime C fluxes to simple phenological indices suggested spring C uptake and temperatures to be related. The CASTANEA process‐based model was used to disentangle the seasonality of climatic drivers (incoming radiation, air and soil temperatures) and biological drivers (canopy dynamics, thermal acclimation of photosynthesis to low temperatures) on spring C fluxes along the latitudinal gradient. A sensitivity analysis of model simulations evidenced the roles of (i) an exceptional early budburst combined with elevated air temperature in deciduous sites, and (ii) an early relief of winter thermal acclimation in coniferous sites for the promotion of 2007 spring assimilation.     

Understanding Fluctuations in Bobcat Harvest at the Northern Limit of Their Range

ABSTRACT In Quebec, Canada, harvest of bobcats (Lynx rufus) started to decline in 1985 and by 1991, harvest seasons were closed due to concerns of a perceived population decline. Since the closing of harvest season in 1991, the average temperature has increased, snow quantity has decreased, and important changes in agriculture and forest management have occurred. In light of changing conditions, the situation of Quebec bobcats needed reassessment. Thus, we analyzed harvest data to clarify the current status of bobcat populations in Quebec. From 1980 to 1991, bobcat harvest in Quebec was strongly correlated with bobcat harvest in Maine (USA), Nova Scotia (Canada), Ontario (Canada), and Vermont (USA). Extrapolations of harvest in Quebec relative to harvest in Maine, Ontario, Vermont, and Nova Scotia suggested an increase in number of bobcats after 1991. Mass of male and female bobcats before 1991 was less than mass of animals captured after 1991. Percentage of juveniles in the reported harvest before 1991 was higher than after 1991. However, percentage of males and litter sizes in the harvest did not differ before and after 1991. The geographic distribution of bobcats captured has gradually expanded after the closure of the harvest season. Our findings suggest that bobcat populations in Quebec have recovered from the 1985–1991 decline, and that the harvest season for this species could resume. This study also illustrates how managers can rely on data from neighboring jurisdiction to manage species when local harvest data is unavailable.     

Circadian and Social Cues Regulate Ion Channel Trafficking

Electric fish generate and sense electric fields for navigation and communication. These signals can be energetically costly to produce and can attract electroreceptive predators. To minimize costs, some nocturnally active electric fish rapidly boost the power of their signals only at times of high social activity, either as night approaches or in response to social encounters. Here we show that the gymnotiform electric fish Sternopygus macrurus rapidly boosts signal amplitude by 40% at night and during social encounters. S. macrurus increases signal magnitude through the rapid and selective trafficking of voltage-gated sodium channels into the excitable membranes of its electrogenic cells, a process under the control of pituitary peptide hormones and intracellular second-messenger pathways. S. macrurus thus maintains a circadian rhythm in signal amplitude and adapts within minutes to environmental events by increasing signal amplitude through the rapid trafficking of ion channels, a process that directly modifies an ongoing behavior in real time.