Effect of NaCl on the exothermic and endothermic components of the inverse temperature transition of a model elastin-like polymer

TMDSC data have been employed to observe the effect of NaCl on the inverse temperature transition of the model elastin-like polymer (GVGVP)251. NaCl causes a decrease in Tt and an increase in DeltaH. The increase in enthalpy appears both in the enthalpy related with the folding of the polymer and in the contribution associated with disruption of the structured water of hydrophobic hydration. It has been suggested that the presence of NaCl may cause a better formation of water structures surrounding the apolar polymer chains.     

2-Alkynoic fatty acids inhibit topoisomerase IB from Leishmania donovani

2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC(50)=5.3±0.7μM. The potency of LdTopIB inhibition follows the trend 2-ODA>2-HDA>2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC(50)=11.0μM), but it was less effective against other protozoa, Trypanosoma cruzi (IC(50)=48.1μM) and Trypanosoma brucei rhodesiense (IC(50)=64.5μM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA>2-HDA>2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound.     

Reversible control of biofilm formation by Cellulomonas spp. in response to nitrogen availability

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments.     

The lizard Psammodromus algirus (Squamata: Lacertidae) is darker at high altitudes

Altitudinal gradients offer a good opportunity to study organisms' adaptations to clinal environmental variables. Regarding altitude, the most influential variables on organisms are temperature and ultraviolet (UV) solar radiation, the first decreasing and the second increasing with altitude. Both variables affect ectotherms' biology, as ectotherms depend on environmental temperature for thermoregulation, frequently being heliotherms. Here, we studied dorsal coloration in the lizard Psammodromus algirus (Linnaeus, 1758) along a wide altitudinal gradient (2200 m) in Sierra Nevada (south‐east Spain). We hypothesize that the skin will be darker with altitude, i.e. in environments with lower temperatures and higher UV radiation intensity. Results show that individual dorsal colorations became darker at high altitude. We propose two non‐mutually exclusive explanations for this result: (1) darker dorsal surface would favour faster warming at high altitudes, where temperature is lower, and (2) darker dorsal surface would protect against UV radiation, stronger at high altitudes. We found significant relationships between both temperature and UV radiation and population dorsal darkness, giving mixed support for the two explanations. Moreover, dorsal hue was positively correlated with substrate hue, suggesting that hue evolved to maximize crypsis. Our study therefore suggests that geographical variation in dorsal coloration in this lizard is adaptive, and darkness coloration might have evolved in response to adverse conditions (low temperature and high UV radiation) at high altitudes. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 132–141.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

In Vivo Intracellular pH Measurements in Tobacco and Arabidopsis Reveal an Unexpected pH Gradient in the Endomembrane System

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH −1.5) and Arabidopsis thaliana root cells (ΔpH −2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H⁺ ATPase–dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.     

Stress-Induced Cytokinin Synthesis Increases Drought Tolerance through the Coordinated Regulation of Carbon and Nitrogen Assimilation in Rice

The effects of water deficit on carbon and nitrogen metabolism were investigated in flag leaves of wild-type and transgenic rice (Oryza sativa japonica ‘Kitaake’) plants expressing ISOPENTENYLTRANSFERASE (IPT; encoding the enzyme that mediates the rate-limiting step in cytokinin synthesis) under the control of P_SARK, a maturation- and stress-induced promoter. While the wild-type plants displayed inhibition of photosynthesis and nitrogen assimilation during water stress, neither carbon nor nitrogen assimilation was affected by stress in the transgenic P_SARK::IPT plants. In the transgenic plants, photosynthesis was maintained at control levels during stress and the flag leaf showed increased sucrose (Suc) phosphate synthase activity and reduced Suc synthase and invertase activities, leading to increased Suc contents. The sustained carbon assimilation in the transgenic P_SARK::IPT plants was well correlated with enhanced nitrate content, higher nitrate reductase activity, and sustained ammonium contents, indicating that the stress-induced cytokinin synthesis in the transgenic plants played a role in maintaining nitrate acquisition. Protein contents decreased and free amino acids increased in wild-type plants during stress, while protein content was preserved in the transgenic plants. Our results indicate that the stress-induced cytokinin synthesis in the transgenic plants promoted sink strengthening through a cytokinin-dependent coordinated regulation of carbon and nitrogen metabolism that facilitates an enhanced tolerance of the transgenic plants to water deficit.Plant hormones control many aspects of plant growth and development and the responses of plants to abiotic and biotic stresses. Cytokinins (CKs) have been shown to regulate plant cell differentiation, leaf senescence, and other key developmental processes (Sakakibara, 2006). It has also been shown that CKs regulate assimilate partitioning (Ronzhina and Mokronosov, 1994), sink strength (Kuiper, 1993), and source/sink relationships (Roitsch, 1999). The localized expression in tobacco (Nicotiana tabacum) of a promoterless ISOPENTENYLTRANSFERASE (IPT), a gene encoding the enzyme that catalyzes the rate-limiting step in CK synthesis, enhanced the local sink strength and quickly mobilized nutrients to the tissues with elevated CK (Guivarc’h et al., 2002). Changes in sink/source relationships were also observed in CK-deficient tobacco shoots and roots (Werner et al., 2008). Elevated CK levels enhanced the survival of plants under water-stress conditions (Rivero et al., 2007). The overexpression of IPT under the control of SENESCENCE-ASSOCIATED RECEPTOR KINASE (SARK; a maturation- and stress-induced promoter) improved the drought tolerance of both eudicots (Rivero et al., 2007; Qin et al., 2011) and monocots (Peleg et al., 2011). After a water-stress episode during the reproductive stages (pre and post anthesis), transgenic P_SARK::IPT rice (Oryza sativa) plants displayed higher grain yield than the wild type (Peleg et al., 2011). The transgenic P_SARK::IPT rice exhibited a differential expression of genes encoding enzymes associated with hormone synthesis and hormone-regulated pathways. These results suggested that changes in hormone homeostasis induced the modification of source/sink relationships in the transgenic plants, resulting in higher grain yields under stress conditions (Peleg et al., 2011).The maintenance of carbon (C) and nitrogen (N) assimilation is of paramount importance to ensure sink strength and improve stress tolerance without yield penalties. The interactions between C and N metabolism are vital for plant growth and development, and complex mechanisms operate in the plant to coordinate C assimilation with N metabolism (Nunes-Nesi et al., 2010). Thus, plants respond to changes in C and N metabolites through the regulation of translation and posttranslational modification mechanisms. C and N metabolites activate signaling pathways that regulate enzyme and transporter activities that control C and N fluxes, optimizing the plant response to developmental and environmental cues changing source/sink relationships (Coruzzi and Zhou, 2001).Plant hormones affect, either directly or indirectly, these pathways and can act antagonistically or synergistically when responding to environmental stress (Wilkinson et al., 2012). The exposure of plants to water-limiting conditions results in abscisic acid (ABA) synthesis that induces ABA-dependent gene expression (Yamaguchi-Shinozaki and Shinozaki, 2006), triggering the closure of stomata and reducing water loss during drought (Wilkinson and Davies, 2010). Other hormones, in particular CK, salicylic acid, ethylene, and jasmonic acid, also play direct or indirect roles in the plant responses to abiotic stress (Peleg and Blumwald, 2011). Under drought stress, plant CK content decreases, and the reduction in CK increases the plant responses to increasing ABA (Davies and Zhang, 1991), inducing stomata closure and inhibiting photosynthesis (Rivero et al., 2010). Our previous results suggested that the stress-induced CK synthesis, driven by a stress-induced promoter, protected against the deleterious effects of water deficit on the photosynthetic apparatus, allowing higher photosynthetic rates and higher yields after water deficit in tobacco (Rivero et al., 2009) and cotton (Gossypium hirsutum; Kuppu et al., 2013) plants grown in the greenhouse and peanut (Arachis hypogaea) plants grown under field conditions (Qin et al., 2011).Here, we analyzed gene expression profiles, metabolites, and enzymatic and photosynthetic activities of flag leaves of wild-type and transgenic rice expressing P_SARK::IPT exposed to water deficit during the reproductive stage and identified metabolic processes associated with the enhanced tolerance of the transgenic plants to water deficit. Our results indicate that the stress-induced CK synthesis in the transgenic plants promoted sink strengthening through the maintenance and coordination of N and C assimilation during water stress.     

Structural Bases of Coronavirus Attachment to Host Aminopeptidase N and Its Inhibition by Neutralizing Antibodies

The coronaviruses (CoVs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10–20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN), a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S) mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs) of two closely related CoV strains, transmissible gastroenteritis virus (TGEV) and porcine respiratory CoV (PRCV), in complex with their receptor, porcine APN (pAPN), or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.