A dominant role for CD8+-T-lymphocyte selection in simian immunodeficiency virus sequence variation

CD8(+) T lymphocytes (CD8-TL) select viral escape variants in both human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. The frequency of CD8-TL viral escape as well as the contribution of escape to overall virus diversification has not been assessed. We quantified CD8-TL selection in SIV infections by sequencing viral genomes from 35 SIVmac239-infected animals at the time of euthanasia. Here we show that positive selection for sequences encoding 46 known CD8-TL epitopes is comparable to the positive selection observed for the variable loops of env. We also found that >60% of viral variation outside of the viral envelope occurs within recognized CD8-TL epitopes. Therefore, we conclude that CD8-TL selection is the dominant cause of SIV diversification outside of the envelope.     

Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta)

To determine the physiological roles of peroxisome proliferator-activated receptor beta (PPARbeta), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARbeta gene. Homozygous PPARbeta-null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARbeta-null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARbeta-null mice. PPARbeta was not required for induction of mRNAs involved in epidermal differentiation induced by O-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARbeta-null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARbeta-null mice. These results are the first to provide in vivo evidence of significant roles for PPARbeta in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.     

Voltage-Sensor Transitions of the Inward-Rectifying K+ Channel KAT1 Indicate a Latching Mechanism Biased by Hydration within the Voltage Sensor

The Kv-like (potassium voltage-dependent) K⁺ channels at the plasma membrane, including the inward-rectifying KAT1 K⁺ channel of Arabidopsis (Arabidopsis thaliana), are important targets for manipulating K⁺ homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K⁺ channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (Phe) residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the context of the effects on hydration of amino acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel.Plant cells utilize the potassium ion (K⁺) to maintain hydrostatic (turgor) pressure, to drive irreversible cell expansion for growth, and to facilitate reversible changes in cell volume during stomatal movements. Potassium uptake and its circulation throughout the plant relies both on high-affinity, H⁺-coupled K⁺ transport (Quintero and Blatt, 1997; Rubio et al., 2008) and on K⁺ channels to facilitate K⁺ ion transfer across cell membranes. Uptake via K⁺ channels is thought to be responsible for roughly 50% of the total K⁺ content of the plant under most field conditions (Spalding et al., 1999; Rubio et al., 2008; Amtmann and Blatt, 2009). K⁺ channels confer on the membranes of virtually every tissue distinct K⁺ conductances and regulatory characteristics (Véry and Sentenac, 2003; Dreyer and Blatt, 2009). Their characteristics are thus of interest for engineering directed to manipulating K⁺ flux in many aspects of plant growth and cellular homeostasis. The control of K⁺ channel gating has been identified as the most promising target for the genetic engineering of stomatal responsiveness (Lawson and Blatt, 2014; Wang et al., 2014a), based on the recent development of quantitative systems models of guard cell transport and metabolism (Chen et al., 2012b; Hills et al., 2012; Wang et al., 2012). By contrast, modifying the expression and, most likely, the population of native K⁺ channels at the membrane was found to have no substantial effect on stomatal physiology (Wang et al., 2014b).The Kv-like K⁺ channels of the plant plasma membrane (Pilot et al., 2003; Dreyer and Blatt, 2009) share a number of structural features with the Kv superfamily of K⁺ channels characterized in animals and Drosophila melanogaster (Papazian et al., 1987; Pongs et al., 1988). The functional channels assemble from four homologous subunits and surround a central transmembrane pore that forms the permeation pathway (Daram et al., 1997). Each subunit comprises six transmembrane α-helices, designated S1 to S6, and both N and C termini are situated on the cytosolic side of the membrane (Uozumi et al., 1998). The pore or P loop between the S5 and S6 α-helices incorporates a short α-helical stretch and the highly conserved amino acid sequence TxGYGD, which forms a selectivity filter for K⁺ (Uozumi et al., 1995; Becker et al., 1996; Nakamura et al., 1997). The carbonyl oxygen atoms of these residues in all four K⁺ channel subunits face inward to form coordination sites for K⁺ ions between them (Doyle et al., 1998; Jiang et al., 2003; Kuo et al., 2003; Long et al., 2005) and a multiple-ion pore (Thiel and Blatt, 1991) such that K⁺ ions pass through the selectivity filter as if in free solution. The plant channels are also sensitive to a class of neurotoxins that exhibit high specificity in binding around the mouth of the channel pore (Obermeyer et al., 1994).These K⁺ channels also share a common gating mechanism. Within each subunit, the first four α-helices form a quasiindependent unit, the voltage sensor domain (VSD), with the S4 α-helix incorporating positively charged (Arg or Lys) residues regularly positioned across the lipid bilayer and transmembrane electric field. Voltage displaces the S4 α-helix within the membrane and couples rotation of the S5 and S6 α-helices lining the pore, thereby opening or closing the channel (Sigworth, 2003; Dreyer and Blatt, 2009). For outward-rectifying channels, such as the mammalian Kv1.2 and the D. melanogaster Shaker K⁺ channels, an inside-positive electric field drives the positively charged, S4 α-helix outward (the up position), which draws on the S4-S5 linker to open the pore. This simple expedient of a lever and string secures current flow in one direction by favoring opening at positive, but not negative, voltages. This same model applies to the Arabidopsis (Arabidopsis thaliana) Kv-like K⁺ channels, including outward rectifiers that exhibit sensitivity to external K⁺ concentration (Blatt, 1988; Blatt and Gradmann, 1997; Johansson et al., 2006), and it serves equally in the gating of inward-rectifying K⁺ channels such as KAT1, which gates open at negative voltages (Dreyer and Blatt, 2009).Studies of KAT1 gating (Latorre et al., 2003; Lai et al., 2005) have indicated that the S4 α-helix of the channel most likely undergoes very similar conformational changes with voltage as those of the mammalian and Shaker K⁺ channels. These findings conform with the present understanding of the evolution of VSD structure (Palovcak et al., 2014) and the view of a common functional dynamic to its molecular design. It is likely, therefore, that a similar electrostatic network occurs in KAT1 to stabilize the VSD. Crucially, however, experimental evidence in support of such a network has yet to surface. Electrostatic countercharges and the hydration of amino acid side chains between the α-helices within the VSDs of mammalian and Shaker K⁺ channel models are important for the latch-like stabilization of the so-called down and up states of these channels (Tao et al., 2010; Pless et al., 2011). Nonetheless, some studies (Gajdanowicz et al., 2009; Riedelsberger et al., 2010) have pointed to subtle differences in the structure of KAT1 that relate to the VSD.We have explored the electrostatic network of the KAT1 VSD through site-directed mutagenesis to manipulate the voltage dependence of KAT1, combining these studies with molecular dynamic simulations previously shown to accommodate the plant VSDs and their hydration during gating transitions (Gajdanowicz et al., 2009; Garcia-Mata et al., 2010). We report here that gating of KAT1 is sensitive to manipulations affecting a set of electrostatic charge transfer centers. These findings conform in large measure to the mammalian and Shaker models. However, virtually all manipulations affecting a highly conserved, central Phe favor the up state of the VSD and the closed KAT1 channel, whereas mutations affecting the electrostatic networks on either side of this Phe favor the down state of the VSD and the open channel. These and additional observations suggest that hydration within the VSD is a major determinant of KAT1 gating.     

Inter-varietal differences in xylem exudate composition and growth under contrasting forms of N supply in cucumber

Variations in the inorganic and organic composition of xylem exudate, growth and N content under contrasting forms of N supply in three cucumber cultivars (Hyclos, Medusa and Victory) were studied in glasshouse conditions. The plants were grown hydroponically with two NO₃ ^-:NH₄ ⁺ ratios (100:0 and 60:40).The xylem sap of Medusa grown with both N sources displayed an increase of organic N and carboxylate concentrations and a decrease of cations, inorganic anions and carbohydrates compared with that of those grown with NO₃ ^- alone, showing a higher growth and N content in tissues and thus better utilization of N supplied as NO₃ ^- and NH₄ ⁺. Mixed N nutrition in Hyclos caused the greatest amounts of NO₃ ^- and NH₄ ⁺ in xylem sap, lower root weight and N levels in the leaves, while its root was unable to generate an adequate supply of organic N compounds. Despite the levels of cations, inorganic and organic anions were reduced by the NH₄ ⁺ supplied to Victory, the ionic balance in the xylem sap, growth and N content remained similar to that of those supplied with NO₃ ^- alone. Finally, the cucumber cultivars studied here, responded differently to the form of N supplied, it may partly be due to their ability of assimilating N in the roots and partly to the form in which the N is translocated to the shoot.     

The interaction between the adaptor protein APS and Enigma is involved in actin organisation

APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.     

Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia.

We examined the genetic variability in the pig–human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.     

Are endophytic microorganisms involved in the stereoselective reduction of ketones by Daucus carota root?

Four strains of endophytic microorganisms isolated from carrot root were able to carry out the reduction of the carbonyl group with diverse degree of enantio-, and diasteroselectivity. Furthermore, biotransformation in the presence of bacterial inhibitor affects the stereochemical outcome of the reaction, and the concomitant addition of a yeast inhibitor results in a large decrease in the conversion percentage. These results indicate that endophytic microorganisms might be involved in the enantioselective reduction of ketones and ketoesters with fresh carrot root pieces.     

Comparative Growth of Natural Bacterial Isolates on Various Lignin-Related Compounds

Bacterial strains were isolated on the basis of their ability to proliferate in a minimal medium containing one of a series of lignin-related compounds as the sole carbon and energy source. These included the aromatic monomers guaiacol, vanillic and coumaric acids, a dimer and a trimer possessing the arylglycerol-β-aryl ether linkage, anisoin, and both the ether-soluble and -insoluble fractions of kraft lignin. The growth of the strains on each of these compounds was measured. The results showed that the metabolic properties of the strains varied according to the structure of the carbon sources used for their selection. Spectrophotometric tracings of the culture medium during the log phase of growth of one of the strains on the β-O-4 dimer revealed decomposition with the release of guaiacol.