Effects of inorganic phosphate on endothermic force generation in muscle.

Using a rapid (ca. 0.2 ms) laser temperature jump technique, the rate of endothermic force generation was examined in single-skinned (rabbit psoas) muscle fibres when they were exposed to different levels of inorganic phosphate (a product released during ATP hydrolysis in active muscle). The steady force is reduced by increased phosphate but the apparent rate constant of force generation induced by a standard temperature jump (from ca. 9 degrees C to ca. 12 degrees C) increases two- to threefold when the phosphate added is increased from zero to ca. 25 mM. The increase in the apparent rate constant also exhibits saturation at higher phosphate levels and the relation is hyperbolic. Detailed examination of the data, particularly in relation to our pressure release experiments, leads to a scheme for the molecular steps involved in phosphate release and force generation in active muscle fibres, where phosphate release from attached cross-bridges involves three reversible and sequentially faster molecular steps. Step one is a moderately slow, pre-force generation step that probably represents a transition of cross-bridges from non-specific to stereospecific attached states. Step two is moderately fast and represents endothermic cross-bridge force generation (temperature sensitive) and step three is a very rapid phosphate release. Such a scheme accommodates findings from a variety of different studies, including pressure perturbation experiments and other studies where the effect of phosphate on muscle force was studied.     

Synthesis of GSK3β mimetic inhibitors of Akt featuring a novel extended dipeptide surrogate

Akt is a cardinal nodal point in PI3K signaling pathway and confers resistance to apoptosis through inactivation of regulatory substrates such as GSK3β. Efforts to inhibit the kinase activity of Akt have largely focused on targeting the ATP-binding domain of Akt. Here, we present the design and synthesis of conformationally constrained GSK3β mimics featuring a novel extended dipeptide surrogate core. This effort resulted in the identification of a novel substrate mimetic Akt inhibitor (11) with low micromolar activity in vitro (Akt1 IC(50)=3.1 μM).     

A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution

The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops, despite size being an important agronomic trait. Here we show that the expression level of a microRNA gene (miRNA172) influences fruit size in apple. A transposon insertional allele of miRNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over‐expression of miRNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to be co‐located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective sweeps are normally weaker in perennial crops than in annual crops.     

A protective role for human IL-10-expressing CD4+ T cells in colitis

IL-10 is an immunoregulatory cytokine expressed by numerous cell types. Studies in mice confirm that different IL-10-expressing cell subsets contribute differentially to disease phenotypes. However, little is known about the relationship between cell- or tissue-specific IL-10 expression and disease susceptibility in humans. In this study, we used the previously described human (h)IL10BAC transgenic model to examine the role of hIL-10 in maintaining intestinal homeostasis. Genomically controlled hIL-10 expression rescued Il10(-/-) mice from Helicobacter-induced colitis and was associated with control of proinflammatory cytokine expression and Th17 cell accumulation in gut tissues. Resistance to colitis was associated with an accumulation of hIL-10-expressing CD4(+)Foxp3(+) regulatory T cells specifically within the lamina propria but not other secondary lymphoid tissues. Cotransfer of CD4(+)CD45RB(lo) cells from Il10(-/-)/hIL10BAC mice rescued Rag1(-/-) mice from colitis, further suggesting that CD4(+) T cells represent a protective source of hIL-10 in the colon. In concordance with an enhanced capacity to express IL-10, CD4(+)CD44(+) T cells isolated from the lamina propria exhibited lower levels of the repressive histone mark H3K27Me3 and higher levels of the permissive histone mark acetylated histone H3 in both the human and mouse IL10 locus compared with the spleen. These results provide experimental evidence verifying the importance of T cell-derived hIL-10 expression in controlling inflammation within the colonic mucosa. We also provide molecular evidence suggesting the tissue microenvironment influences IL-10 expression patterns and chromatin structure in the human (and mouse) IL10 locus.     

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl⁻-HCO₃⁻ exchanger and Cl⁻ channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl⁻-HCO₃⁻ exchange and Cl⁻ currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-ΔSTAS) virtually eliminated the Cl⁻ currents with only a modest affect on nCl⁻-HCO₃⁻ exchange activity. Co-expression of a9-ΔSTAS and the (R)CFTR fragment did not alter the residual a9-ΔSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.Slc26 genes and proteins have attracted the attention of physiologists and geneticists. Why? Slc26a1 (Sat-1) was characterized as a Na⁺-independent SO₄²⁻ transporter (1). Given the transport characteristics of the founding member of the gene family, Slc26 proteins were assumed to be sulfate transporters. Disease phenotypes, clone characterization, and family additions demonstrate that the Slc26 proteins are anion transporters or channels (2–4). These proteins have varied tissue expression patterns. At one extreme, Slc26a5 in mammals is found in the hair cells of the inner ear (5), whereas Slc26a2 (DTDST) is virtually ubiquitous in epithelial tissues (2).Several Slc26 proteins are found in the epithelia of the lung, intestine, stomach, pancreas, and kidney, usually in apical membranes. Interestingly these are also tissues and membranes in which the cystic fibrosis transmembrane conductance regulator (CFTR)⁵ has been found functionally or by immunohistochemistry. Ko and co-workers (6–8) examined the distribution of Slc26a3 and Slc26a6 in HCO₃⁻ secretory epithelia, and asked if an interaction might occur between these Slc26 proteins and CFTR. In particular, these studies indicate that in expression systems, there is a reciprocal-stimulatory interaction of the STAS (sulfate transporter anti-sigma) domains of Slc26a3 and Slc26a6 with the regulatory region (R-region) of CFTR. These investigators hypothesized that this stimulatory interaction could account for the differences in pancreatic insufficiency and sufficiency observed in cystic fibrosis patients. Nevertheless, knock-out Slc26a6 mouse studies reveal more complicated cell and tissue physiology (see “Discussion”).Slc26a9 has been reported to be a Cl⁻-HCO₃⁻ exchanger (9, 10) or a large Cl⁻ conductance (3, 11, 12). Loriol and co-workers (12) indicated that SLC26A9 has a Cl⁻ conductance that may be stimulated by HCO₃⁻. Two other groups have indicated that the Cl⁻ conductance is not affected by the presence of HCO₃⁻ (10, 11). We have recently demonstrated that Slc26a9 functions as both an electrogenic nCl⁻-HCO₃⁻ exchanger and a Cl⁻ channel (10). Dorwart and colleagues (11) found that WNK kinases inhibited the SLC26A9 Cl⁻ conductance but that this effect was independent of kinase activity. One group has a preliminary report indicating that WNK3 decreased Cl⁻ uptake, whereas WNK4 increased Cl⁻ uptake via Slc26a9 expressed in Xenopus oocytes (13).Slc26a9 and CFTR are also co-expressed in several tissues. Slc26a9 protein has been localized to epithelia of the stomach and lung (9, 10, 14), although mRNA is also detectable in brain, heart, kidney, small intestine, thymus, and ovary (10). The R-region of CFTR was previously shown to increase the activity of Slc26a3 and Slc26a6 by interaction with STAS domains (6, 15, 16). Because Slc26a9 displays several different modes of ion transport, we asked if the R-region of CFTR would also increase the activity of Slc26a9. Our results indicate that the R-region of CFTR does interact with the STAS domain of Slc26a9. However, in the case of Slc26a9 this apparently similar interaction results in inhibition of Slc26a9 ion transport.     

Human RAD52 protein has extreme thermal stability.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 associate into seven-membered rings. These rings can form higher order complexes. RAD52 binds to DNA breaks, and recent studies suggest that the higher order self-association of the rings promotes DNA end joining. Monomers of the RAD52(1--192) deletion mutant also associate into ring structures but do not form higher order complexes. The thermal stability of wild-type and mutant RAD52 was studied by differential scanning calorimetry. Three thermal transitions (labeled A, B, and C) were observed with melting temperatures of 38.8, 73.1, and 115.2 degrees C. The RAD52(1--192) mutant had only two thermal transitions at 47.6 and 100.9 degrees C (labeled B and C). Transitions were labeled such that transition C corresponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic light scattering. From these data a four-state hypothetical model was developed to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A was attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transition C to complete unfolding. The ring-shaped quaternary structure of RAD52 and the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are stable at physiological temperatures in vitro.     

Genome mapping of three major resistance genes to woolly apple aphid (Eriosoma lanigerum Hausm.)

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) can be a major economic problem to apple growers in most parts of the world, and resistance breeding provides a sustainable means to control this pest. We report molecular markers for three genes conferring WAA resistance and placing them on two linkage groups (LG) on the genetic map of apple. The Er1 and Er2 genes derived from ‘Northern Spy’ and ‘Robusta 5,’ respectively, are the two major genes that breeders have used to date to improve the resistance of apple rootstocks to this pest. The gene Er3, from ‘Aotea 1’ (an accession classified as Malus sieboldii), is a new major gene for WAA resistance. Genetic markers linked to the Er1 and Er3 genes were identified by screening random amplification of polymorphic deoxyribonucleic acid (DNA; RAPD) markers across DNA bulks from resistant and susceptible plants from populations segregating for these genes. The closest RAPD markers were converted into sequence-characterized amplified region markers and the genome location of these two genes was assigned to LG 08 by aligning the maps around the genes with a reference map of ‘Discovery’ using microsatellite markers. The Er2 gene was located on LG 17 of ‘Robusta 5’ using a genetic map developed in a M.9 × ‘Robusta 5’ progeny. Markers for each of the genes were validated for their usefulness for marker-assisted selection in separate populations. The potential use of the genetic markers for these genes in the breeding of apple cultivars with durable resistance to WAA is discussed.     

Homology modeling and molecular dynamics simulation studies of an inward rectifier potassium channel

A homology model has been generated for the pore-forming domain of Kir6.2, a component of an ATP-sensitive K channel, based on the x-ray structure of the bacterial channel KcsA. Analysis of the lipid-exposed and pore-lining surfaces of the model reveals them to be compatible with the known features of membrane proteins and Kir channels, respectively. The Kir6.2 homology model was used as the starting point for nanosecond-duration molecular dynamics simulations in a solvated phospholipid bilayer. The overall drift from the model structure was comparable to that seen for KcsA in previous similar simulations. Preliminary analysis of the interactions of the Kir6.2 channel model with K(+) ions and water molecules during these simulations suggests that concerted single-file motion of K(+) ions and water through the selectivity filter occurs. This is similar to such motion observed in simulations of KcsA. This suggests that a single-filing mechanism is conserved between different K channel structures and may be robust to changes in simulation details. Comparison of Kir6.2 and KcsA suggests some degree of flexibility in the filter, thus complicating models of ion selectivity based upon a rigid filter.     

Human RAD52 exhibits two modes of self-association

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 self-associate into rings that can then form higher order complexes. RAD52 binds to double-strand DNA ends, and recent studies suggest that the higher order self-association of the rings promotes DNA end-joining. Earlier studies defined the self-association domain of RAD52 to a unique region in the N-terminal half of the protein. Here we show that there are in fact two experimentally separable self-association domains in RAD52. The N-terminal self-association domain mediates the assembly of monomers into rings, and the previously unidentified domain in the C-terminal half of the protein mediates higher order self-association of the rings.     

A Cross-Bridge Cycle with Two Tension-Generating Steps Simulates Skeletal Muscle Mechanics

We examined whether cross-bridge cycle models with one or two tension-generating steps can account for the force-velocity relation of and tension response to length steps of frog skeletal muscle. Transition-state theory defined the strain dependence of the rate constants. The filament stiffness was non-Hookean. Models were refined against experimental data by simulated annealing and downhill simplex runs. Models with one tension-generating step were rejected, as they had a low efficiency and fitted the experimental data relatively poorly. The best model with two tension-generating steps (stroke distances 5.6 and 4.6 nm) and a cross-bridge stiffness of 1.7 pN/nm gave a good account of the experimental data. The two tensing steps allow an efficiency of up to 38% during shortening. In an isometric contraction, 54.7% of the attached heads were in a pre-tension-generating state, 44.5% of the attached heads had undergone the first tension-generating step, and only 0.8% had undergone both tension-generating steps; they bore 34%, 64%, and 2%, respectively, of the isometric tension. During slow shortening, the second tensing step made a greater contribution. During lengthening, up to 93% of the attached heads were in a pre-tension-generating state yet bore elevated tension by being dragged to high strains before detaching.