Structural implications into dsRNA binding and RNA silencing suppression by NS3 protein of Rice Hoja Blanca Tenuivirus

Rice Hoja Blanca Tenuivirus (RHBV), a negative strand RNA virus, has been identified to infect rice and is widely transmitted by the insect vector. NS3 protein encoded by RHBV RNA3 was reported to be a potent RNAi suppressor to counterdefense RNA silencing in plants, insect cells, and mammalian cells. Here, we report the crystal structure of the N-terminal domain of RHBV NS3 (residues 21–114) at 2.0 Å. RHBV NS3 N-terminal domain forms a dimer by two pairs of α-helices in an anti-parallel mode, with one surface harboring a shallow groove at the dimension of 20 Å × 30 Å for putative dsRNA binding. In vitro RNA binding assay and RNA silencing suppression assay have demonstrated that the structural conserved residues located along this shallow groove, such as Arg50, His51, Lys77, and His85, participate in dsRNA binding and RNA silencing suppression. Our results provide the initial structural implications in understanding the RNAi suppression mechanism by RHBV NS3.     

Erratum to: Characterization of a KCS-like KASII from<Emphasis Type="Italic"> Jessenia bataua</Emphasis> that Elongates Saturated and Monounsaturated Stearic Acids in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the “fuel vs food” debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.     

First somatic mutation of E2F1 in a critical DNA binding residue discovered in well-differentiated papillary mesothelioma of the peritoneum

Background

Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP.

Results

WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector.

Conclusions

The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1.     

siRNA, miRNA and HIV: promises and challenges

Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).     

Physiological responses to single versus double stepping pattern of ascending the stairs

The aim of this study was to compare the physiological responses and energy cost between two ascending patterns, the single-step (SS) and the double-step (DS), in climbing a public staircase. In the SS pattern, a person climbs one step at a time whilst in the double-step (DS) pattern, the individual traverses two steps in a single stride. Advocates of each stepping pattern claimed that their type of ascent is physically more taxing and expends more calories. Thirty subjects (10 males and 20 females) climbed a typical 11-storey flat (each step height of 0.15 m, a total of 180 steps and a vertical displacement of 27.0 m). The subjects climbed using either the SS pattern at a tempo of 100 steps x min(-1) or the DS pattern at 50 steps x min(-1). The prescribed stepping frequencies ensured that an equal amount of total work was performed between the SS and DS patterns. The climbing patterns were performed in random order. Physiological measures during the last 30 s of the climbs were used in the comparative analysis. The results showed that ventilation, oxygen uptake and heart rate values were significantly higher (all p < 0.01) in the SS as compared to the DS pattern. However, the caloric expenditure during the SS pattern was calculated to be only marginally higher than the DS pattern. In conclusion, ascending with the SS pattern led to significantly higher physiological responses compared to the DS pattern. The higher calorie expended with the SS compared to the DS pattern was deemed to be of little practical significance.     

The low affinity Fc receptor for IgG functions as an effective cytolytic receptor for self-specific CD8 T cells

We have recently described a population of self-Ag-specific murine CD8(+) T cells with a memory phenotype that use receptors of both the adaptive and innate immune systems in the detection of transformed and infected cells. In this study we show that upon activation with IL-2 with or without Ag, between 10 and 20% of the activated self-specific CD8(+) T cells express the low affinity FcR for IgG. By contrast, all IL-2-activated NK cells express high levels of this FcR. The FcR comprises the FcgammaRIIIalpha and FcRgamma subunits. However, the FcRgamma subunit also associates with the CD3 complex, and this association probably contributes to the low expression of FcR in activated cells. Although the FcR is expressed at a low level on activated self-specific CD8(+) T cells, it functions very efficiently as a cytolytic receptor in ADCC. FcR-dependent killing occurred in the absence of TCR stimulation, but could be augmented by concurrent stimulation of the TCR. In addition to mediating ADCC, engagement of the FcR on self-specific CD8(+) T cells results in the production of both IFN-gamma and TNF-alpha. This is the first report of an activating FcR on self-specific murine CD8(+)alphabeta TCR(+) T cells and establishes the importance of innate immune system receptors in the function of these self-specific CD8(+) T cells.     

Critical role of TNF receptor type-2 (p75) as a costimulator for IL-2 induction and T cell survival: a functional link to CD28

CD28 provides important signals that lower the threshold of T cell activation, augment the production of IL-2, and promote T cell survival. The recent identification of a second family of costimulatory molecules within the TNFR family has reshaped the "two-signal" model of T cell activation. In this study the role of p75 as a T cell costimulatory molecule in controlling cell fate during TCR/CD28-mediated stimulation was examined. We found that p75-deficient T cells possess a profound defect in IL-2 production in response to TCR/CD28-mediated stimulation. Examination of key signaling intermediates revealed that TCR proximal events such as global tyrosine phosphorylation and ZAP70 phosphorylation, as well as downstream MAPK cascades are unperturbed in p75-deficient T cells. In contrast, p75 is nonredundantly coupled to sustained AKT activity and NF-kappaB activation in response to TCR/CD28-mediated stimulation. Moreover, p75-deficient T cells possess a defect in survival during the early phase of T cell activation that is correlated with a striking defect in Bcl-x(L) expression. These data indicate discrete effects of p75 on the intracellular signaling milieu during T cell activation, and reveal the synergistic requirement of TCR, CD28, and p75 toward optimal IL-2 induction and T cell survival. We propose that p75 acts as one of the earliest of the identified costimulatory members of the TNFR family, and is functionally linked to CD28 for initiating and determining T cell fate during activation.     

Alpneumines A–H,new anti-melanogenic indole alkaloids from Alstonia pneumatophora

Eight new indole alkaloids, alpneumines A–H (1–8) were isolated from the Malaysian Alstonia pneumatophora (Apocynaceae) and their structures were determined by MS and 2D NMR spectroscopic methods. Alpneumines E and G (5 and 7), vincamine, and apovincamine showed anti-melanogenesis in B16 mouse melanoma cells.     

Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo

Self-specific CD8 T cells, which are selected by high-affinity interactions with self-Ags, develop into a lineage distinct from conventional CD8 T cells. We have previously shown that these self-specific cells acquire phenotypic and functional similarities to cells of the innate immune system including the expression of functional receptors associated with NK cells. In this study, we show that these self-specific cells have the ability to produce large amounts of IFN-gamma in response to infection with Listeria monocytogenes in a bystander fashion. The rapid production of IFN-gamma is associated with a dramatic reduction in the number of viable bacteria at the peak of infection. Self-specific CD8 T cells provide only marginal innate protection in the absence of self-Ag; however, the presence of self-Ag dramatically increases their protective ability. Exposure to self-Ag is necessary for the maintenance of the memory phenotype and responsiveness to inflammatory cytokines such as IL-15. Significantly, self-specific CD8 T cells are also more efficient in the production of IFN-gamma and TNF-alpha, thus providing more cytokine-dependent protection against bacterial infection when compared with NK cells. These findings illustrate that self-reactive CD8 T cells can play an important innate function in the early defense against bacterial infection.