Yeast mRNA Poly(A) tail length control can be reconstituted in vitro in the absence of Pab1p-dependent Poly(A) nuclease activity

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.     

Evolution of specialization and ecological character displacement of herbivores along a gradient of plant quality

We study the combined evolutionary dynamics of herbivore specialization and ecological character displacement, taking into account foraging behavior of the herbivores, and a quality gradient of plant types. Herbivores can adapt by changing two adaptive traits: their level of specialization in feeding efficiency and their point of maximum feeding efficiency along the plant gradient. The number of herbivore phenotypes, their levels of specialization, and the amount of character displacement among them are the result of the evolutionary dynamics, which is driven by the underlying population dynamics, which in turn is driven by the underlying foraging behavior. Our analysis demonstrates broad conditions for the diversification of a herbivore population into many specialized phenotypes, for basically any foraging behavior focusing use on highest gains while also including errors. Our model predicts two characteristic phases in the adaptation of herbivore phenotypes: a fast character-displacement phase and a slow coevolutionary niche-shift phase. This two-phase pattern is expected to be of wide relevance in various consumer-resource systems. Bringing together ecological character displacement and the evolution of specialization in a single model, our study suggests that the foraging behavior of herbivorous arthropods is a key factor promoting specialist radiation.     

Resveratrol and curcumin reduce the respiratory burst of Chlamydia-primed THP-1 cells

The intracellular bacterium Chlamydia pneumoniae is involved in the inflammation process of atherosclerosis. We previously demonstrated that C. pneumonia infected monocytes (THP-1 cells) responded to stimulation by an increased respiratory burst linked to an increased NADPH oxidase (NOX) activity. We now tested agents acting on the assembly of the NOX subunits or on protein kinase C, a trigger of NOX activity. Apocynin, resveratrol, rutin, quercetin, curcumin, and tocopherols were tested. The cells were pre-incubated with Chlamydia and the agent for 19 h, and then stimulated with phorbol myristate acetate. The NOX activity was monitored by measuring the hydrogen peroxide production. Resveratrol and curcumin (10(-4)-10(-6) M) were better inhibitors than apocynin. alpha-Tocopherol was inactive, and gamma-tocopherol inhibitor at 10(-4) M only. Quercetin was inactive, and rutin a moderate but significant inhibitor. The inhibition by resveratrol was increased by 10(-6) M rutin or quercetin. Resveratrol and curcumin thus appeared to be interesting for atherosclerosis treatment.     

The pnhA gene of Pasteurella multocida encodes a dinucleoside oligophosphate pyrophosphatase member of the Nudix hydrolase superfamily

The pnhA gene of Pasteurella multocida encodes PnhA, which is a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. PnhA hydrolyzes diadenosine tetra-, penta-, and hexaphosphates with a preference for diadenosine pentaphosphate, from which it forms ATP and ADP. PnhA requires a divalent metal cation, Mg(2+) or Mn(2+), and prefers an alkaline pH of 8 for optimal activity. A P. multocida strain that lacked a functional pnhA gene, ACP13, was constructed to further characterize the function of PnhA. The cellular size of ACP13 was found to be 60% less than that of wild-type P. multocida, but the growth rate of ACP13 and its sensitivity to heat shock conditions were similar to those of the wild type, and the wild-type cell size was restored in the presence of a functional pnhA gene. Wild-type and ACP13 strains were tested for virulence by using the chicken embryo lethality model, and ACP13 was found to be up to 1,000-fold less virulent than the wild-type strain. This is the first study to use an animal model in assessing the virulence of a bacterial strain that lacked a dinucleoside oligophosphate pyrophosphatase and suggests that the pyrophosphatase PnhA, catalyzing the hydrolysis of diadenosine pentaphosphates, may also play a role in facilitating P. multocida pathogenicity in the host.     

Thymic selection and peripheral activation of CD8 T cells by the same class I MHC/peptide complex

Thymic selection is controlled by the interaction between TCR and MHC/peptide. Strength and quality of the signal determine whether thymocytes are selected or deleted. The factors that contribute to this signal remain poorly defined. Here we show that fetal thymic organ cultures (FTOCs) derived from OT-I transgenic mice (the OT-I TCR is restricted by K(b)-SIINFEKL) on a K(b)D(b-/-) background support positive selection, but only when provided with soluble H-2K(b)-SIINFEKL complexes. Selection of CD8 T cells is independent of the valency of the ligand or its capability to coengage CD8 molecules. Both CD8alphaalpha and CD8alphabeta T cells are selected by H-2K(b)-SIINFEKL, but only CD8alphabeta cells are capable of releasing IFN-gamma in response to the same ligand. The alpha(4)beta(7) integrin is up-regulated on postselection thymocytes from FTOCs. After adoptive transfer, FTOC-derived OT-I CD8 T cells divide in response to the agonist peptide SIINFEKL. These results establish that CD8 T cells responsive to their nominal peptide-Ag can be generated in FTOC supplemented with soluble MHC class I molecules equipped with the same peptide.     

Structural studies of the Nudix hydrolase DR1025 from Deinococcus radiodurans and its ligand complexes

We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution). In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.     

Perchlorate-induced conformational transition of Staphylococcal nuclease: evidence for an equilibrium unfolding intermediate

The sodium perchlorate-induced conformational transition of Staphylococcal nuclease has been monitored by both circular dichroism (CD) and fluorescence spectroscopy. The perchlorate-induced transition is cooperative as observed by both spectroscopic signals. However, the protein loses only about one-third of its native far-UV CD signal at high perchlorate concentrations, indicating that a significant amount of secondary structure remains in the post-transition state. The remaining CD signal can be further diminished in a cooperative manner by the addition of the strong denaturant, urea. Near-UV CD spectra clearly show that the protein loses its tertiary structure in the perchlorate-induced denatured state. The perchlorate-induced transition curves were fit to the standard two-state model and the standard free energy change and m value of the transition are 2.3kcal/mol and 1.8kcal/(molM), respectively. By comparison, the urea-induced unfolding of Staphylococcal nuclease (in the absence of perchlorate) yields an unfolding free energy change, DeltaG(0,un), of 5.6kcal/mol and an m value of 2.3kcal/(molM). Thus, the thermodynamic state obtained in the post-transition region of perchlorate-induced conformation transition has a significantly lower free energy change, a high content of secondary structure, and diminished tertiary structure. These results suggest that the perchlorate-induced denatured state is a partially folded equilibrium state. Whether this intermediate is relevant to the folding/unfolding path under standard conditions is unknown at this time.