Activation of G-protein Galpha subunits by receptors through Galpha-Gbeta and Galpha-Ggamma interactions

Activation of the Galpha subunit of heterotrimeric GTP-binding proteins by transmembrane receptors requires the propagation of structural signals from the receptor-binding site to the nucleotide-binding site at the opposite side of the protein. In a previous model, it was suggested that the Gbeta-Ggamma dimer is tilted away from Galpha by a lever-arm motion of the Galpha N-terminal helix. Here, we propose that the motion occurs in the opposite direction, close-packing the Galpha-Gbeta interface and creating a novel interface between the helical domain of Galpha and the N terminus of Ggamma, which determines the specificity of activation.     

In vivo impact of CpG1826 oligodeoxynucleotide on CD8 T cell primary responses and survival

CpG oligodeoxynucleotide (ODN) promotes maturation of APCs in vivo and induces strong type 1 T cell responses in mice. In this study, we have investigated the ability of CpG1826 to modulate peptide-specific CD8 T cell responses in a context where CD4 T cells are likely to play a minor role. The effects of CpG1826 were evaluated in a system where a population of NP68-specific F5 TCR transgenic CD8 T cells is diluted into a polyclonal host following adoptive transfer into C57BL/10 syngeneic recipients. Using this approach, we found that CpG1826 enhanced the ability of F5 CD8 T cells to undergo multiple divisions in vivo, to express IFN-gamma ex vivo, and to up-regulate memory-associated cell surface markers such as CD122 (IL-2Rbeta) and Ly-6C. Moreover, CpG1826 greatly increased in vivo cytotoxic activity. Using tetramer detection, we found that CpG1826 promoted long-term survival of Ag-specific CD8 T cells after immunization while no NP68-specific cells were detected when the cognate peptide was injected alone. These results indicate that CpG1826 acts as an adjuvant which increases CD8 T cell effector responses and promotes long-term survival of NP68 peptide-specific cells in vivo. They also suggest that this adjuvant can modulate CD8 T cell responses in a system which is likely to be independent of CD4 T cell help.     

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.     

Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.     

p116Rip is a novel filamentous actin-binding protein

p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA in a yeast two-hybrid screen. Overexpression of p116Rip in neuroblastoma cells inhibits RhoA-mediated cell contraction induced by lysophosphatidic acid (LPA); so far, however, the function of p116Rip is unknown. Here we report that p116Rip localizes to filamentous actin (F-actin)-rich structures, including stress fibers and cortical microfilaments, in both serum-deprived and LPA-stimulated cells, with the N terminus (residues 1-382) dictating cytoskeletal localization. In addition, p116Rip is found in the nucleus. Direct interaction or colocalization with RhoA was not detected. We find that p116Rip binds tightly to F-actin (Kd approximately 0.5 microm) via its N-terminal region, while immunoprecipitation assays show that p116Rip is complexed to both F-actin and myosin-II. Purified p116Rip and the F-actin-binding region can bundle F-actin in vitro, as shown by electron microscopy. When overexpressed in NIH3T3 cells, p116Rip disrupts stress fibers and promotes formation of dendrite-like extensions through its N-terminal actin-binding domain; furthermore, overexpressed p116Rip inhibits growth factor-induced lamellipodia formation. Our results indicate that p116Rip is an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton.     

Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.     

The FAR protein family of the nematode Caenorhabditis elegans. Differential lipid binding properties,structural characteristics,and developmental regulation

Parasitic nematodes of humans and plants secrete a structurally novel type of fatty acid- and retinol-binding protein, FAR, into the tissues they occupy. These proteins may interfere with intercellular lipid signaling to manipulate the defense reactions of the host or acquire essential lipids for the parasites. The genome of the nematode Caenorhabditis elegans encodes eight FAR-like proteins (Ce-FAR-1 to -8). These fall into three discrete groups as indicated by phylogenetic sequence comparisons and intron positions, the proteins from parasitic nematodes falling into group A. Recombinant Ce-FAR-1 to -7 were produced in Escherichia coli and tested for lipid binding in fluorescence-based assays. Ce-FAR-1 to -6 bound DAUDA (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid), cis-parinaric acid, and retinol with dissociation constants in the micromolar range, whereas Ce-FAR-7 bound the latter two lipids relatively poorly. Each protein produced a characteristic shift in peak fluorescence emission of DAUDA, and one (Ce-FAR-5) produced a shift greater than has been observed previously for any lipid-binding protein. Selected Ce-FAR proteins were analyzed by circular dichroism (CD) and differential scanning calorimetry, were found to be helix-rich, and exhibited high thermal stability (transition midpoint, 82.7 degrees C). CD and secondary structure predictions, however, both indicated that Ce-FAR-7 possesses substantially less helix than the other FAR proteins. The genes encoding the Ce-FAR proteins were found to be transcribed differentially through the life cycle of C. elegans, such that Ce-far-4 was transcribed at highest levels in the fourth larval stage, and Ce-far-3 and -7 predominated in males.     

Development,survival and fitness performance of Helicoverpa zea (Lepidoptera: Noctuidae) in MON810 Bt field corn

Helicoverpa zea (Boddie) development, survival, and feeding injury in MON810 transgenic ears of field corn (Zea mays L.) expressing Bacillus thuringiensis variety kurstaki (Bt) Cry1Ab endotoxins were compared with non-Bt ears at four geographic locations over two growing seasons. Expression of Cry1Ab endotoxin resulted in overall reductions in the percentage of damaged ears by 33% and in the amount of kernels consumed by 60%. Bt-induced effects varied significantly among locations, partly because of the overall level and timing of H. zea infestations, condition of silk tissue at the time of egg hatch, and the possible effects of plant stress. Larvae feeding on Bt ears produced scattered, discontinuous patches of partially consumed kernels, which were arranged more linearly than the compact feeding patterns in non-Bt ears. The feeding patterns suggest that larvae in Bt ears are moving about sampling kernels more frequently than larvae in non-Bt ears. Because not all kernels express the same level of endotoxin, the spatial heterogeneity of toxin distribution within Bt ears may provide an opportunity for development of behavioral responses in H. zea to avoid toxin. MON810 corn suppressed the establishment and development of H. zea to late instars by at least 75%. This level of control is considered a moderate dose, which may increase the risk of resistance development in areas where MON810 corn is widely adopted and H. zea overwinters successfully. Sublethal effects of MON810 corn resulted in prolonged larval and prepupal development, smaller pupae, and reduced fecundity of H. zea. The moderate dose effects and the spatial heterogeneity of toxin distribution among kernels could increase the additive genetic variance for both physiological and behavioral resistance in H. zea populations. Implications of localized population suppression are discussed.