Primary infection by a human immunodeficiency virus with atypical coreceptor tropism

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.     

Excision and episomal replication of cauliflower mosaic virus integrated into a plant genome

Transgenic Arabidopsis (Arabidopsis thaliana) plants containing a monomeric copy of the cauliflower mosaic virus (CaMV) genome exhibited the generation of infectious, episomally replicating virus. The circular viral genome had been split within the nonessential gene II for integration into the Arabidopsis genome by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were assessed for episomal infections at flowering, seed set, and/or senescence. The infections were confirmed by western blot for the CaMV P6 and P4 proteins, electron microscopy for the presence of icosahedral virions, and through polymerase chain reaction across the recombination junction. By the end of the test period, a majority of the transgenic Arabidopsis plants had developed episomal infections. The episomal form of the virus was infectious to nontransgenic plants, indicating that no essential functions were lost after release from the Arabidopsis chromosome. An analysis of the viral genomes recovered from either transgenic Arabidopsis or nontransgenic turnip (Brassica rapa var rapa) revealed that the viruses contained deletions within gene II, and in some cases, the deletions extended to the beginning of gene III. In addition, many of the progeny viruses contained small regions of nonviral sequence derived from the flanking transformation vector. The nature of the nucleotide sequences at the recombination junctions in the circular progeny virus indicated that most were generated by nonhomologous recombination during the excision event. The release of the CaMV viral genomes from an integrated copy was not dependent upon the application of environmental stresses but occurred with greater frequency with either age or the late stages of plant maturation.     

Age- and task-dependent foraging gene expression in the bumblebee Bombus terrestris

In eusocial insects, the division of labor within a colony, based on either age or size, is correlated with a differential foraging (for) gene expression and PKG activity. This article presents in the first part a study on the for gene, encoding a cGMP-dependent protein kinase (PKG) in the bumblebee Bombus terrestris. Cloning of the open reading frame allowed phylogenetic tracing, which showed conservation of PKGs among social insects. Our results confirm the proposed role for PKGs in division of labor. Btfor gene expression is significantly higher in the larger foragers compared with the smaller sized nurses. More importantly, we discovered an age-related decrease in Btfor expression in both nursing and foraging bumblebees. We therefore speculate that the presence of BtFOR is required for correct adaptation to new external stimuli and rapid learning for foraging. In a second series of experiments, worker bumblebees of B. terrestris were treated with two insecticides imidacloprid and kinoprene, which have shown to cause impaired foraging behavior. Compared with controls, only the latter treatment resulted in a decreased Btfor expression, which concurs with a stimulation of ovarian growth and a shift in labor toward nest-related tasks. The data are discussed in relation to Btfor expression in the complex physiological event of foraging and side-effects by pesticides.     

Similar requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in diverse cell types

During animal development, a complex of Par3, Par6 and atypical protein kinase C (aPKC) plays a central role in cell polarisation. The small G protein Cdc42 also functions in cell polarity and has been shown in some cases to act by regulating the Par3 complex. However, it is not yet known whether Cdc42 and the Par3 complex widely function together in development or whether they have independent functions. For example, many studies have implicated Cdc42 in cell migrations, but the Par3 complex has only been little studied, with conflicting results. Here we examine the requirements for CDC-42 and the PAR-3/PAR-6/PKC-3 complex in a range of different developmental events. We found similar requirements in all tissues examined, including polarised growth of vulval precursors and seam cells, migrations of neuroblasts and axons, and the development of the somatic gonad. We also propose a novel role for primordial germ cells in mediating coalescence of the Caenorhabditis elegans gonad. These results indicate that CDC-42 and the PAR-3/PAR-6/aPKC complex function together in diverse cell types.     

Development of SPECT imaging agents for the norepinephrine transporters: [123I]INER

A series of reboxetine analogs was synthesized and evaluated for in vitro binding as racemic mixtures. The best candidate (INER) was synthesized as the optically pure (S,S) enantiomer, labeled with iodine-123 and its in vivo binding determined by SPECT imaging in baboons. The in vivo specificity, selectivity, and kinetics of [123I]INER make it a promising agent for imaging NET in vivo by noninvasive SPECT imaging.     

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.     

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in?vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs?form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.     

Molecular systematics, character evolution, and pollen morphology of Cistus and Halimium (Cistaceae)

Pollen analysis and parsimony-based phylogenetic analyses of the genera Cistus and Halimium, two Mediterranean shrubs typical of Mediterranean vegetation, were undertaken, on the basis of cpDNA sequence data from the trnL-trnF, and trnS-trnG regions, to evaluate limits between the genera. Neither of the two genera examined formed a monophyletic group. Several monophyletic clades were recognized for the ingroup. (1) The ??white and whitish pink Cistus??, where most of the Cistus sections were present, with very diverse pollen ornamentations ranging from striato-reticulate to largely reticulate, sometimes with supratectal elements; (2) The ??purple pink Cistus?? clade grouping all the species with purple pink flowers belonging to the Macrostylia and Cistus sections, with rugulate or microreticulate pollen. Within this clade, the pink-flowered endemic Canarian species formed a monophyletic group, but with weak support. (3) Three Halimium clades were recovered, each with 100% bootstrap support; all Halimium species had striato-reticulate pollen. Two Halimium clades were characterized by yellow flowers, and the other by white flowers.     

Redox control of iron regulatory protein 2 stability

Iron regulatory protein 2 (IRP2) is a critical switch for cellular and systemic iron homeostasis. In iron-deficient or hypoxic cells, IRP2 binds to mRNAs containing iron responsive elements (IREs) and regulates their expression. Iron promotes proteasomal degradation of IRP2 via the F-box protein FBXL5. Here, we explored the effects of oxygen and cellular redox status on IRP2 stability. We show that iron-dependent decay of tetracycline-inducible IRP2 proceeds efficiently under mild hypoxic conditions (3% oxygen) but is compromised in severe hypoxia (0.1% oxygen). A treatment of cells with exogenous H(2)O(2) protects IRP2 against iron and increases its IRE-binding activity. IRP2 is also stabilized during menadione-induced oxidative stress. These data demonstrate that the degradation of IRP2 in iron-replete cells is not only oxygen-dependent but also sensitive to redox perturbations.     

Community-wide assessment of protein-interface modeling suggests improvements to design methodology

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder.