Metabolic signalling and carbon partitioning: role of Snf1-related (SnRK1) protein kinase

A protein kinase that plays a key role in the global control of plant carbon metabolism is SnRK1 (sucrose non-fermenting-1-related protein kinase 1), so-called because of its homology and functional similarity with sucrose non-fermenting 1 (SNF1) of yeast. This article reviews studies on the characterization of SnRK1 gene families, SnRK1 regulation and function, interacting proteins, and the effects of manipulating SnRK1 activity on carbon metabolism and development.     

The avian retrovirus avian sarcoma/leukosis virus subtype A reaches the lipid mixing stage of fusion at neutral pH

We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at >or=22 degrees C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and >or=22 degrees C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37 degrees C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.     

Genetic dissection of hybrid incompatibilities between Drosophila simulans and D. mauritiana. I. Differential accumulation of hybrid male sterility effects on the X and autosomes

The genetic basis of hybrid incompatibility in crosses between Drosophila mauritiana and D. simulans was investigated to gain insight into the evolutionary mechanisms of speciation. In this study, segments of the D. mauritiana third chromosome were introgressed into a D. simulans genetic background and tested as homozygotes for viability, male fertility, and female fertility. The entire third chromosome was covered with partially overlapping segments. Many segments were male sterile, while none were female sterile or lethal, confirming previous reports of the rapid evolution of hybrid male sterility (HMS). A statistical model was developed to quantify the HMS accumulation. In comparison with previous work on the X chromosome, we estimate that the X has approximately 2.5 times the density of HMS factors as the autosomes. We also estimate that the whole genome contains approximately 15 HMS "equivalents"-i.e., 15 times the minimum number of incompatibility factors necessary to cause complete sterility. Although some caveats for the quantitative estimate of a 2.5-fold density difference are described, this study supports the notion that the X chromosome plays a special role in the evolution of reproductive isolation. Possible mechanisms of a "large X" effect include selective fixation of new mutations that are recessive or partially recessive and the evolution of sex-ratio distortion systems.     

Adenoviral Bid overexpression induces caspase-dependent cleavage of truncated Bid and p53-independent apoptosis in human non-small cell lung cancers

Proapoptotic gene transfer to promote death or to augment killing by DNA-damaging agents represents a promising strategy for cancer therapy. We have constructed an adenoviral Tet-Off trade mark vector with tightly controlled expression of Bid (Ad-Bid) (Clontech, Palo Alto, CA). Using the non-small cell lung cancer cell lines H460, H358, and A549, low dose Ad-Bid was shown to induce high levels of full-length Bid as well as caspase-3 and -9 activity. Although only a small fraction of Bid was processed to truncated Bid (a step inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), Ad-Bid gene transfer resulted in mitochondrial changes consistent with apoptosis (mitochondrial depolarization, cytochrome c release), DNA fragmentation, and a dramatic loss of cell viability. The proapoptotic effects of Ad-Bid were independent of p53 status and were augmented markedly by caspase-8 activators such as the DNA-damaging agent cisplatin. When Ad-Bid and cisplatin were used together, chemosensitivity was restored in p53-null H358 cells, increasing death from 35% following treatment with cisplatin and Ad-LacZ to >90% death with Ad-Bid and cisplatin (Ad-Bid alone induced 50% cell death under these conditions). Ad-Bid can induce apoptosis in malignant cells and enhance chemosensitivity in the absence of p53, suggesting this approach as a potential cancer therapy.     

Genetic algorithms and parallel processing in maximum-likelihood phylogeny inference

We investigated the usefulness of a parallel genetic algorithm for phylogenetic inference under the maximum-likelihood (ML) optimality criterion. Parallelization was accomplished by assigning each "individual" in the genetic algorithm "population" to a separate processor so that the number of processors used was equal to the size of the evolving population (plus one additional processor for the control of operations). The genetic algorithm incorporated branch-length and topological mutation, recombination, selection on the ML score, and (in some cases) migration and recombination among subpopulations. We tested this parallel genetic algorithm with large (228 taxa) data sets of both empirically observed DNA sequence data (for angiosperms) as well as simulated DNA sequence data. For both observed and simulated data, search-time improvement was nearly linear with respect to the number of processors, so the parallelization strategy appears to be highly effective at improving computation time for large phylogenetic problems using the genetic algorithm. We also explored various ways of optimizing and tuning the parameters of the genetic algorithm. Under the conditions of our analyses, we did not find the best-known solution using the genetic algorithm approach before terminating each run. We discuss some possible limitations of the current implementation of this genetic algorithm as well as of avenues for its future improvement.     

Hepatocyte growth factor inhibits anoikis by induction of activator protein 1-dependent cyclooxygenase-2. Implication in head and neck squamous cell carcinoma progression

Anoikis, also called suspension-induced apoptosis, plays an important role in tumor development, progression, and metastasis. Recently we found that hepatocyte growth factor (HGF) inhibited anoikis of human head and neck squamous cell carcinoma (HNSCC) cells by activating the extracellular signal-regulated kinase (ERK)-signaling pathway. However, the anti-apoptotic effectors that were regulated by the ERK-signaling pathway were unknown. Here we report that HGF-mediated inhibition of anoikis was dependent on activator protein-1 activity through the activation of the ERK-signaling pathway. Using a combination of microarray analysis and Northern blot analysis, we found that an anti-apoptotic gene cyclooxygenase-2 (cox-2) was induced by HGF in an activator protein-1-dependent fashion. Inhibition of Cox-2 activity partially abolished HGF-mediated cell survival, and overexpression of Cox-2 in HNSCC cells provided resistance against anoikis. Moreover, HNSCC cells stably expressing Cox-2 had aggressive tumor growth in a nude mouse model compared with control cells. Taken together, our results demonstrate that Cox-2 plays an important role in HGF-mediated anoikis resistance. HGF may stimulate the progression and growth of HNSCC in vivo by induction of Cox-2.     

Parathyroid hormone and parathyroid hormone-related protein exert both pro- and anti-apoptotic effects in mesenchymal cells

During bone formation, multipotential mesenchymal cells proliferate and differentiate into osteoblasts, and subsequently many die because of apoptosis. Evidence suggests that the receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), the PTH-1 receptor (PTH-1R), plays an important role in this process. Multipotential mesenchymal cells (C3H10T1/2) transfected with normal or mutant PTH-1Rs and MC3T3-E1 osteoblastic cells were used to explore the roles of PTH, PTHrP, and the PTH-1R in cell viability relative to osteoblastic differentiation. Overexpression of wild-type PTH-1R increased cell numbers and promoted osteocalcin gene expression versus inactivated mutant receptors. Furthermore, the effects of PTH and PTHrP on apoptosis were dramatically dependent on cell status. In preconfluent C3H10T1/2 and MC3T3-E1 cells, PTH and PTHrP protected against dexamethasone-induced reduction in cell viability, which was dependent on cAMP activation. Conversely, PTH and PTHrP resulted in reduced cell viability in postconfluent cells, which was also dependent on cAMP activation. Further, the proapoptotic-like effects were associated with an inhibition of Akt phosphorylation. These data suggest that parathyroid hormones accelerate turnover of osteoblasts by promoting cell viability early and promoting cell departure from the differentiation program later in their developmental scheme. Both of these actions occur at least in part via the protein kinase A pathway.     

Human genome search in celiac disease: mutated gliadin T-cell-like epitope in two human proteins promotes T-cell activation

Discovery of a number of novel and known human genes whose protein products bear striking similarity to two or more wheat gliadin domains raised the possibility that human intestinal non-HLA peptides homologous to celiac T-cell epitopes could play a role in non-HLA gene specification in celiac disease. Database searching of the entire human genome identified only 11 gut-expressed proteins with high T-cell epitope homology, particularly to the DQ2-gamma-I-gliadin epitope (i.e. TFIIA, FOXJ2 and IgD; mean BestFit quality score=40 versus random value of 24). Others were similar to DQ2-alpha-I-gliadin (i.e. PAX9; BestFit quality 46 versus 20 for random), or DQ2-alpha-II-gliadin (PHLDA1, known in mice as the T-cell death-associated gene; BestFit quality 43 versus 30 for random) epitopes. Among proteins previously screened for gliadin homology, noteworthy was achaete scute homologous protein (DQ2-alpha-I-gliadin; BestFit quality 41 versus 22 for random). With the exception of IgD, all are nuclear factors. Paying particular attention to the position of potential major histocompatibility complex (MHC) anchor residues, several were selected for testing in a DQ2-gamma-I-gliadin-restricted T-cell system. All native 10-mer peptides were inactive, even when deamidated, but V96F substitution of deamidated TFIIA amino acid residues 91-100 stimulated IL-2 release at levels exceeding the wheat gliadin positive control. Also active, but only slightly, was L1009F substitution of AIB3 amino acid residues 1004-1013. PlotSimilarity alignment of TFIIAs from eight species revealed subthreshold similarity score in the peptide region, in contrast to the highly conserved amino and carboxy termini. Molecular modeling of TFIIA[V96F] peptide points to an important juxtaposition of an upwardly projecting phenylalanine residue at peptide position 6 that likely contacts a receptor complementarity-determining region, and a downwardly projecting glutamic acid residue that fits into the shallow MHC P7 pocket. These observations tentatively point to a new multi-gene hypothesis for the initiation of celiac disease in which deamidated free human peptides with T-cell epitope homology (particularly those made more homologous by mutation) escape negative selection, as per deamidation of the HEL(48-62) peptide in the hen egg lysozyme model of autoimmunity. Deamidation following peptide release due to injury triggers inflammation, thereafter repeatedly provoked by dietary gliadin immunodominant peptides concentrated in the proximal small intestine.