Analysis of expressed sequence tags derived from pea leaves infected by Peronospora viciae f. sp. pisi

A cDNA library was constructed from field pea leaves infected by the downy mildew pathogen, Peronospora viciae f. sp. pisi, using a suppression subtractive hybridisation approach. The library consists of 399 expressed sequence tags, from which 207 unisequences were obtained after sequence assembly. Of the unisequences, six were shown to be of Peronospora viciae f. sp. pisi origin. The remaining unisequences were subjected to gene ontology analysis and their functions were predicted in silico. Eleven of these unisequences (representing 24 clones) shared significant sequence similarities with Arabidopsis genes known to be involved in downy mildew resistance, including the well‐characterised genes RPP5, RPP6 and RPP27. Expression analysis of five selected unisequences by real‐time PCR indicated that all five were up‐regulated during downy mildew pathogenesis, suggesting a significant role for these genes in the host response to downy mildew infection.     

Genome comparisons indicate recent transfer of wRi‐like Wolbachia between sister species Drosophila suzukii and D. subpulchrella

Wolbachia endosymbionts may be acquired by horizontal transfer, by introgression through hybridization between closely related species, or by cladogenic retention during speciation. All three modes of acquisition have been demonstrated, but their relative frequency is largely unknown. Drosophila suzukii and its sister species D. subpulchrella harbor Wolbachia, denoted wSuz and wSpc, very closely related to wRi, identified in California populations of D. simulans. However, these variants differ in their induced phenotypes: wRi causes significant cytoplasmic incompatibility (CI) in D. simulans, but CI has not been detected in D. suzukii or D. subpulchrella. Our draft genomes of wSuz and wSpc contain full‐length copies of 703 of the 734 single‐copy genes found in wRi. Over these coding sequences, wSuz and wSpc differ by only 0.004% (i.e., 28 of 704,883 bp); they are sisters relative to wRi, from which each differs by 0.014%–0.015%. Using published data from D. melanogaster, Nasonia wasps and Nomada bees to calibrate relative rates of Wolbachia versus host nuclear divergence, we conclude that wSuz and wSpc are too similar—by at least a factor of 100—to be plausible candidates for cladogenic transmission. These three wRi‐like Wolbachia, which differ in CI phenotype in their native hosts, have different numbers of orthologs of genes postulated to contribute to CI; and the CI loci differ at several nucleotides that may account for the CI difference. We discuss the general problem of distinguishing alternative modes of Wolbachia acquisition, focusing on the difficulties posed by limited knowledge of variation in absolute and relative rates of molecular evolution for host nuclear genomes, mitochondria, and Wolbachia.     

Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice

Background

Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Met^fl/fl;Alb-Cre^+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.

Methodology/Principal Finding

The priming events appeared to be intact in Met^fl/fl;Alb-Cre^+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Met^fl/fl;Alb-Cre^+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.

Conclusion/Significance

In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.     

Effects of aqueous sulfur dioxide on cellulose

Summary The reaction of aqueous sulfur dioxide with cellulose pulp and cotton linters was investigated to determine the effect of this lignocellulosic pretreatment method on the cellulosic portion. Sulfur dioxide treatment dramatically reduced the DP of the cellulose but did not affect its enzymatic digestibility, and did not decrystallize the cellulose as recently reported by Leeet al. (1979).Maintained in cooperation with the University of Wisconsin, Madison, Wis.     

Interactions of cytochrome P450s with their ligands

Cytochrome P450s (CYPs) are heme-containing monooxygenases that contribute to an enormous range of enzymatic function including biosynthetic and detoxification roles. This review summarizes recent studies concerning interactions of CYPs with ligands including substrates, inhibitors, and diatomic heme-ligating molecules. These studies highlight the complexity in the relationship between the heme spin state and active site occupancy, the roles of water in directing protein–ligand and ligand–heme interactions, and the details of interactions between heme and gaseous diatomic CYP ligands. Both kinetic and thermodynamic aspects of ligand binding are considered.     

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.     

Herpes simplex virus type 1 strain HSV1716 grown in baby hamster kidney cells has altered tropism for nonpermissive Chinese hamster ovary cells compared to HSV1716 grown in vero cells

Chinese hamster ovary (CHO) cells are traditionally regarded as nonpermissive cells for herpes simplex virus type 1 (HSV-1) infection as they lack the specific entry receptors, and modified CHO cells have been instrumental in the identification of HSV-1 receptors in numerous studies. In this report we demonstrate that the HSV-1 strain 17+ variant HSV1716 is able to infect unmodified CHO cells but only if the virus is propagated in baby hamster kidney (BHK) cells. Infection of CHO cells by BHK-propagated HSV1716 results in expression of immediate-early, early, and late viral genes, and infectious progeny virions are produced. In normally cultured CHO cells, up to a maximum of 50% of cells were permissive for BHK-propagated HSV1716 infection, with 24 h of serum starvation increasing this to 100% of CHO cells, suggesting that the mechanism used by BHK-propagated virus to infect CHO cells was cell cycle dependent. The altered tropism of HSV1716 was also evident in another nonpermissive mouse melanoma cell line and is an exclusive property resulting from propagation of the virus using BHK cells, as viruses propagated on Vero, C8161 (a human melanoma cell line), or indeed, CHO cells were completely unable to infect either CHO or mouse melanoma cells.     

Early response to rotavirus infection involves massive B cell activation

Rotavirus is an acute enteric pathogen which induces severe diarrhea in infants and children. To determine the immune response to rotavirus in vivo, we used a mouse model of rotavirus infection. We observed dramatic increases in the sizes of both Peyer's patches and mesenteric lymph nodes, but not spleen, between 1 and 6 days after infection with a homologous strain of murine rotavirus, EC wild type. Histological analysis showed large increases in the numbers of lymphocytes in these same tissues in rotavirus-infected mice. Flow cytometric analysis confirmed the increase in numbers of lymphocytes and revealed a large increase in the percentage of activated B, but not T, lymphocytes in both Peyer's patches and mesenteric lymph nodes of rotavirus-infected mice compared with control mice. Fragment cultures from these tissues established at 3-4 days postinfection contain rotavirus-specific IgM but not IgA Ab. A similar degree of lymphoid hyperplasia and percentage of activated B cells were observed in rotavirus-infected TCR knockout mice. Taken together, our findings show that rotavirus infection, in the context of a normal immune response, induces a large increase in the percentages of activated B cells in the absence of any detectable increase in the percentage of activated T cells, implicating a T cell-independent B cell response as the primary mechanism for initial rotavirus clearance.     

The release of genetically modified crops into the environment. Part II. Overview of ecological risk assessment

Despite numerous future promises, there is a multitude of concerns about the impact of GM crops on the environment. Key issues in the environmental assessment of GM crops are putative invasiveness, vertical or horizontal gene flow, other ecological impacts, effects on biodiversity and the impact of presence of GM material in other products. These are all highly interdisciplinary and complex issues. A crucial component for a proper assessment is defining the appropriate baseline for comparison and decision. For GM crops, the best and most appropriately defined reference point is the impact of plants developed by traditional breeding. The latter is an integral and accepted part of agriculture. In many instances, the putative impacts identified for GM crops are very similar to the impacts of new cultivars derived from traditional breeding. When assessing GM crops relative to existing cultivars, the increased knowledge base underpinning the development of GM crops will provide greater confidence in the assurances plant science can give on the risks of releasing such crops.