Nonredundant role of Bax and Bak in Bid-mediated apoptosis

Animal models suggest that Bax and Bak play an essential role in the implementation of apoptosis and as a result can hinder tumorigenesis. We analyzed the expression of these proteins in 50 human glioblastoma multiforme (GBM) tumors. We found that all the tumors expressed Bak, while three did not express Bax. In vitro, Bax-deficient GBM (BdGBM) exhibited an important resistance to various apoptogenic stimuli (e.g., UV, staurosporine, and doxorubicin) compared to the Bax-expressing GBM (BeGBM). Using an antisense strategy, we generated Bak(-) BeGBM and Bak(-) BdGBM, which enabled us to show that the remaining sensitivity of the BdGBM to apoptosis was due to the overexpression of Bak. Bax/Bak single or double deficiency had no influence on either the clonogenicity or the growth of tumors in Swiss nude mice. Of note, Bak(-) BeGBM cells were resistant to apoptosis induced by caspase 8 (C8) but not to that induced by granzyme B (GrB). Cells lacking both Bax and Bak (i.e., Bak(-) BdGBM) were completely resistant to all stimuli including the microinjection of C8 and GrB. We show that GrB-cleaved Bid and C8-cleaved Bid differ in size and utilize preferentially Bax and Bak, respectively, to promote cytochrome c release from mitochondria. Our results suggest that Bax deficiency is compensated by an increase of the expression of Bak in GBM and show, for the first time in human cancer, that the double Bax and Bak deficiency severely impairs the apoptotic program.     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.     

Management of early-instar Japanese beetle (Coleoptera: Searabaeidae) in field-grown nursery crops.

Numerous field studies were conducted in commercial nurseries in Tennessee from 1996 through 1999 to evaluate chemical and biological treatments, application timing and rates, and method of application for control of early instars of Japanese beetle, Popillia japonica Newman. Insecticide treatments included bifenthrin, bendiocarb, chlorpyrifos, carbaryl, fipronil, halofenozide, imidacloprid, permethrin, tefluthrin, thiamethoxam, and trichlorfon. Biological treatments included entomopathogenic nematodes (Heterorhabditis bacteriophora HP88 or H. marelatus, Bacillus thuringiensis Berliner subspecies japonensis Buibui strain, and Beauveria bassiana (Balsamo) Vuillemin. All treatments were applied on the soil surface or injected into the soil around the base of each tree. Tree type and size varied among and within tests, however, the sampling unit (61-cm-diameter root ball) remained the same throughout all tests. The biological treatments provided poor-to-moderate control (0-75%) of Japanese beetle larvae. Imidacloprid was the most frequently evaluated insecticide and achieved 91-100, 87-100, 83-100, and 41-100% control with applications in May, June, July, and August, respectively. Halofenozide treatments were not significantly different from imidacloprid treatments with one exception. Halofenozide provided 60-87, 85-100, and 82-92 control with applications made in June, July, and August, respectively. Fipronil and thiamethoxam were evaluated to a lesser extent but both performed similarly to imidacloprid. Most other insecticide treatments were less successful in reducing numbers of Japanese beetle larvae and with few exceptions achieved <50% control.     

Pathways of soil genesis in the Coast Range of Oregon, USA

Background and Aims

Soil chronosequences on marine terraces along the Pacific Coast of California and Oregon show evidence of podzolization, though soils ultimately evolve to Ultisols. It is not clear if this pathway of soil evolution can be extended to the humid, inland Oregon Coast Range.

Methods

We analyzed soil properties for a fluvial terrace chronosequence sampled along the Siuslaw River (Oregon, USA) about 50 km from the Pacific coast. The seven terraces ranged in age from <3.5 ky to nearly 1,000 ky.

Results

There was no evidence of early podsolization. Instead, evidence was found that andisolization starts early and occurs even in older soils when pedogenic iron accumulation and clay synthesis and illuviation dominate. Soils develop the morphology characteristic of Ultisols sometime between 20 and 70 ky, but high levels of oxalate extractable iron and aluminum satisfy criteria of an andic subgroup. Alfisols are not formed as an intermediary stage.

Conclusions

The lack of Spodosols inland is due to the inland shift from udic to ustic or xeric moisture regime, which favors summer drying and ripening of short-range order minerals rather than deep leaching or translocation. Other factors are higher pH, different organic chemistry and faster calcium cycling under the Douglas fir inland when compared to the Sitka spruce of the coastal terraces.     

Purple non-sulphur bacteria and plant production: benefits for fertilization,stress resistance and the environment

Purple non-sulphur bacteria (PNSB) are phototrophic microorganisms, which increasingly gain attention in plant production due to their ability to produce and accumulate high-value compounds that are beneficial for plant growth. Remarkable features of PNSB include the accumulation of polyphosphate, the production of pigments and vitamins and the production of plant growth-promoting substances (PGPSs). Scattered case studies on the application of PNSB for plant cultivation have been reported for decades, yet a comprehensive overview is lacking. This review highlights the potential of using PNSB in plant production, with emphasis on three key performance indicators (KPIs): fertilization, resistance to stress (biotic and abiotic) and environmental benefits. PNSB have the potential to enhance plant growth performance, increase the yield and quality of edible plant biomass, boost the resistance to environmental stresses, bioremediate heavy metals and mitigate greenhouse gas emissions. Here, the mechanisms responsible for these attributes are discussed. A distinction is made between the use of living and dead PNSB cells, where critical interpretation of existing literature revealed the better performance of living cells. Finally, this review presents research gaps that remain yet to be elucidated and proposes a roadmap for future research and implementation paving the way for a more sustainable crop production.     

Effects of a retaining wall and an artificial embankment on nearshore littoral habitats and biota in a large Alpine lake

Hydrobiologia - The littoral zones of many Central European lakes are severely altered by lake-side retaining walls. These are suspected to impair littoral biota due to the reflection of incoming...     

Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein,BCR, and Regulates Levels of Rac1-GTP

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

Temporal sampling,resetting, and adaptation orchestrate gradient sensing in sperm

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca²⁺ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca²⁺ response was produced. Additional molecules delivered during a Ca²⁺ response reset the cell by causing a pronounced Ca²⁺ drop that terminated the response; this reset was followed by a new Ca²⁺ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.