Electrostatics of the intracellular vestibule of K+ channels

Previous calculations using continuum electrostatic calculations showed that a fully hydrated monovalent cation is electrostatically stabilized at the center of the cavity of the KcsA potassium channel. Further analysis demonstrated that this cavity stabilization was controlled by a balance between the unfavorable reaction field due to the finite size of the cavity and the favorable electrostatic field arising from the pore helices. In the present study, continuum electrostatic calculations are used to investigate how the stability of an ion in the intracellular vestibular cavity common to known potassium channels is affected as the inner channel gate opens and the cavity becomes larger and contiguous with the intracellular solution. The X-ray structure of the calcium-activated potassium channel MthK, which was crystallized in the open state, is used to construct models of the KcsA channel in the open state. It is found that, as the channel opens, the barrier at the helix bundle crossing decreases to approximately 0 kcal/mol, but that the ion in the cavity is also significantly destabilized. The results are compared and contrasted with additional calculations performed on the KvAP (voltage-activated) and KirBac1.1 (inward rectifier) channels, as well as models of the pore domain of Shaker in the open and closed state. In conclusion, electrostatic factors give rise to energetic constraints on ion permeation that have important functional consequences on the various K+ channels, and partly explain the presence or absence of charged residues near the inner vestibular entry.     

Expression of human FE65 in amyloid precursor protein transgenic mice is associated with a reduction in beta-amyloid load

FE65 is an adaptor protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP). In cultured non-neuronal cells, the formation of the FE65-APP complex is a key element for the modulation of APP processing, signalling and beta-amyloid (Abeta) production. The functions of FE65 in vivo, including its role in the metabolism of neuronal APP, remain to be investigated. In this study, transgenic mice expressing human FE65 were generated and crossbred with APP transgenic mice, known to develop Abeta deposits at 6 months of age. Compared with APP mice, APP/FE65 double transgenic mice exhibited a lower Abeta accumulation in the cerebral cortex as demonstrated by immunohistochemistry and immunoassay, and a lower level of APP-CTFs. The reduced accumulation of Abeta in APP/FE65 double transgenics, compared with APP mice, could be linked to the low Abeta42 level observed at 4 months of age and to the lower APP-CTFs levels. The present work provides evidence that FE65 plays a role in the regulation of APP processing in an in vivo model.     

The dynamics of adaptation: an illuminating example and a Hamilton-Jacobi approach

Our starting point is a selection-mutation equation describing the adaptive dynamics of a quantitative trait under the influence of an ecological feedback loop. Based on the assumption of small (but frequent) mutations we employ asymptotic analysis to derive a Hamilton-Jacobi equation. Well-established and powerful numerical tools for solving the Hamilton-Jacobi equations then allow us to easily compute the evolution of the trait in a monomorphic population when this evolution is continuous but also when the trait exhibits a jump. By adapting the numerical method we can, at the expense of a significantly increased computing time, also capture the branching event in which a monomorphic population turns dimorphic and subsequently follow the evolution of the two traits in the dimorphic population. From the beginning we concentrate on a caricatural yet interesting model for competition for two resources. This provides the perhaps simplest example of branching and has the great advantage that it can be analyzed and understood in detail.     

Adhesion-aggregation and inactivation of poliovirus 1 in groundwater stored in a hydrophobic container

Viral inactivation and adhesion-aggregation in water are often studied as separate phenomena. When the focus is placed on viral adhesion-aggregation, inactivation is neglected because the phenomena under investigation occur over a short period measured in days. When viral inactivation is studied, adhesion-aggregation phenomena are considered to be negligible because viral survival is traced over several days or months. In the present work, we took a global approach, examining the relative contributions of each of these processes in a complex system composed of groundwater, Poliovirus 1, and a hydrophobic container (polypropylene) maintained in a dark environment at 20 degrees C. We demonstrated that infectious viral load fell off 2.8 log(10) during the first 20 days. During this time, adhesion was far from negligible because it accounted for most of the decline, 1.5 log(10). Adhesion was undoubtedly favored by the presence of divalent ions in the groundwater. After 20 days, aggregation may also have been the cause of 0.66 to 0.92 log(10) of viral loss. Finally, viral inactivation was quantitatively the lowest phenomena because it only explained 0.38 to 0.64 log(10) of the viral loss. This study thus clearly demonstrated that estimates of viral survival in a given system must always take into account adhesion-aggregation phenomena which may be responsible for the majority of viral loss in the aqueous phase. Adhesion and aggregation are reversible processes which may lead to an underestimation of viral load in certain studies.     

Gene expression profile in response to <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis> infection in cassava using a cDNA microarray

A cassava cDNA microarray based on a large cassava EST database was constructed and used to study the incompatible interaction between cassava and Xanthomonas axonopodis pv. manihotis (Xam) strain CIO151. For microarray construction, 5700 clones from the cassava unigene set were amplified by polymerase chain reaction (PCR) and printed on glass slides. Microarray hybridization was performed using cDNA from cassava plants (resistant variety MBra685) collected at 12, 24, 48 h and 7 and 15 days post-infection as treatment and cDNA from mock-inoculated plants as control. A total of 199 genes were found to be differentially expressed (126 up-regulated and 73 down-regulated). A greater proportion of differentially-expressed genes was observed at 7 days after inoculation. Expression profiling and cluster analyses indicate that, in response to inoculation with Xam, cassava induces dozens of genes, including principally those involved in oxidative burst, protein degradation and pathogenesis-related (PR) genes. In contrast, genes encoding proteins that are involved in photosynthesis and metabolism were down regulated. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. Quantitative real time PCR experiments confirmed the reliability of our microarray data. In addition we showed that some genes are induced more rapidly in the resistant than in the susceptible cultivar.These authors made equal contributions to this work.     

pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

One channel: open and closed

Structural information about the prokaryotic KirBac3.1 inward rectifier family K(+) channel from Magnetospirillum magnetotacticum is reported. These results from two-dimensional electron cryomicroscopy (EM) shed light on the gating mechanism of members of the Kir channel family.     

Inhibition of phosphatidylethanolamine and phosphatidylinositol synthesis, alteration of the G0 and G1 cell cycle phases and spontaneous apoptosis in lymphocytes from patients under immunosuppressive treatment

Lymphocytes from patients treated with immunosuppressive agents were cultured for 48 hours either with or without concanavalin A. Phospholipid synthesis was then studied using 32p pulse-incorporation (5-hours pulses). Phosphatidylethanolamine synthesis was strongly decreased under immunosuppressive treatment: 1.5-fold in resting lymphocytes and 3- to 4-fold in concanavalin A-stimulated lymphocytes. Phosphatidylinositol synthesis also decreased about 2-fold in stimulated lymphocytes. These results indicate a loss of sensitivity of immunodeficient lymphocytes to the mitogen and an alteration of the G0 and the late G1 cell cycle phases. In parallel, but after a 72-hours incubation, lymphocytes were analysed by flow-cytofluorimetry with propidium iodide. Under concanavalin A-triggered stimulation, the entry into the S phase was much lower in immunodeficient lymphocytes as compared to standard. The characteristics of the G0-G1 population of lymphocytes were also modified. More importantly, after incubation in the culture medium in the absence of mitogen, we observed, among the immunodeficient lymphocytes, a high level of apoptotic cells, about 20 to 30%. This susceptibility to spontaneous apoptosis seems inherent to the status of immunodeficiency itself, whatever its origin. It may be related to the inhibition of phosphatidylethanolamine synthesis in the G0 phase.     

The Salmonella genomic island 1 is an integrative mobilizable element

Salmonella genomic island 1 (SGI1) is a genomic island containing an antibiotic resistance gene cluster identified in several Salmonella enterica serovars. The SGI1 antibiotic resistance gene cluster, which is a complex class 1 integron, confers the common multidrug resistance phenotype of epidemic S. enterica Typhimurium DT104. The SGI1 occurrence in S. enterica serovars Typhimurium, Agona, Paratyphi B, Albany, Meleagridis and Newport indicates the horizontal transfer potential of SGI1. Here, we report that SGI1 could be conjugally transferred from S. enterica donor strains to non-SGI1 S. enterica and Escherichia coli recipient strains where it integrated into the recipient chromosome in a site-specific manner. First, an extrachromosomal circular form of SGI1 was identified by PCR which forms through a specific recombination of the left and right ends of the integrated SGI1. Chromosomal excision of SGI1 was found to require SGI1-encoded integrase which presents similarities to the lambdoid integrase family. Second, the conjugal transfer of SGI1 required the presence of a helper plasmid. The conjugative IncC plasmid R55 could thus mobilize in trans SGI1 which was transferred from the donor to the recipient strains. By this way, the conjugal transfer of SGI1 occurred at a frequency of 10(-5)-10(-6) transconjugants per donor. No transconjugants could be obtained for the SGI1 donor lacking the int integrase gene. Third, chromosomal integration of SGI1 occurred via a site-specific recombination between a 18 bp sequence found in the circular form of SGI1 and a similar 18 bp sequence at the 3' end of thdF gene in the S. enterica and E. coli chromosome. SGI1 appeared to be transmissible only in the presence of additional conjugative functions provided in trans. SGI1 can thus be classified within the group of integrative mobilizable elements (IMEs).