Suppressor of cytokine signaling 1 interacts with the macrophage colony-stimulating factor receptor and negatively regulates its proliferation signal

Macrophage colony-stimulating factor receptor (M-CSF-R) is a tyrosine kinase that regulates proliferation, differentiation, and cell survival during monocytic lineage development. Upon activation, M-CSF-R dimerizes and autophosphorylates on specific tyrosines, creating binding sites for several cytoplasmic SH2-containing signaling molecules that relay and modulate the M-CSF signal. Here we show that M-CSF-R interacts with suppressor of cytokine signaling 1 (Socs1), a negative regulator of various cytokine and growth factor signaling pathways. Using the yeast two-hybrid system, in vitro glutathione S-transferase-M-CSF-R pull-down, and in vivo coimmunoprecipitation experiments, we demonstrated a direct interaction between the SH2 domain of Socs1 and phosphorylated tyrosines 697 or 721 of the M-CSF-R kinase insert region. Moreover, Socs1 is tyrosine-phosphorylated in response to M-CSF. Ectopic expression of Socs1 in FDC-P1/MAC and EML hematopoietic cell lines decreased their growth rates in the presence of limiting concentrations of M-CSF. However, Socs1 expression did not totally suppress long term cell growth in the presence of saturating M-CSF concentrations, in contrast to other cytokines such as stem cell factor and interleukin 3. Taken together, these results suggest that Socs1 is an M-CSF-R-binding partner involved in negative regulation of proliferation signaling and that it differentially affects cytokine receptor signals.     

New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria

A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.     

Spiroquinazolinones as novel, potent, and selective PDE7 inhibitors. Part 1

The synthesis and SAR studies of spiroquinazolinones as novel PDE7 inhibitors are discussed. The best compounds from the series displayed nanomolar inhibitory affinity and were selective versus other PDE isoenzymes.     

Electroencephalographic brain dynamics following manually responded visual targets

Scalp-recorded electroencephalographic (EEG) signals produced by partial synchronization of cortical field activity mix locally synchronous electrical activities of many cortical areas. Analysis of event-related EEG signals typically assumes that poststimulus potentials emerge out of a flat baseline. Signals associated with a particular type of cognitive event are then assessed by averaging data from each scalp channel across trials, producing averaged event-related potentials (ERPs). ERP averaging, however, filters out much of the information about cortical dynamics available in the unaveraged data trials. Here, we studied the dynamics of cortical electrical activity while subjects detected and manually responded to visual targets, viewing signals retained in ERP averages not as responses of an otherwise silent system but as resulting from event-related alterations in ongoing EEG processes. We applied infomax independent component analysis to parse the dynamics of the unaveraged 31-channel EEG signals into maximally independent processes, then clustered the resulting processes across subjects by similarities in their scalp maps and activity power spectra, identifying nine classes of EEG processes with distinct spatial distributions and event-related dynamics. Coupled two-cycle postmotor theta bursts followed button presses in frontal midline and somatomotor clusters, while the broad postmotor "P300" positivity summed distinct contributions from several classes of frontal, parietal, and occipital processes. The observed event-related changes in local field activities, within and between cortical areas, may serve to modulate the strength of spike-based communication between cortical areas to update attention, expectancy, memory, and motor preparation during and after target recognition and speeded responding.     

Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8⁺ T-cells and the use of an in vitro model of naïve CD8⁺ T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8⁺ T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8⁺ T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8⁺ and CD4⁺ T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.     

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Removal of exhaled air from total body emanations or artificially standardising carbon dioxide (CO2) outputs has previously been shown to eliminate differential attractiveness of humans to certain blackfly (Simuliidae) and mosquito (Culicidae) species. Whether or not breath contributes to between-person differences in relative attractiveness to the highly anthropophilic malaria vector Anopheles gambiae sensu stricto remains unknown and was the focus of the present study. METHODS: The contribution to and possible interaction of breath (BR) and body odours (BO) in the attraction of An. gambiae s.s. to humans was investigated by conducting dual choice tests using a recently developed olfactometer. Either one or two human subjects were used as bait. The single person experiments compared the attractiveness of a person's BR versus that person's BO or a control (empty tent with no odour). His BO and total emanations (TE = BR+BO) were also compared with a control. The two-person experiments compared the relative attractiveness of their TE, BO or BR, and the TE of each person against the BO of the other. RESULTS: Experiments with one human subject (P1) as bait found that his BO and TE collected more mosquitoes than the control (P = 0.005 and P < 0.001, respectively), as did his BO and the control versus his BR (P < 0.001 and P = 0.034, respectively). The TE of P1 attracted more mosquitoes than that of another person designated P8 (P < 0.021), whereas the BR of P8 attracted more mosquitoes than the BR of P1 (P = 0.001). The attractiveness of the BO of P1 versus the BO of P8 did not differ (P = 0.346). The BO from either individual was consistently more attractive than the TE from the other (P < 0.001). CONCLUSIONS: We demonstrated for the first time that human breath, although known to contain semiochemicals that elicit behavioural and/or electrophysiological responses (CO2, ammonia, fatty acids) in An. gambiae also contains one or more constituents with allomonal (~repellent) properties, which inhibit attraction and may serve as an important contributor to between-person differences in the relative attractiveness of humans to this important malaria vector.     

Activation of a Dab1/CrkL/C3G/Rap1 pathway in Reelin-stimulated neurons

During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype. Reelin action requires tyrosine phosphorylation of the intracellular protein Dab1 by Src-family kinases. However, little is known about signaling pathways downstream of Dab1. Here, we identify several proteins in embryonic brain extract that bind to tyrosine-phosphorylated, but not non-phosphorylated, Dab1. Of these, the Crk-family proteins (CrkL, CrkI, and CrkII ), bind significant quantities of Dab1 when embryonic cortical neurons are exposed to Reelin. CrkL binding to Dab1 involves two tyrosine phosphorylation sites, Y220 and 232, that are critical for proper positioning of migrating cortical plate neurons. CrkL also binds C3G, an exchange factor (GEF) for the small GTPase Rap1 that is activated in other systems by tyrosine phosphorylation. We report that Reelin stimulates tyrosine phosphorylation of C3G and activates Rap1. C3G and Rap1 regulate adhesion of fibroblasts and other cell types. Regulation of Crk/CrkL, C3G, and Rap1 by Reelin may be involved in coordinating neuron migrations during brain development.     

Genetic assignment methods for the direct, real-time estimation of migration rate: a simulation-based exploration of accuracy and power

Genetic assignment methods use genotype likelihoods to draw inference about where individuals were or were not born, potentially allowing direct, real-time estimates of dispersal. We used simulated data sets to test the power and accuracy of Monte Carlo resampling methods in generating statistical thresholds for identifying F0 immigrants in populations with ongoing gene flow, and hence for providing direct, real-time estimates of migration rates. The identification of accurate critical values required that resampling methods preserved the linkage disequilibrium deriving from recent generations of immigrants and reflected the sampling variance present in the data set being analysed. A novel Monte Carlo resampling method taking into account these aspects was proposed and its efficiency was evaluated. Power and error were relatively insensitive to the frequency assumed for missing alleles. Power to identify F0 immigrants was improved by using large sample size (up to about 50 individuals) and by sampling all populations from which migrants may have originated. A combination of plotting genotype likelihoods and calculating mean genotype likelihood ratios (DLR) appeared to be an effective way to predict whether F0 immigrants could be identified for a particular pair of populations using a given set of markers.