Cloning of a gene encoding an antigen associated with the centrosome in Drosophila

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10³ and 66 x 10³, on Western blots. We have used this antibody to isolate five clones (λcs1, -2, -3, -4 and λj63) from λgt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone λcs3, we have demonstrated that the fusion proteins encoded by four of these clones (λcs1-4) share at least two epitopes with the 185 x 10³ M_r centrosomal antigen. This indicates that clones λcs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10³ bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10³ and 66 x 10³, on Western blots. We have used this antibody to isolate five clones (λcs1, -2, -3, -4 and λj63) from λgt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone λcs3, we have demonstrated that the fusion proteins encoded by four of these clones (λcs1-4) share at least two epitopes with the 185 x 10³ M_r centrosomal antigen. This indicates that clones λcs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10³ bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle

A technique for the determination of protein concentration by neutron activation analysis of silver binding

A method for the quantitative determination of small amounts of protein samples was developed employing neutron activation analysis. Current methods of protein concentration determination are severely limited as a result of differences in the specific characteristics of each protein. Silver binding has been used as a sensitive colorimetric method to indicate the presence of protein. However, silver-protein complexes can have a variety of absorbance spectra unique to each protein, which complicate the analysis. Various amounts of specific proteins were equilibrated in an excess of silver nitrate prior to the reduction of the silver by the addition of NaBH 4 , HCHO, and NaOH. The protein-silver complex was rapidly separated from the unbound silver by centrifugation chromatography and the amount of bound silver was determined by INAA. The amount of silver was proportional to the amount of protein present in each sample. When the silver was not reduced prior to removal of the unbound silver by chromatography, only negligible amounts of silver remained bound to the protein. The stoichiometry of bound silver to protein on a molar basis showed relatively small differences for the proteins that were examined. This ratio was found to depend on the conditions of the binding and reduction of the silver. The results suggest that the binding of silver is not specific to any charged or polar groups on these proteins and may, therefore, provide a means of determination of the concentration of protein that has general application for all proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43109/1/10967_2005_Article_BF02037279.pd

α7 Nicotinic Acetylcholine Receptor Signaling Modulates Ovine Fetal Brain Astrocytes Transcriptome in Response to Endotoxin

Neuroinflammation in utero may result in lifelong neurological disabilities. Astrocytes play a pivotal role in this process, but the mechanisms are poorly understood. No early postnatal treatment strategies exist to enhance neuroprotective potential of astrocytes. We hypothesized that agonism on α7 nicotinic acetylcholine receptor (α7nAChR) in fetal astrocytes will augment their neuroprotective transcriptome profile, while the inhibition of α7nAChR will achieve the opposite. Using an in vivo–in vitro model of developmental programming of neuroinflammation induced by lipopolysaccharide (LPS), we validated this hypothesis in primary fetal sheep astrocytes cultures re-exposed to LPS in the presence of a selective α7nAChR agonist or antagonist. Our RNAseq findings show that a pro-inflammatory astrocyte transcriptome phenotype acquired in vitro by LPS stimulation is reversed with α7nAChR agonistic stimulation. Conversely, α7nAChR inhibition potentiates the pro-inflammatory astrocytic transcriptome phenotype. Furthermore, we conducted a secondary transcriptome analysis against the identical α7nAChR experiments in fetal sheep primary microglia cultures. Similar to findings in fetal microglia, in fetal astrocytes we observed a memory effect of in vivo exposure to inflammation, expressed in a perturbation of the iron homeostasis signaling pathway (hemoxygenase 1, HMOX1), which persisted under pre-treatment with α7nAChR antagonist but was reversed with α7nAChR agonist. For both glia cell types, common pathways activated due to LPS included neuroinflammation signaling and NF-κB signaling in some, but not all comparisons. However, overall, the overlap on the level of signaling pathways was rather minimal. Astrocytes, not microglia—the primary immune cells of the brain, were characterized by unique inhibition patterns of STAT3 pathway due to agonistic stimulation of α7nAChR prior to LPS exposure. Lastly, we discuss the implications of our findings for fetal and postnatal brain development

Information Transmission in Cercal Giant Interneurons Is Unaffected by Axonal Conduction Noise

What are the fundamental constraints on the precision and accuracy with which nervous systems can process information? One constraint must reflect the intrinsic “noisiness” of the mechanisms that transmit information between nerve cells. Most neurons transmit information through the probabilistic generation and propagation of spikes along axons, and recent modeling studies suggest that noise from spike propagation might pose a significant constraint on the rate at which information could be transmitted between neurons. However, the magnitude and functional significance of this noise source in actual cells remains poorly understood. We measured variability in conduction time along the axons of identified neurons in the cercal sensory system of the cricket Acheta domesticus, and used information theory to calculate the effects of this variability on sensory coding. We found that the variability in spike propagation speed is not large enough to constrain the accuracy of neural encoding in this system

Endotoxaemia in Haemodialysis: A Novel Factor in Erythropoetin Resistance?

Background/Objectives Translocated endotoxin derived from intestinal bacteria is a driver of systemic inflammation and oxidative stress. Severe endotoxaemia is an underappreciated, but characteristic finding in haemodialysis (HD) patients, and appears to be driven by acute repetitive dialysis induced circulatory stress. Resistance to erythropoietin (EPO) has been identified as a predictor of mortality risk, and associated with inflammation and malnutrition. This study aims to explore the potential link between previously unrecognised endotoxaemia and EPO Resistance Index (ERI) in HD patients. Methodology/Principal Findings 50 established HD patients were studied at a routine dialysis session. Data collection included weight, BMI, ultrafiltration volume, weekly EPO dose, and blood sampling pre and post HD. ERI was calculated as ratio of total weekly EPO dose to body weight (U/kg) to haemoglobin level (g/dL). Mean haemoglobin (Hb) was 11.3±1.3 g/dL with a median EPO dose of 10,000 [IQR 7,500–20,000] u/wk and ERI of 13.7 [IQR 6.9–23.3] ((U/Kg)/(g/dL)). Mean pre-HD serum ET levels were significantly elevated at 0.69±0.30 EU/ml. Natural logarithm (Ln) of ERI correlated to predialysis ET levels (r = 0.324, p = 0.03) with a trend towards association with hsCRP (r = 0.280, p = 0.07). Ln ERI correlated with ultrafiltration volume, a driver of circulatory stress (r = 0.295, p = 0.046), previously identified to be associated with increased intradialytic endotoxin translocation. Both serum ET and ultrafiltration volume corrected for body weight were independently associated with Ln ERI in multivariable analysis. Conclusions This study suggests that endotoxaemia is a significant factor in setting levels of EPO requirement. It raises the possibility that elevated EPO doses may in part merely be identifying patients subjected to significant circulatory stress and suffering the myriad of negative biological consequences arising from sustained systemic exposure to endotoxin

Beryllium increases the CD14^dimCD16+ subset in the lung of chronic beryllium disease

CD14dimCD16+ and CD14brightCD16+ cells, which compose a minor population of monocytes in human peripheral blood mononuclear cells (PBMC), have been implicated in several inflammatory diseases. The aim of this study was to investigate whether this phenotype was present as a subset of lung infiltrative alveolar macrophages (AMs) in the granulomatous lung disease, chronic beryllium disease (CBD). The monocytes subsets was determined from PBMC cells and bronchoalveolar lavage (BAL) cells from CBD, beryllium sensitized Non-smoker (BeS-NS) and healthy subjects (HS) using flow cytometry. The impact of smoking on the AMs cell phenotype was determined by using BAL cells from BeS smokers (BeS-S). In comparison with the other monocyte subpopulations, CD14dimCD16+ cells were at decreased frequency in PBMCs of both BeS-NS and CBD and showed higher HLA-DR expression, compared to HS. The AMs from CBD and BeS-NS demonstrated a CD14dimCD16+phenotype, while CD14brightCD16+ cells were found at increased frequency in AMs of BeS, compared to HS. Fresh AMs from BeS-NS and CBD demonstrated significantly greater CD16, CD40, CD86 and HLA-DR than HS and BeS-S. The expression of CD16 on AMs from both CBD and BeS-NS was downregulated significantly after 10μM BeSO4 stimulation. The phagocytic activity of AMs decreased after 10μM BeSO4 treatment in both BeS-NS and CBD, although was altered or reduced in HS and BeS-S. These results suggest that Be increases the CD14dimCD16+ subsets in the lung of CBD subjects. We speculate that Be-stimulates the compartmentalization of a more mature CD16+ macrophage phenotype and that in turn these macrophages are a source of Th1 cytokines and chemokines that perpetuate the Be immune response in CBD. The protective effect of cigarette smoking in BeS-S may be due to the low expression of co-stimulatory markers on AMs from smokers as well as the decreased phagocytic function

Molecular dynamics simulation of the early stages of the synthesis of periodic mesoporous silica

We present results of detailed atomistic modeling of the early stages of the synthesis of periodic mesoporous silica using molecular dynamics. Our simulations lead to the proposal of a mechanism that validates several previous experimental and modeling studies and answers many controversial issues regarding the synthesis of mesoporous silicas. In particular, we show that anionic silicates interact very strongly with cationic surfactants and, significantly adsorb on the surface of micelles, displacing a fraction of previously bound bromide counterions. This induces an increase in micelle size and also enhances silica condensation at the micelle surface. The presence of larger silica aggregates in solution further promotes the growth of micelles and, by binding to surfactant molecules in different micelles, their aggregation. This work demonstrates the crucial role played by silica in influencing, by way of a cooperative templating mechanism, the structure of the eventual liquid-crystal phase, which in turn determines the structure of the porous material

Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation

10.1371/journal.pgen.1000106PLoS Genetics46