Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [(3)H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110 degrees C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [(3)H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N(2)-acetylserine and N(2)-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.     

Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis.

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea--6 mM 2-mercaptoethanol--20 mM glycine--20 mM Tris--HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.     

Multimeric Recombinant M2e Protein-Based ELISA: A Significant Improvement in Differentiating Avian Influenza Infected Chickens from Vaccinated Ones

Killed avian influenza virus (AIV) vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA) in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e) of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e) for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006) was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.     

A Morpho-Density Approach to Estimating Neural Connectivity

Neuronal signal integration and information processing in cortical neuronal networks critically depend on the organization of synaptic connectivity. Because of the challenges involved in measuring a large number of neurons, synaptic connectivity is difficult to determine experimentally. Current computational methods for estimating connectivity typically rely on the juxtaposition of experimentally available neurons and applying mathematical techniques to compute estimates of neural connectivity. However, since the number of available neurons is very limited, these connectivity estimates may be subject to large uncertainties. We use a morpho-density field approach applied to a vast ensemble of model-generated neurons. A morpho-density field (MDF) describes the distribution of neural mass in the space around the neural soma. The estimated axonal and dendritic MDFs are derived from 100,000 model neurons that are generated by a stochastic phenomenological model of neurite outgrowth. These MDFs are then used to estimate the connectivity between pairs of neurons as a function of their inter-soma displacement. Compared with other density-field methods, our approach to estimating synaptic connectivity uses fewer restricting assumptions and produces connectivity estimates with a lower standard deviation. An important requirement is that the model-generated neurons reflect accurately the morphology and variation in morphology of the experimental neurons used for optimizing the model parameters. As such, the method remains subject to the uncertainties caused by the limited number of neurons in the experimental data set and by the quality of the model and the assumptions used in creating the MDFs and in calculating estimating connectivity. In summary, MDFs are a powerful tool for visualizing the spatial distribution of axonal and dendritic densities, for estimating the number of potential synapses between neurons with low standard deviation, and for obtaining a greater understanding of the relationship between neural morphology and network connectivity.     

World Trade Center Disaster Exposure-Related Probable Posttraumatic Stress Disorder among Responders and Civilians: A Meta-Analysis

The World Trade Center (WTC) disaster on September 11, 2001 was an unprecedented traumatic event with long-lasting health consequences among the affected populations in the New York metropolitan area. This meta-analysis aimed to estimate the risk of probable posttraumatic stress disorder (PTSD) associated with specific types of WTC exposures. Meta-analytical findings from 10 studies of 3,271 to 20,294 participants yielded 37 relevant associations. The pooled summary odds ratio (OR) was 2.05 (95% confidence interval (CI): 1.82, 2.32), with substantial heterogeneity linked to exposure classification, cohort type, data source, PTSD assessment instrument/criteria, and lapse time since 9/11. In general, responders (e.g. police, firefighters, rescue/recovery workers and volunteers) had a lower probable PTSD risk (OR = 1.61; 95% CI: 1.39, 1.87) compared to civilians (e.g. residents, office workers, and passersby; OR = 2.71, 95% CI: 2.35, 3.12). The differences in ORs between responders and civilians were larger for physical compared to psychosocial exposure types. We also found that injury, lost someone, and witnessed horror were the three (out of six) most pernicious exposures. These findings suggest that these three exposures should be a particular focus in psychological evaluation and treatment programs in WTC intervention and future emergency preparedness efforts.     

Recombinant M2e Protein-Based ELISA: A Novel and Inexpensive Approach for Differentiating Avian Influenza Infected Chickens from Vaccinated Ones

Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test.     

Cytoskeletal disruption induces T cell apoptosis by a caspase-3 mediated mechanism

T cell apoptosis can be triggered by different mechanisms that lead to distinctive features such as cell shrinkage, membrane blebbing, phosphatidylserine externalization, and internucleosomal DNA fragmentation. Prevailing models for the induction of apoptosis place the cytoskeleton as a distal target of the death effector molecules ('executioners'). However, the cytoskeleton can also play a role in the induction of apoptosis as suggested by the finding that cytoskeletal disruption can induce apoptosis. The mechanism by which this occurs is unknown. Here, we report that T cell apoptosis by cytoskeletal disruption involves a protein synthesis-independent mechanism leading to up-regulation of caspase-3 protease activity and increased accessibility of active caspase-3 to its substrate. Thus, cytoskeleton integrity may regulate the subcellular compartmentalization of death effector molecules.     

Alterations in γ-aminobutyric acid content in the rat superior cervical ganglion and pineal gland

The endogenous γ-aminobutyric acid (GABA) content of the rat pineal gland and superior cervical ganglion (SCG) was measured by high pressure liquid chromatography. It was found that GABA levels in both tissues increased after decapitation of the animals. The GABA content of tissues frozen within 20 seconds after decapitation was the same as that of tissues removed from animals killed by microwave irradiation. Amino-oxyacetic acid, a GABA-transaminase inhibitor, increased the endogenous GABA content of both of these tissues. Dimethylphenylpiperizinium or isoniazid administration did not alter GABA levels in these tissues. Isoproterenol increased the GABA content of the SCG but did not change the pineal gland GABA levels. The ability of the pineal gland to take up and accumulate ³H-GABA was significantly reduced in rats that had been ganglionectomized. A fluctuation in endogenous GABA levels in the pineal gland was seen to occur when measures were taken at different times of the day. These results tend to suggest that GABA may have some functional role in the pineal gland and the superior cervical ganglion.     

Independently Outgrowing Neurons and Geometry-Based Synapse Formation Produce Networks with Realistic Synaptic Connectivity

Neuronal signal integration and information processing in cortical networks critically depend on the organization of synaptic connectivity. During development, neurons can form synaptic connections when their axonal and dendritic arborizations come within close proximity of each other. Although many signaling cues are thought to be involved in guiding neuronal extensions, the extent to which accidental appositions between axons and dendrites can already account for synaptic connectivity remains unclear. To investigate this, we generated a local network of cortical L2/3 neurons that grew out independently of each other and that were not guided by any extracellular cues. Synapses were formed when axonal and dendritic branches came by chance within a threshold distance of each other. Despite the absence of guidance cues, we found that the emerging synaptic connectivity showed a good agreement with available experimental data on spatial locations of synapses on dendrites and axons, number of synapses by which neurons are connected, connection probability between neurons, distance between connected neurons, and pattern of synaptic connectivity. The connectivity pattern had a small-world topology but was not scale free. Together, our results suggest that baseline synaptic connectivity in local cortical circuits may largely result from accidentally overlapping axonal and dendritic branches of independently outgrowing neurons.