In vitro cytotoxicity, chemotactic effect, and cellular uptake of branched polypeptides with poly[L-lys] backbone by J774 murine macrophage cell line

Branched polypeptides with polylysine backbone are promising candidates for selective delivery of drugs, epitopes. or reporter molecules. We reported earlier that polylysine-based polypeptides with polyanionic character were internalized by murine bone marrow derived macrophages via class A scavenger receptor. In the present studies, our investigations were extended to seven polypeptides with different amino acid composition and charge properties. We report on our findings on the concentration-dependent influence of these compounds on survival and chemotaxis of the murine macrophage-like cell line J774 and internalization properties of the polypeptides by J774 cells. Our observations indicate that the polypeptides regardless of their charge properties were essentially nontoxic and did not alter significantly the chemotaxis of J774 cells; therefore, the polypeptides suit the requirements for nontoxic and "neutral" carrier molecules. We also demonstrated that the polypeptides were internalized efficiently by J774 cells, depending on their chemical structure and charge properties. Using the scavenger receptor-ligand fucoidan as inhibitor, we established that the scavenger receptor played a role-in accordance with findings on murine bone marrow derived macrophages in the internalization only of the polyanionic polypeptides.     

Molecular dissociation of the role of PSD-95 in regulating synaptic strength and LTD

The postsynaptic density protein PSD-95 influences synaptic AMPA receptor (AMPAR) content and may play a critical role in LTD. Here we demonstrate that the effects of PSD-95 on AMPAR-mediated synaptic responses and LTD can be dissociated. Our findings suggest that N-terminal-domain-mediated dimerization is important for PSD-95's effect on basal synaptic AMPAR function, whereas the C-terminal SH(3)-GK domains are also necessary for localizing PSD-95 to synapses. We identify PSD-95 point mutants (Q15A, E17R) that maintain PSD-95's influence on basal AMPAR synaptic responses yet block LTD. These point mutants increase the proteolysis of PSD-95 within its N-terminal domain, resulting in a C-terminal fragment that functions as a dominant negative likely by scavenging critical signaling proteins required for LTD. Thus, the C-terminal portion of PSD-95 serves a dual function. It is required to localize PSD-95 at synapses and as a scaffold for signaling proteins that are required for LTD.     

On the derivation and tuning of phase oscillator models for lamprey central pattern generators

Using phase response curves and averaging theory, we derive phase oscillator models for the lamprey central pattern generator from two biophysically-based segmental models. The first one relies on network dynamics within a segment to produce the rhythm, while the second contains bursting cells. We study intersegmental coordination and show that the former class of models shows more robust behavior over the animal's range of swimming frequencies. The network-based model can also easily produce approximately constant phase lags along the spinal cord, as observed experimentally. Precise control of phase lags in the network-based model is obtained by varying the relative strengths of its six different connection types with distance in a phase model with separate coupling functions for each connection type. The phase model also describes the effect of randomized connections, accurately predicting how quickly random network-based models approach the determinisitic model as the number of connections increases.     

A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information

Background  

The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.     

Overexpression of human apolipoprotein B-100 induces severe neurodegeneration in transgenic mice

Recent studies showed correlation between increased serum apolipoprotein B-100 (apoB-100) level and Alzheimer's disease. To reveal the possible role of apoB-100 in neurodegeneration, we analyzed the serum lipoprotein and cerebral protein profiles, amyloid plaque formation, apoptosis and brain morphology of transgenic mice overexpressing the human apoB-100 protein. Serum lipoprotein profile showed significant increase of the plasma triglyceride level, while no alteration in total cholesterol was detected. The antibody microarray experiment revealed upregulation of several cytoskeletal, neuronal proteins and proteins that belong to the mitogen activated protein kinase pathway, indicating active apoptosis in the brain. Histochemical experiments showed formation of amyloid plaques and extensive neuronal death. Biochemical changes severely affected brain morphology; a dramatic genotype-dependent enlargement of the third and lateral ventricles in the brain was detected. On the basis of earlier and present results, we conclude that overexpressed human apoB-100 protein significantly increases the level of serum lipids (triglyceride upon normal chow diet and cholesterol on cholesterol-rich diet) which leads to cerebrovascular lesions and subsequently induces apoptosis and neurodegeneration.     

Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells

The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15 min to 5h did not cause any changes in the expression and localization of principal junctional proteins. One day of treatment with a relatively high concentration of nicotine induced a decrease in the expression of the tight junction protein ZO-1, occludin, and the adherens junction protein, cadherin. Treatment with 3 x 10(-5)M phenanthrene for 24h caused a redistribution of occludin from the Triton X-100 insoluble to the Triton X-100 soluble fraction. Transendothelial electrical resistance was not significantly affected by 24h treatments with nicotine, methylanthracene or phenanthrene. However, 24h nicotine treatment increased transendothelial permeability in CECs exposed to oxidative stress. Both nicotine and phenanthrene were able to regulate the expression of a large number of proteins as revealed by 2D electrophoresis. Our experiments suggest that tobacco smoking may affect the junctional complex of CECs, and that this effect is enhanced by oxidative stress.     

Diversity of Sulfate-Reducing Bacteria Inhabiting the Rhizosphere of Phragmites australis in Lake Velencei (Hungary) Revealed by a Combined Cultivation-based and Molecular approach

The community structure of sulfate-reducing bacteria (SRB) associated with reed (Phragmites australis) rhizosphere in Lake Velencei (Hungary) was investigated by using cultivation-based and molecular methods. The cultivation methods were restricted to recover lactate-utilizing species with the exclusion of Desulfobacter and some Desulfobacterium species presumably not being dominant members of the examined community. The most-probable-number (MPN) estimations of lactate-utilizing SRB showed that the cell counts in reed rhizosphere were at least one order of magnitude higher than that in the bulk sediment. The number of endospores was low compared to the total SRB counts. From the highest positive dilution of MPN series, 47 strains were isolated and grouped by restriction fragment length polymorphism (RFLP) analysis of the amplified 16S ribosomal RNA (rRNA) and dsrAB (dissimilatory sulfite reductase) genes. Contrary to the physiological diversity of the isolates, the combined results of RFLP analysis revealed higher diversity at species as well as at subspecies level. Based on the partial 16S rRNA sequences, the representative strains were closely affiliated with the genera Desulfovibrio and Desulfotomaculum. The partial dsrAB sequences of the clones, recovered after isolation and PCR amplification of the community DNA, were related to hitherto uncultured species of the genera Desulfovibrio and Desulfobulbus. Nevertheless, the representative of the second largest clone group was shown to be closely affiliated with the sequenced dsrAB gene of a strain isolated from the same environment and identified as Desulfovibrio alcoholivorans. Another clone sequence was closely related to a possible novel species also isolated within the scope of this work.     

Detection and quantification of group C rotaviruses in communal sewage

Group C rotaviruses have been recognized as a cause of acute gastroenteritis in humans, cattle, and swine, although the true epidemiologic and clinical importance of this virus in these hosts has not yet been fully established. A real-time PCR assay based on a broadly reactive primer pair was developed and used to quantitatively determine the viral load of group C rotaviruses in environmental samples. A total of 35 raw and 35 treated sewage samples collected at the same sampling time in four Hungarian sewage treatment plants during a survey in 2005 were tested for the presence of group C rotaviruses. The overall detection rates were 91% (32 of 35) for the influent and 57% (20 of 35) for the effluent samples. Molecular characterization of the amplified partial VP6 gene revealed the cocirculation of human and animal (i.e., bovine and porcine) strains that were easily distinguishable by melting curve analysis. Human strains yielded relatively high viral loads (mean, 1.2 x 10(7); median, 6.9 x 10(5) genome equivalents per liter influent sewage) and appeared to display seasonal activity over the study period, whereas animal strains appeared to circulate throughout the year at much lower average titers (bovine strains mean, 9.9 x 10(4); median, 3.0 x 10(4); porcine strains mean, 3.9 x 10(4); median, 3.1 x 10(4) genome equivalents per liter influent sewage). Our findings suggest that monitoring of communal sewage may provide a good surrogate for investigating the epidemiology and ecology of group C rotaviruses in humans and animals.