H2A.Z stabilizes chromatin in a way that is dependent on core histone acetylation

The functional and structural chromatin roles of H2A.Z are still controversial. This work represents a further attempt to resolve the current functional and structural dichotomy by characterizing chromatin structures containing native H2A.Z. We have analyzed the role of this variant in mediating the stability of the histone octamer in solution using gel-filtration chromatography at different pH. It was found that decreasing the pH from neutral to acidic conditions destabilized the histone complex. Furthermore, it was shown that the H2A.Z-H2B dimer had a reduced stability. Sedimentation velocity analysis of nucleosome core particles (NCPs) reconstituted from native H2A.Z-containing octamers indicated that these particles exhibit a very similar behavior to that of native NCPs consisting of canonical H2A. Sucrose gradient fractionation of native NCPs under different ionic strengths indicated that H2A.Z had a subtle tendency to fractionate with more stabilized populations. An extensive analysis of the salt-dependent dissociation of histones from hydroxyapatite-adsorbed chromatin revealed that, whereas H2A.Z co-elutes with H3-H4, hyperacetylation of histones (by treatment of chicken MSB cells with sodium butyrate) resulted in a significant fraction of this variant eluting with the canonical H2A. These studies also showed that the late elution of this variant (correlated to enhanced binding stability) was independent of the chromatin size and of the presence or absence of linker histones.     

Structure of a capsular polysaccharide isolated from Salmonella enteritidis

Salmonella enteritidis is a food-borne enteric human pathogen that can form a complex protective extracellular matrix. We describe here a component of this matrix which is distinct from other known salmonella extracellular polysaccharides such as cellulose and colanic acid. We have used glycosyl composition and linkage analysis, as well as 1D and 2D NMR spectroscopy to determine the structure of this polysaccharide. We propose that the primary saccharide in the S. enteritidis capsule has a branched tetrasaccharide repeating unit having the following structure: -->3)-alpha-D-Galp-(1-->2)-[alpha-Tyvp-(1-->3)]-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->. This structure is partially substituted on both tyvelose and galactose with a glucose-containing side chain. It further bears considerable similarity to the O antigen from this organism, a feature found in a number of other capsules from Gram-negative bacteria. In addition, we have detected fatty acids at levels that indicate the presence of a lipid anchor.     

Advances toward new antidepressants beyond SSRIs: 1-aryloxy-3-piperidinylpropan-2-ols with dual 5-HT1A receptor antagonism/SSRI activities. Part 5

A series of 1-aryloxy-3-piperidinylpropan-2-ols possessing potent dual 5-HT1A receptor antagonism and serotonin reuptake inhibition was discovered. 1-(1H-Indol-4-yloxy)-3-(4-benzo[b]thiophen-2-ylpiperidinyl)propan-2-ols exhibited selective and high affinities at the 5-HT1A receptor and serotonin reuptake site in vitro. In vivo evaluation of this series of compounds demonstrated elevated extracellular serotonin levels from the basal and quick recovery of neuron firing that was presumably suppressed by the initial acute activation of 5-HT1A somatodendritic autoreceptors.     

Aminopyridine carboxamides as c-Jun N-terminal kinase inhibitors: targeting the gatekeeper residue and beyond

The structure-activity relationships of 5,6-positions of aminopyridine carboxamide-based c-Jun N-terminal Kinase (JNK) inhibitors were explored to expand interaction with the kinase specificity and ribose-binding pockets. The syntheses of analogues and the impact of structural modification on in vitro potency and cellular activity are described.     

Automated purification of recombinant proteins: combining high-throughput with high yield

Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli (E. coli). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni-NTA-Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 microg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 microg to 45 mg per purification run, as well as strategies for optimizing these protocols.     

Pure albumin is a potent trigger of calcium signalling and proliferation in microglia but not macrophages or astrocytes

Microglial activation is implicated in the neurotoxicity of neurodegenerative diseases. Raised intracerebral levels of albumin are associated with the pathology of Alzheimer's disease, multiple sclerosis, and stroke where blood-brain barrier damage is evident. We report here that treatment of primary cultured microglia and the N9 microglial cell line with pure albumin, or albumin in which fatty acids and immunoglobulins remain attached (fraction V), induced a rise in intracellular calcium. This rise in intracellular calcium was mediated via Src tyrosine kinase and phospholipase C. The albumin-induced calcium response was coupled to microglial proliferation, which was prevented by BAPTA, U73122 or PP2 but not mimicked by thapsigargin. In contrast, peritoneal macrophages were resistant to albumin- or fraction V-induced calcium responses and proliferation, whilst primary cultured astrocytes or the TSA-3 astrocyte cell line were responsive to fraction V albumin but not pure albumin. Furthermore, cerebellar granule neurones did not respond to albumin. These data suggest that albumin may play a role in microglial activation in pathological situations involving blood-brain barrier impairment, and that the specific responses of microglia to albumin allow a distinction to be made between the signalling responses of microglia, blood-borne macrophages, astrocytes and neurones.     

SmeltCam: Underwater Video Codend for Trawled Nets with an Application to the Distribution of the Imperiled Delta Smelt

Studying rare and sensitive species is a challenge in conservation biology. The problem is exemplified by the case of the imperiled delta smelt Hypomesus transpacificus, a small delicate fish species endemic to the San Francisco Estuary, California. Persistent record-low levels of abundance and relatively high sensitivity to handling stress pose considerable challenges to studying delta smelt in the wild. To attempt to overcome these and other challenges we have developed the SmeltCam, an underwater video camera codend for trawled nets. The SmeltCam functions as an open-ended codend that automatically collects information on the number and species of fishes that pass freely through a trawled net without handling. We applied the SmeltCam to study the fine-scale distribution of juvenile delta smelt in the water column in the upper San Francisco Estuary. We learned that during flood tides delta smelt were relatively abundant throughout the water column and that during ebb tides delta smelt were significantly less abundant and occurred only in the lower half and sides of the water column. The results suggest that delta smelt manipulate their position in the water column to facilitate retention in favorable habitats. With the application of the SmeltCam we increased the survival of individual delta smelt by 72% compared to using a traditional codend, where all of the fish would have likely died due to handling stress. The SmeltCam improves upon similar previously developed silhouette photography or video recording devices and demonstrates how new technology can be developed to address important questions in conservation biology as well as lessen the negative effects associated with traditional sampling methods on imperiled species.     

Comparison of the genetic activity of aflatoxins B1 and G1 in Escherichia coli and Saccharomyces cerevisiae

The ability of aflatoxins B₁ and G₁ to induce back mutations to arg⁺ in Escherichia coli K-12/343/113 was compared with induction of mitotic gene conversion to ade⁺ in the diploid yeast strain Saccharomyces cerevisiae D4, ade₂^?. In analogy to previous results with other microorganisms, the compounds were not genetically active per se, indicating that under the experimental conditions employed none of the tester strains were able to activate the compounds to mutagenic products.In experiments using liver homegenates (S-9 fraction) of male Golden Syrian hamsters previously treated with phenobarbital, aflatoxin B₁ exhibited strong genetic activity both in E. coli and in S. cerevisiae, whereas the mutagenic activity of aflatoxin G₁ was markedly lower and could be detected only in the E. coli tester strain. These results correlate the findings that aflatoxin G₁ is a less potent carcinogen and mutagen than aflatoxin B₁.