Interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments containing isolated ligand binding sites

We have examined the interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments which contain isolated ligand binding sites. A 145 base pair fragment was prepared on the basis of the sequence of tyrT DNA, which contained no CpG or (A/T)(4) binding sites for these ligands. Isolated binding sites were introduced into this fragment at discrete locations where the minor groove is known to face toward or away from the protein core when reconstituted onto nucleosome core particles. The interaction of ligands with target sites on these nucleosomal DNA fragments was assessed by DNase I footprinting. We find that Hoechst 33258 can bind to single nucleosomal sites which face both toward and away from the protein core, without affecting the nucleosome structure. Hoechst binding is also observed on nucleosomal fragments which contain two or more drug binding sites, though in these cases the footprints are accompanied by the presence of new cleavage products in positions which suggest that the ligand has caused a proportion of the DNA molecules to adopt a new rotational positioning on the protein surface. Hoechst 33258 does not affect nucleosome reconstitution with any of these fragments. In contrast, the bifunctional intercalating antibiotic echinomycin is not able to bind to single nucleosomal CpG sites. Echinomycin footprints are observed on nucleosomal fragments containing two or more CpG sites, but there are no changes in the cleavage patterns in the remainder of the fragment. Echinomycin abolishes nucleosome reconstitution when included in the reconstitution mixture.     

The palaeoecology of the Upper Ndolanya Beds at Laetoli,Tanzania

The palaeoecology of the fauna from the Ndolanya Beds, Laetoli, Tanzania, has been analysed to reconstruct the environment of this 2.6 Ma site. Community profiles have been constructed in relation to three variables that carry ecological meaning: body weight, locomotor adaptations and feeding preferences. Comparing the similarities and differences in the structure of the fossil faunas with those of modern environments allows us to draw inferences about palaeoenvironmental conditions, and this is based on a comparative dataset of 15 modern environments (44 localities) covering a wide range of climatic and ecological conditions across Asia, Africa and Central America. In addition, 16 fossil sites in East and South Africa have been analysed in the same way, and both sets of comparative data have been used as the basis for comparison with an ecological diversity analysis of the fauna from the Upper Ndolanya Beds. The Ndolanya fauna is characterized by a predominance of medium to large sized terrestrial and herbivorous species.There is evidence of taphonomic bias that eliminated many of the smaller species. A comparison of multivariate analyses of 23 selected modern localities conducted both with and without the small species included, indicates that the loss of these species does not adversely influence the results of an ecological diversity analysis. The evidence suggests that at the time of the deposition of the Ndolanya Beds the Laetoli region was a semi-arid bushland. This is considerably drier and more open than is suggested for the earlier Laetolil Beds.     

Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica

Root-knot nematodes (Meloidogynejavanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.     

PIP(2)-PDZ domain binding controls the association of syntenin with the plasma membrane

PDZ proteins organize multiprotein signaling complexes. According to current views, PDZ domains engage in protein-protein interactions. Here we show that the PDZ domains of several proteins bind phosphatidylinositol 4,5-bisphosphate (PIP(2)). High-affinity binding of syntenin to PIP(2)-containing lipid layers requires both PDZ domains of this protein. Competition and mutagenesis experiments reveal that the protein and the PIP(2) binding sites in the PDZ domains overlap. Overlay assays indicate that the two PDZ domains of syntenin cooperate in binding to cognate peptides and PIP(2). Experiments on living cells demonstrate PIP(2)-dependent and peptide-dependent modes of plasma membrane association of the PDZ domains of syntenin. These observations suggest that local changes in phosphoinositide concentration control the association of PDZ proteins with their target receptors at the plasma membrane.     

Molecular cloning and characterization of fox testis kinectin

Kinectin was isolated and characterized from a fox testis cDNA library using a monoclonal antibody (FTA-1) raised against testis surface proteins. The cDNA sequence of 4,479 nucleotides encodes an ORF of 1,330 amino acids (aa) with high homology to mouse, human, and chicken kinectins (GenBank Accession Number AF095786). Southern analysis was used to show that genes homologous to kinectin are present in several mammal species and in at least one marsupial, but not in bacteria. Alternatively spliced forms of fox kinectin were identified, and one of these is uniquely expressed in brain and spleen tissues. Kinectin expression was highest in testis relative to other tissues examined. Sequence analysis and comparisons between species revealed that kinectin encodes multiple alpha-helical coiled coils predicted to form dimers, and is, therefore, likely to exist as a dimer. The results presented in this article suggest that kinectin is required for spermatogenesis, but is not a likely candidate for use in immunocontraceptive vaccines.     

Assessment of electroporation by flow cytometry

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.     

Anodal tDCS over the Primary Motor Cortex Facilitates Long-Term Memory Formation Reflecting Use-Dependent Plasticity

Previous research suggests that anodal transcranial direct current stimulation (tDCS) over the primary motor cortex (M1) modulates NMDA receptor dependent processes that mediate synaptic plasticity. Here we test this proposal by applying anodal versus sham tDCS while subjects practiced to flex the thumb as fast as possible (ballistic movements). Repetitive practice of this task has been shown to result in performance improvements that reflect use-dependent plasticity resulting from NMDA receptor mediated, long-term potentiation (LTP)-like processes. Using a double-blind within-subject cross-over design, subjects (n=14) participated either in an anodal or a sham tDCS session which were at least 3 months apart. Sham or anodal tDCS (1 mA) was applied for 20 min during motor practice and retention was tested 30 min, 24 hours and one week later. All subjects improved performance during each of the two sessions (p < 0.001) and learning gains were similar. Our main result is that long term retention performance (i.e. 1 week after practice) was significantly better when practice was performed with anodal tDCS than with sham tDCS (p < 0.001). This effect was large (Cohen’s d=1.01) and all but one subject followed the group trend. Our data strongly suggest that anodal tDCS facilitates long-term memory formation reflecting use-dependent plasticity. Our results support the notion that anodal tDCS facilitates synaptic plasticity mediated by an LTP-like mechanism, which is in accordance with previous research.     

DYn-2 Based Identification of Arabidopsis Sulfenomes

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H₂O₂) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H₂O₂ dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H₂O₂ treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.Among the different amino acids, the sulfur containing amino acids like cysteine are particularly susceptible to oxidation by reactive oxygen species (ROS)¹ (1, 2). Recent studies suggest that the sulfenome, the initial oxidation products of cysteine residues, functions as an intermediate state of redox signaling (3 –5). Thus, identifying the sulfenome under oxidative stress is a way to detect potential redox sensors (6, 7).This central role of the sulfenome in redox signaling provoked chemical biologists to develop strategies for sensitive detection and identification of sulfenylated proteins. The in situ trapping of the sulfenome is challenging because of two major factors: (1) the highly reactive, transient nature of sulfenic acids, which might be over-oxidized in excess of ROS, unless immediately protected by disulfide formation (7); (2) the intracellular compartmentalization of the redox state that might be disrupted during extraction procedures, resulting in artificial non-native protein oxidations (8, 9). Having a sulfur oxidation state of zero, sulfenic acids can react as both electrophile and nucleophile, however, direct detection methods are based on the electrophilic character of sulfenic acid (10). In 1974, Allison and coworkers reported a condensation reaction between the electrophilic sulfenic acid and the nucleophile dimedone (5,5-dimethyl-1,3-cyclohexanedione), producing a corresponding thioether derivative (11). This chemistry is highly selective and, since then, has been exploited to detect dimedone modified sulfenic acids using mass spectrometry (12). However, dimedone has limited applications for cellular sulfenome identification because of the lack of a functional group to enrich the dimedone tagged sulfenic acids. Later, dimedone-biotin/fluorophores conjugates have been developed, which allowed sensitive detection and enrichment of sulfenic acid modified proteins (13 –15). This approach, however, was not always compatible with in vivo cellular sulfenome analysis, because the biotin/fluorophores-conjugated dimedone is membrane impermeable (9) and endogenous biotinylated proteins might appear as false positives.More recently, the Carroll lab has developed DYn-2, a sulfenic acid specific chemical probe. This chemical probe consists of two functional units: a dimedone scaffold for sulfenic acid recognition and an alkyne chemical handle for enrichment of labeled proteins (9). Once the sulfenic acids are tagged with the DYn-2 probe, they can be biotinylated through click chemistry (16). The click reaction used here is a copper (I)-catalyzed azide-alkyne cycloaddition reaction (17), also known as azide-alkyne Huisgen cycloaddition (16). With this chemistry, a complex is formed between the alkyne functionalized DYn-2 and the azide functionalized biotin. This biotin functional group facilitates downstream detection, enrichment, and mass spectrometry based identification (Fig. 1). In an evaluation experiment, DYn-2 was found to efficiently detect H₂O₂-dependent sulfenic acid modifications in recombinant glutathione peroxidase 3 (Gpx3) of budding yeast (18). Moreover, it was reported that DYn-2 is membrane permeable, non-toxic, and a non-influencer of the intracellular redox balance (17, 18). Therefore, DYn-2 has been suggested as a global sulfenome reader in living cells (17, 18), and has been applied to investigate epidermal growth factor (EGF) mediated protein sulfenylation in a human epidermoid carcinoma A431 cell line and to identify intracellular protein targets of H₂O₂ during cell signaling (17).Open in a separate windowFig. 1.Schematic views of the molecular mechanism of the DYn-2 probe and the strategy to identify DYn-2 trapped sulfenylated proteins. A, DYn-2 specifically detects sulfenic acid modifications, but no other thiol modifications. B, Biotinylation of the DYn-2 tagged proteins by click reaction. C, Once DYn-2 tagged proteins are biotinylated, a streptavidin-HRP (Strep-HRP) blot visualizes sulfenylation, or alternatively, after enrichment on avidin beads, proteins are identified by mass spectrometry analysis.Here, we selected the DYn-2 probe to identify the sulfenome in plant cells under oxidative stress. Through a combination of biochemical, immunoblot and mass spectrometry techniques, and TAIR10 database and SUBA3-software predictions, we can claim that DYn-2 is able to detect sulfenic acids on proteins located in different subcellular compartments of plant cells. We identified 226 sulfenylated proteins in response to an H₂O₂ treatment of Arabidopsis cell suspensions, of which 123 proteins are new candidates for cysteine oxidative post-translational modification (PTM) events.