Acidic Domain Research Articles

Identification of novel liquid biopsy biomarker for monitoring the immune set point in both solid tumor and hematological malignancy patients.

52 Background: We identified an extracellular pathway that plays a role in modulating T cell activity in vivo, which we call the Resokine Pathway. The pathway specifically involves a 60 amino acid domain of the tRNA Synthetase for Histidine, which is an essential gene in all cells, including tumor cells, vis-a-vis protein synthesis. Methods: Circulating Resokine levels were measured in 466 plasma samples from patients bearing a number of different tumor types, as well as healthy individuals, using an ECLIA format with sensitivity to 5pM. Serum Resokine levels were also measured in tumor-bearing mice. Results: Low, Circulating Resokine Levels in Cancer Patients are Negligible Compared to Healthy Individuals: Resokine levels in healthy volunteers (n = 148) ranged from 8pM - > 2333pM with 18% of the individuals possessing a level below 30pM. In contrast, levels measured across patients with all tumor types tested ranged from 20pM to > 2333pM (above the upper limit of quantification) with only 4% of the patients possessing low levels, defined as < 30pM; (P < 0.0001). Tumors in Mice Induce Higher Circulating Resokine Levels: Resokine levels in serum from normal C57Bl6 mice ranged from 70 to 250pM (n = 10). Significantly higher levels (450-3000pM) were found in the serum of B16F10 tumor-bearing mice with up to a 10-fold increase in the mean serum level compared to healthy mice (p < 0.001). Resokine levels were correlated with tumor size. Conclusions: Resokine levels correlate with tumor size in mice, and are elevated in human cancer patients. This is consistent with the hypothesis that tumors secrete Resokine, to avoid detection by the immune system. Measurement of levels of the Resokine protein may offer a new liquid biopsy biomarker for tracking immune cell activity in cancer patients. It may also be a target for therapeutic intervention but the pathway may be challenging to inhibit given the presumably higher levels at the tumor and that there are many splice variants of HARS with potential activity (Lo et al. Science 2014, 345:328-32). Acknowledgements: We would like to thank Jedd Wolchok and Phil Wong of MSKCC, New York for access to patient samples.

Newly identified type II crustin (SpCrus2) in Scylla paramamosain contains a distinct cysteine distribution pattern exhibiting broad antimicrobial activity

Type II crustins are the most abundant type of crustins in shrimps that exhibit remarkable sequence diversities and broad antibacterial activities. This study characterized a novel type II crustin, SpCrus2, in the mud crab Scylla paramamosain. The SpCrus2 cDNA sequence is 620-bp long with a 495-bp open reading frame encoding a 164-amino acid protein. In the deduced protein, a 17-amino acid signal peptide, a glycine-rich hydrophobic region (GRR), and a cysteine-rich region (CRR) containing a whey acidic protein domain were predicted. SpCrus2 shares high similarity with most type II crustins (types IIa and IIb crustins) in shrimps but has a novel distribution pattern of cysteine residues that is distinct from most crustins. SpCrus2 and PlCrus3 from Pacifastacus leniusculus share high similarity and the same distribution pattern of cysteine residues. Thus, we proposed them as type IIc crustins. SpCrus2 is mainly distributed in the gills and can be up-regulated through Vibrio parahemolyticus or Staphylococcus aureus challenge. To investigate the biological functions of SpCrus2 and the underlying mechanisms, SpCrus2, GRR, CRR, and the mutant of CRR (CRR-M, the cysteine distribution pattern is mutated into that in most conventional crustins) were all overexpressed and purified. SpCrus2 GRR itself, as a glycine-rich amphiphilic peptide, exhibited evident antibacterial ability against Gram-negative bacteria, whereas CRR possessed potent antibacterial activity against Gram-positive bacteria. Either GRR or CRR exhibited weaker antibacterial activity than the whole protein of SpCrus2, indicating that GRR and CRR synergized to exert their potential antibacterial functions. In addition, CRR exhibited slightly stronger antimicrobial activity than CRR-M, suggesting that SpCrus2 containing this novel cysteine distribution pattern may exhibit stronger antimicrobial activity than most type II crustins with the conventional distribution pattern of cysteine residues. The likely antimicrobial ability of SpCrus2 may result from its microbial polysaccharide-binding and agglutination activities. Overall, this study characterized the first type II crustin in crabs and provided new insights into understanding the sequence and functional diversity of crustins and their immune functions in crustaceans.

Complex Structures of Human MDM2 with Interacting Proteins Reveal the Regulation Mechanism of p53

Human MDM2 (murine double minute 2), amplified and overexpressed frequently in most of tumors and cancers, directly interacts with and inhibits the tumor suppressor protein p53. Understanding the regulation mechanism of MDM2 has recently been highlighted because of its roles in the regulation of p53 in cancer biology. MDM2 contains three domains. The N–terminal region interacts with p53 and downregulates its activity. The RING finger domain has ubiquitin ligase activity that can mediate p53 ubiquitination and degradation. Many studies showed that the central region of MDM2 is critical for p53 activation and tumor suppression. To date, more than twenty proteins have been shown to bind to this central region and further to be involved in the regulations of p53. However, how these interacting proteins precisely interact with MDM2 remains to be elucidated. We solved the first structure of the human MDM2–RPL11 complex. Both the acidic domain and C4 zinc finger of MDM2 are essential for RPL11 binding. Moreover, another unexpectedly zinc finger that forms inter-molecularly within the complex. A small molecule imidazole was found to be chelated in this zinc finger, which might be a promising start of drug design. These structural and functional information combining with earlier studies of MDM2 with other interacting proteins establish MDM2 as a novel class of therapeutic targets in human diseases such as cancers, yield insights into the MDM2–p53 surveillance pathway and could also yield useful anticancer strategies.

TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.

The shelterin protein TRF2 assembles protective T-loops at chromosome ends by stimulating intramolecular invasion of the telomeric G-rich ssDNA overhang into the duplex telomeric array. The other shelterin factor TRF1 is thought to mainly facilitate telomeric dsDNA replication without directly participating in end protection. Here we show that in vitro human TRF2 stimulates invasion of G-rich TERRA-like RNA into telomeric dsDNA leading to formation of telomeric RNA:DNA hybrids (telR-loops). The N-terminal basic domain of TRF2 binds to TERRA-like RNA and enables TRF2 to promote efficient RNA invasion. TRF1, through its N-terminal acidic domain, counteracts TRF2-mediated RNA but not ssDNA invasion. In vivo, when TRF1 is depleted or replaced with a variant lacking the acidic domain, TRF2 induces formation of telR-loops, which in turn cause telomere loss. Hence, uncontrolled TRF2 threatens telomere integrity and TRF1 directly supports end protection by suppressing harmful telR-loops.

Acyl-CoA-binding protein family members in laticifers are possibly involved in lipid and latex metabolism of Hevea brasiliensis (the Para rubber tree)

BackgroundAcyl-CoA-binding proteins (ACBPs) are mainly involved in acyl-CoA ester binding and trafficking in eukaryotic cells, and their various functions have been characterized in model plants, such as Arabidopsis thaliana (A. thaliana), Oryza sativa (rice), and other plant species. In the present study, genome-wide mining and expression analysis of ACBP genes was performed on Hevea brasiliensis (the para rubber tree), the most important latex-producing crop in the world.ResultsSix members of the H. brasiliensis ACBP family genes, designated HbACBP1-HbACBP6, were identified from the H. brasiliensis genome. They can be categorized into four classes with different amino acid sequences and domain structures based on the categorization of their A. thaliana counterparts. Phylogenetic analysis shows that the HbACBPs were clustered with those of other closely related species, such as Manihot esculenta, Ricinus communis, and Jatropha carcas, but were further from those of A. thaliana, a distantly related species. Expression analysis demonstrated that the HbACBP1 and HbACBP2 genes are more prominently expressed in H. brasiliensis latex, and their expression can be significantly enhanced by bark tapping (a mechanical wound) and jasmonic acid stimulation, whereas HbACBP3-HbACBP6 had almost the same expression patterns with relatively high levels in mature leaves and male flowers, but a markedly low abundance in the latex. HbACBP1 and HbACBP2 may have crucial roles in lipid and latex metabolism in laticifers, so their subcellular location was further investigated and the results indicated that HbACBP1 is a cytosol protein, whereas HbACBP2 is an endoplasmic reticulum-associated ACBP.ConclusionsIn this study, the H. brasiliensis ACBP family genes were identified. Phylogenetic analyses of the HbABCPs indicate that there is a high conservation and evolutionary relationship between ACBPs in land plants. The HbACBPs are organ/tissue-specifically expressed and have different expression patterns in response to stimulation by bark tapping or ethrel/jasmonic acid. HbACBP1 and HbACBP2 are two important latex ACBPs that might be involved in the lipid and latex metabolism. The results may provide valuable information for further investigations into the biological functions of HbACBPs during latex metabolism and stress responses in H. brasiliensis.

Functional characterization of isolated RNA-binding domains of the GRSF1 protein

The Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the heterogeneous nuclear ribonucleoprotein F/H family and has been implicated in RNA processing, RNA transport and translational regulation. Amino acid alignments and homology modeling suggested the existence of three distinct RNA-binding domains and two auxiliary domains. Unfortunately, little is known about the molecular details of GRSF1/RNA interactions. To explore the RNA-binding mechanisms we first expressed full-length human GRSF1 and several truncation mutants, which include the three separated qRRM domains in E. coli, purified the recombinant proteins and quantified their RNA-binding affinity by RNA electrophoretic mobility shift assays. The expression levels varied between 1 and 10mg purified protein per L bacterial liquid culture and for full-length human GRSF1 a binding constant (KD-value) of 0.5μM was determined. In addition, our mechanistic experiments with different truncation mutants allowed the following conclusions: i) Deletion of either of the three RNA-binding domains impaired the RNA-binding affinity suggesting that the simultaneous presence of the three domains is essential for high-affinity RNA-binding. ii) Deletion of the Ala-rich auxiliary domain did hardly affect RNA-binding. Thus, this structural subunit may not be involved in RNA interaction. iii) Deletion of the acidic auxiliary domain improved the RNA-binding suggesting a regulatory role for this structural motif. iv) The isolated RNA-binding domains did not exhibit sizeable RNA-binding affinities. Taken together these data suggest that a cooperative interaction of the three qRRMs is required for high affinity RNA-binding.

Front dynamics of pH oscillators with initially separated reactants

The spatiotemporal dynamics of the Landolt-type pH oscillators are studied both numerically and experimentally with initially separated reagents in space. This configuration results in an A + B → oscillator front type system with localized patterns. The generic Rabai model of the pH-oscillators predicts the formation of an asymmetric acidic domain at the interface of the two zones loaded by different sets of chemicals. This asymmetry is caused by the initial conditions rather than the difference in the diffusivities of the components. As the influence of the negative feedback process increases, this acidic zone becomes to be localized around the interface. At some point, the acidic zone bifurcates, a less acidic zone separates and starts to move forward the oxidant rich zone. In a limited domain of parameters, spatiotemporal oscillations are found due to the instability of the main acidic zone. The appropriate conditions for the development of this periodic behavior is characterized by simulations. The numerically predicted phenomena are supported by experiments performed with the bromate–sulfite–ferrocyanide and with the hydrogen peroxide–sulfite–ferrocyanide systems, except the oscillatory phenomena.

Characterization of LhSorTGA2, a novel TGA2-like protein that interacts with LhSorNPR1 in oriental hybrid lily Sorbonne

BackgroundNon-expressor of pathogenesis-related genes 1 (NPR1) regulates expression of pathogenesis-related (PR) genes by interacting with TGA family proteins during systemic acquired resistance (SAR). However, no TGA-like proteins or their interacting partners have been characterized in lily.ResultsIn the present study, LhSorTGA2, a novel TGA-like protein, was identified as an interacting partner of LhSorNPR1 (an NPR-like protein) by bimolecular fluorescence complementation (BIFC) and yeast two-hybrid assay (Y2H). Subcellular localization of GFP-tagged proteins targeted LhSorTGA2 to the nucleus, whereas GFP-labeled LhSorNPR1 was observed both in the nucleus and at the cytomembrane. Sequence alignment revealed that LhSorTGA2 was featured with a basic leucine zipper (bZIP) domain and two glutamine rich acid domains (QI and QII). Further phylogenetic analysis showed that TGA family proteins can be grouped into three subclades, within which LhSorTGA2 was clustered into subclade I, together with AtTGA2/5/6. Expression of LhSorTGA2 was investigated in different tissues by qPCR, and the highest expression level was observed in stem. Besides, when treated with phytohormones (SA, MeJA, ETH and ABA) or fungal pathogen Botrytis elliptica, LhSorTGA2 expression was also induced at different time points post treatments.ConclusionsCollectively, these results suggested that LhSorTGA2 was an interacting partner of LhSorNPR1, which might function in regulating expression of PR genes in lily during SAR.

Purification of small-size acidic proteoglycan-like domain of carbonic anhydrase IX fused with thioredoxine expressed in Escherichia coli for structural characterization

AbstractProteoglycan-like (PG) domain of carbonic anhydrase IX (CA IX) is a small-sized acidic domain (8.3 kDa; pI ∼ 3.5) with a very unusual amino acid composition. More than one third of its sequence consists of acidic amino acids and it contains no aromatic residues. It has been revealed that it holds one of the key roles in oncogenetic mechanisms, in which CA IX participates. However, it has not been structurally characterized yet. With these prospects, we developed an expression system of recombinant PG domain production in the form of a fusion protein using thioredoxine (Trx) as the fusion partner in

Dimerization and Long-Range Repulsion Established by Both Termini of the Microtubule-Associated Protein Tau.

Tau is a microtubule-associated protein found in neuronal axons that has several well-known functions, such as promoting microtubule polymerization, stabilizing microtubules against depolymerization, and spatially organizing microtubules in axons. Two contrasting models have been previously described to explain tau's ability to organize the spacing between microtubules: complementary dimerization of the projection domains of taus on adjacent microtubules or tau's projection domain acting as a polyelectrolyte brush. In this study, atomic force microscopy was used to interrogate intermolecular interactions between layers of tau protein immobilized on mica substrates and on silicon nitride atomic force microscope tips. On these surfaces, tau adopts an orientation comparable to that when bound to microtubules, with the basic microtubule binding domain immobilized and the acidic domains extending into solution. Force-distance curves collected via atomic force microscopy reveal that full length human tau, when assembled into dense surface-bound layers, can participate in attractive electrostatic interactions consistent with the previously reported dimerization model. However, modulating the ionic strength of the surrounding solution can change the structure of these layers to produce purely repulsive interactions consistent with a polyelectrolyte brush structure, thus providing biophysical evidence to support both the zipper and brush models. In addition, a pair of projection domain deletion mutants were examined to investigate whether the projection domain of the protein is essential for the dimerization and brush models. Force-distance curves collected on layers of these proteins demonstrate that the C-terminus can play a role analogous to that of the projection domain.

Comparative profiling of HLA-DR and HLA-DQ associated factor VIII peptides presented by monocyte-derived dendritic cells.

The development of anti-factor VIII antibodies is a major complication of the treatment of patients with hemophilia A. Generation of high affinity anti-factor VIII antibodies is dependent on help provided by CD4+ T cells that recognize factor VIII-derived peptides presented on class II major histocompatibility complex on the surface of antigen-presenting cells. In order to identify the immune-dominant epitopes that can be presented to CD4+ T cells, we previously developed a mass spectrometry-based method to identify factor VIII-derived peptides that are presented on human leukocyte antigen (HLA)-DR. In the present work, we compared the repertoire of FVIII-derived peptide presented on HLA-DR and HLA-DQ. Monocyte-derived dendritic cells from nine HLA-typed healthy donors were pulsed with recombinant factor VIII. HLA-DR and HLA-DQ molecules were purified using monoclonal antibodies. Our data show that HLA-DQ and HLA-DR present a similar repertoire of factor VIII-derived peptides. However, the number of peptides associated with HLA-DQ was lower than that with HLA-DR. We also identified a peptide, within the acidic a3 domains of factor VIII, which is presented with higher frequency on HLA-DQ. Interestingly, this peptide was found to have a higher predicted affinity for HLA-DQ than for HLA-DR. Taken together, our data suggest that HLA-DQ participates in the presentation of factor VIII peptides, thereby contributing to the development of inhibitory antibodies in a proportion of patients with severe hemophilia A.

Cancer-mutated ribosome protein L22 (RPL22/eL22) suppresses cancer cell survival by blocking p53-MDM2 circuit.

Several ribosomal proteins (RPs) in response to various ribosomal stressors have been shown to play a critical role in p53-dependent regulation of cell cycle arrest, apoptosis and tumor suppression. Here, we report ribosomal protein L22 (RPL22/eL22) as a novel p53 activator highly mutated (mostly deletion mutation) in various types of human cancers, but not essential for ribosomal biogenesis in normal cells. Ectopic expression of RPL22/eL22 suppressed the colony formation of cancer cells in a p53-dependent manner, whereas knockdown of RPL22/eL22 significantly compromised p53 activation by Actinomycin D, rescuing p53-induced G1/G0 cell cycle arrest. Interestingly, human tumors with RPL22/eL22 deletion appeared to sustain wild type p53. Mechanistically, RPL22/eL22 bound to MDM2 acidic domain and inhibited MDM2-mediated p53 ubiquitination and degradation, hence extending the half-life of p53. Ribosome-profiling analysis revealed that induction of ribosomal stress by Actinomycin D leads to the increase of ribosome-free RPL22/eL22 pool. Also, RPL22/eL22 formed a complex with MDM2/RPL5/uL18/RPL11/uL5 and synergized with RPL11/uL5 to activate p53. Furthermore, the N terminus of RPL22/eL22 bound to MDM2, while the C terminus interacted with RPL5/uL18/RPL11/uL5; both of these two fragments activated p53 by inhibiting MDM2. Our study indicates that RPL22/eL22 highly mutated in human cancers plays an anti-cancer role likely through regulation of the MDM2-p53 feedback loop, and also suggests that targeting the RPL22/eL22-MDM2-p53 pathway could be a potential strategy for future development of anti-cancer therapy.

The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria

BackgroundThe parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface.ResultsHere we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions.ConclusionsWe demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

Two Archaeal RecJ Nucleases from Methanocaldococcus jannaschii Show Reverse Hydrolysis Polarity: Implication to Their Unique Function in Archaea.

Bacterial nuclease RecJ, which exists in almost all bacterial species, specifically degrades single-stranded (ss) DNA in the 5′ to 3′ direction. Some archaeal phyla, except Crenarchaea, also encode RecJ homologs. Compared with bacterial RecJ, archaeal RecJ exhibits a largely different amino acid sequence and domain organization. Archaeal RecJs from Thermococcus kodakarensis and Pyrococcus furiosus show 5′→3′ exonuclease activity on ssDNA. Interestingly, more than one RecJ exists in some Euryarchaeota classes, such as Methanomicrobia, Methanococci, Methanomicrobia, Methanobacteria, and Archaeoglobi. Here we report the biochemical characterization of two RecJs from Methanocaldococcus jannaschii, the long RecJ1 (MJ0977) and short RecJ2 (MJ0831) to understand their enzymatic properties. RecJ1 is a 5′→3′ exonuclease with a preference to ssDNA; however, RecJ2 is a 3′→5′ exonuclease with a preference to ssRNA. The 5′ terminal phosphate promotes RecJ1 activity, but the 3′ terminal phosphate inhibits RecJ2 nuclease. Go-Ichi-Ni-San (GINS) complex does not interact with two RecJs and does not promote their nuclease activities. Finally, we discuss the diversity, function, and molecular evolution of RecJ in archaeal taxonomy. Our analyses provide insight into the function and evolution of conserved archaeal RecJ/eukaryotic Cdc45 protein.

Functional Analysis of the Pepper Ethylene-Responsive Transcription Factor, CaAIEF1, in Enhanced ABA Sensitivity and Drought Tolerance.

Abscisic acid (ABA) is a plant hormone that plays a critical role in the response to environmental stress conditions, especially regulation of the stomatal aperture under water-deficit conditions. The signal transduction occurring during the stress response is initiated by transcription of defense-related genes. Here, we isolated the pepper ethylene-responsive transcription factor CaAIEF1 (Capsicum annuum ABA Induced ERF 1). The CaAIEF1 gene was significantly induced after exposure to ABA, drought, and high salinity. Fusion of the acidic domain in the C-terminal region of CaAIEF1 to the GAL4 DNA-binding domain had a transactivation effect on the reporter gene in yeast. Further, the CaAIEF1-GFP fusion constructs localized in the nucleus. We used CaAIEF1-silenced plants and CaAIEF1-overexpressing (OX) plants to elucidate the biological function of CaAIEF1 in response to ABA and drought stress. CaAIEF1-silenced pepper plants and CaAIEF1-OX Arabidopsis plants displayed drought-sensitive and -tolerant phenotypes, respectively, which were characterized by regulation of transpirational water loss and stomatal aperture. In drought stress condition, quantitative RT-PCR analyses revealed that the expression levels of pepper stress-related genes were higher in CaAIEF1-silenced pepper plants than control plants. Moreover, expression levels of Arabidopsis stress-related genes were significantly reduced in CaAIEF1-OX plants compared with control plants in drought stress condition. Our findings suggest that CaAIEF1 positively regulates the drought stress response and the ABA signaling.

A Lysine Desert Protects a Novel Domain in the Slx5-Slx8 SUMO Targeted Ub Ligase To Maintain Sumoylation Levels in Saccharomyces cerevisiae.

Protein modification by the small ubiquitin-like modifier (SUMO) plays important roles in genome maintenance. In Saccharomyces cerevisiae, proper regulation of sumoylation is known to be essential for viability in certain DNA repair mutants. Here, we find the opposite result; proper regulation of sumoylation is lethal in certain DNA repair mutants. Yeast cells lacking the repair factors TDP1 and WSS1 are synthetically lethal due to their redundant roles in removing Top1-DNA covalent complexes (Top1ccs). A screen for suppressors of tdp1∆ wss1∆ synthetic lethality isolated mutations in genes known to control global sumoylation levels including ULP1, ULP2, SIZ2, and SLX5 The results suggest that alternative pathways of repair become available when sumoylation levels are altered. Curiously, both suppressor mutations that were isolated in the Slx5 subunit of the SUMO-targeted Ub ligase created new lysine residues. These "slx5-K" mutations localize to a 398 amino acid domain that is completely free of lysine, and they result in the auto-ubiquitination and partial proteolysis of Slx5. The decrease in Slx5-K protein leads to the accumulation of high molecular weight SUMO conjugates, and the residual Ub ligase activity is needed to suppress inviability presumably by targeting polysumoylated Top1ccs. This "lysine desert" is found in the subset of large fungal Slx5 proteins, but not its smaller orthologs such as RNF4. The lysine desert solves a problem that Ub ligases encounter when evolving novel functional domains.

Magnetic bead based assays for complement component C5

Two novel magnetic agarose bead based assays have been developed to measure complement component C5 interaction with C3b and the Factor I Modules (FIMs) of C7. One innovation was to couple C3b onto the magnetic agarose bead using the alternative pathway C3 convertase, which resulted in a linkage of the ligand by a covalent ester bond. A second innovation was to employ nickel ion charged N,N,N′-tris(carboxymethyl)ethylene-diamine-magnetic agarose to capture recombinantly prepared C7 FIMs that were expressed with an oligo-histidine linker followed by an acidic domain that provided a spacer enabling the C7 modules exposure to C5. Detection was brought about by peroxidase coupled to C5. Both assays exhibited adequate statistics suitable for screening. As examples of the utility of these new methods, we chose to examine influence of natural products on C5 interaction. Fucoidan and β-glucans were observed to inhibit C3b-C5 interaction, and dextran sulfate was similarly active; however, rosmarinic acid had no measurable effect. In contrast only β-glucans from two species of macrofungi were able to interfere with interaction of C5 with the FIMs of C7.

Release kinetics of vanadium from vanadium titano-magnetite: The effects of pH, dissolved oxygen, temperature and foreign ions

As part of a broader study of the environmental geochemistry behavior of vanadium (V), the release kinetics of V from the dissolution of natural vanadium titano-magnetite under environmentally relevant conditions was investigated. In both the acidic and basic domains, the V release rate was found to be proportional to fractional powers of hydrogen ion and dissolved oxygen activities. The dependence of the rate on dissolved oxygen can also be described in terms of the Langmuir adsorption model. The empirical rate equation is given by: r=k′αH+αKαO21+KαO2 where, α=0.099–0.265, k′=3.2×10−6–1.7×10−5, K=2.7×104–3.9×104mol/L in acid solution (pH4.1), and α=−0.494−(−0.527), k′=2.0×104–2.5×10−11, and K=4.1×103–6.5×103mol/L in basic solution (pH8.8) at 20°C. Based on the effect of temperature on the release rate of V, the activation energies of minerals at pH8.8 were determined to be 148–235kJ/mol, suggesting that the dissolution of vanadium titano-magnetite is a surface-controlled process. The presence of Na+, Ca2+, Mg2+, K+, NO3−, Cl−, SO42− and CO32− was found to accelerate the V release rates. This study improves the understanding of both the V pollution risk in some mine areas and the fate of V in the environment.

Nuclear localization and transactivation by Vitis CBF transcription factors are regulated by combinations of conserved amino acid domains

The highly conserved CBF pathway is crucial in the regulation of plant responses to low temperatures. Extensive analysis of Arabidopsis CBF proteins revealed that their functions rely on several conserved amino acid domains although the exact function of each domain is disputed. The question was what functions similar domains have in CBFs from other, overwintering woody plants such as Vitis, which likely have a more involved regulation than the model plant Arabidopsis. A total of seven CBF genes were cloned and sequenced from V. riparia and the less frost tolerant V. vinifera. The deduced species-specific amino acid sequences differ in only a few amino acids, mostly in non-conserved regions. Amino acid sequence comparison and phylogenetic analysis showed two distinct groups of Vitis CBFs. One group contains CBF1, CBF2, CBF3 and CBF8 and the other group contains CBF4, CBF5 and CBF6. Transient transactivation assays showed that all Vitis CBFs except CBF5 activate via a CRT or DRE promoter element, whereby Vitis CBF3 and 4 prefer a CRT element. The hydrophobic domains in the C-terminal end of VrCBF6 were shown to be important for how well it activates. The putative nuclear localization domain of Vitis CBF1 was shown to be sufficient for nuclear localization, in contrast to previous reports for AtCBF1, and also important for transactivation. The latter highlights the value of careful analysis of domain functions instead of reliance on computer predictions and published data for other related proteins.

Cardiomyocyte specific overexpression of a 37 amino acid domain of regulator of G protein signalling 2 inhibits cardiac hypertrophy and improves function in response to pressure overload in mice

Regulator of G protein signalling 2 (RGS2) is known to play a protective role in maladaptive cardiac hypertrophy and heart failure via its ability to inhibit Gq- and Gs- mediated GPCR signalling. We previously demonstrated that RGS2 can also inhibit protein translation and can thereby attenuate cell growth. This G protein-independent inhibitory effect has been mapped to a 37 amino acid domain (RGS2eb) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed in neonatal rat cardiomyocytes, RGS2eb attenuates both protein synthesis and hypertrophy induced by Gq- and Gs- activating agents. In the current study, we investigated the potential cardioprotective role of RGS2eb by determining whether RGS2eb transgenic (RGS2eb TG) mice with cardiomyocyte specific overexpression of RGS2eb show resistance to the development of hypertrophy in comparison to wild-type (WT) controls. Using transverse aortic constriction (TAC) in a pressure-overload hypertrophy model, we demonstrated that cardiac hypertrophy was inhibited in RGS2eb TG mice compared to WT controls following four weeks of TAC. Expression of the hypertrophic markers atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC-β) was also reduced in RGS2eb TG compared to WT TAC animals. Furthermore, cardiac function in RGS2eb TG TAC mice was significantly improved compared to WT TAC mice. Notably, cardiomyocyte cell size was significantly decreased in TG compared to WT TAC mice. These results suggest that RGS2 may limit pathological cardiac hypertrophy at least in part via the function of its eIF2B-binding domain.