R7BP augments the function of RGS7*Gbeta5 complexes by a plasma membrane-targeting mechanism

The RGS7 (R7) family of G protein regulators, Gbeta5, and R7BP form heterotrimeric complexes that potently regulate the kinetics of G protein-coupled receptor signaling. Reversible palmitoylation of R7BP regulates plasma membrane/nuclear shuttling of R7*Gbeta5*R7BP heterotrimers. Here we have investigated mechanisms whereby R7BP controls the function of the R7 family. We show that unpalmitoylated R7BP undergoes nuclear/cytoplasmic shuttling and that a C-terminal polybasic motif proximal to the palmitoylation acceptor sites of R7BP mediates nuclear localization, palmitoylation, and plasma membrane targeting. These results suggest a novel mechanism whereby palmitoyltransferases and nuclear import receptors both utilize the C-terminal domain of R7BP to determine the trafficking fate of R7*Gbeta5*R7BP heterotrimers. Analogous mechanisms may regulate other signaling proteins whose distribution between the plasma membrane and nucleus is controlled by palmitoylation. Lastly, we show that cytoplasmic RGS7*Gbeta5*R7BP heterotrimers and RGS7*Gbeta5 heterodimers are equivalently inefficient regulators of G protein-coupled receptor signaling relative to plasma membrane-bound heterotrimers bearing palmitoylated R7BP. Therefore, R7BP augments the function of the complex by a palmitoylation-regulated plasma membrane-targeting mechanism.     

VKORC1 pharmacogenetics and pharmacoproteomics in patients on warfarin anticoagulant therapy: transthyretin precursor as a potential biomarker

BACKGROUND: Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke. METHODS AND FINDING: We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation. CONCLUSION: This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism.     

Distribution pattern, endemism, threat status and conservation measures of fishes in the Tunga and Bhadra rivers of Western Ghats, India

This study documents the distribution patterns, endemism and uniqueness (species richness) of fishes in the Tunga and Bhadra rivers of Western Ghats, India. We recorded 77 species represented by 7 orders, 16 families and 44 genera, of which 36 species are endemic to Western Ghats, 12 species endemic to India and 26 species endemic to the Indian Subcontinent. Based on our analysis on the distribution patterns, the Tunga River is richer in diversity and higher in endemism than the Bhadra. We calculated the similarity of the species composition among sites within these two rivers using the Jacquard index. The similarity index between the sampling sites of these rivers revealed that the similarity decreases with increasing distance between the sampling sites. Of the 77 fish species we collected, 8 species (11.1%) are in the Critical category, 10 species (13.8%) are in the High Risk category, 36 species (50%) are in the Moderate category and the remaining 18 species (25%) are at lower risk. The threat status of fishes found in the Tunga and Bhadra rivers strongly suggests the need for effective conservation measures to conserve the fish species richness of these rivers.     

Zn(++) binding disrupts the Asp(23)-Lys(28) salt bridge without altering the hairpin-shaped cross-β Structure of Aβ(42) amyloid aggregates

Observations like high Zn²⁺ concentrations in senile plaques found in the brains of Alzheimer''s patients and evidences emphasizing the role of Zn²⁺ in amyloid-β (Aβ)-induced toxicity have triggered wide interest in understanding the nature of Zn²⁺-Aβ interaction. In vivo and in vitro studies have shown that aggregation kinetics, toxicity, and morphology of Aβ aggregates are perturbed in the presence of Zn²⁺. Structural studies have revealed that Zn²⁺ has a binding site in the N-terminal region of monomeric Aβ, but not much is precisely known about the nature of binding of Zn²⁺ with aggregated forms of Aβ or its effect on the molecular structure of these aggregates. Here, we explore this aspect of the Zn²⁺-Aβ interaction using one- and two-dimensional ¹³C and ¹⁵N solid-state NMR. We find that Zn²⁺ causes major structural changes in the N-terminal and the loop region connecting the two β-sheets. It breaks the salt bridge between the side chains of Asp²³ and Lys²⁸ by driving these residues into nonsalt-bridge-forming conformations. However, the cross-β structure of Aβ₄₂ aggregates remains unperturbed though the fibrillar morphology changes distinctly. We conclude that the salt bridge is not important for defining the characteristic molecular architecture of Aβ₄₂ but is significant for determining its fibrillar morphology and toxicity.     

Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes

The canonical Wnt/β-catenin signaling pathway plays an important role in thymocyte development and T cell migration, but little is known about its role in naive-to-effector differentiation in human peripheral T cells. We show that activation of Wnt/β-catenin signaling arrests human peripheral blood and cord blood T lymphocytes in the naive stage and blocks their transition into functional T effector cells. Wnt signaling was induced in polyclonally activated human T cells by treatment either with the glycogen synthase kinase 3β inhibitor TWS119 or the physiological Wnt agonist Wnt-3a, and these T cells preserved a naive CD45RA(+)CD62L(+) phenotype compared with control-activated T cells that progressed to a CD45RO(+)CD62L(-) effector phenotype, and this occurred in a TWS119 dose-dependent manner. TWS119-induced Wnt signaling reduced T cell expansion, as a result of a block in cell division, and impaired acquisition of T cell effector function, measured by degranulation and IFN-γ production in response to T cell activation. The block in T cell division may be attributed to the reduced IL-2Rα expression in TWS119-treated T cells that lowers their capacity to use autocrine IL-2 for expansion. Collectively, our data suggest that Wnt/β-catenin signaling is a negative regulator of naive-to-effector T cell differentiation in human T lymphocytes. The arrest in T cell differentiation induced by Wnt signaling might have relevant clinical applications such as to preserve the naive T cell compartment in Ag-specific T cells generated ex vivo for adoptive T cell immunotherapy.     

Nature of the amyloid-beta monomer and the monomer-oligomer equilibrium

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-β (Aβ) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aβ(40) and Aβ(42) remain predominantly monomeric up to 3 μm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aβ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.     

Ultrasensitive detection of rare mutations using next-generation targeted resequencing

With next-generation DNA sequencing technologies, one can interrogate a specific genomic region of interest at very high depth of coverage and identify less prevalent, rare mutations in heterogeneous clinical samples. However, the mutation detection levels are limited by the error rate of the sequencing technology as well as by the availability of variant-calling algorithms with high statistical power and low false positive rates. We demonstrate that we can robustly detect mutations at 0.1% fractional representation. This represents accurate detection of one mutant per every 1000 wild-type alleles. To achieve this sensitive level of mutation detection, we integrate a high accuracy indexing strategy and reference replication for estimating sequencing error variance. We employ a statistical model to estimate the error rate at each position of the reference and to quantify the fraction of variant base in the sample. Our method is highly specific (99%) and sensitive (100%) when applied to a known 0.1% sample fraction admixture of two synthetic DNA samples to validate our method. As a clinical application of this method, we analyzed nine clinical samples of H1N1 influenza A and detected an oseltamivir (antiviral therapy) resistance mutation in the H1N1 neuraminidase gene at a sample fraction of 0.18%.     

Plantlet production through high frequency somatic embryogenesis in long term cultures ofEucalyptus citriodora

A highly embryogenic culture ofEucalyptus citriodora was obtained by repetitive embryogenesis from somatic embryos cultured in the dark on a medium containing 500 mg/l each of glutamine and casein hydrolysate, 30 g/l of sucrose and 5 mg/l of 1-napthaleneacetic acid. Cultures retained morphogenetic ability for upto 36 months when maintained at 27°C by subculture at intervals of 4–5 weeks. The subculture period could be extended beyond 9 months if cultures were incubated at 10°C. On a hormone free medium incubated in light 50% of the embryos germinated to plantlets of which 70% survived when transferred to a sand and soil mixture.Abbreviations NAA 1-naphthal eneacetic acid NCL Communication No: 4480     

Gene Flow Technique for Response to Selection under Non-Random Mating

The gene flow technique for predicting response to selection in random mating populations with overlapping generations is extended to cover any system of mating and illustrated with full-sibbing.     

Genetic variability of aflatoxin B<Subscript>1</Subscript> producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains isolated from discolored rice grains

Twenty-two aflatoxin B₁ (AFB1) producing Aspergillus flavus strains were isolated from 1,200 discolored rice grain samples collected from 20 states across India and tested their potential to produce AFB1 on different agar media. Further these isolates were characterized through randomly amplified polymorphic DNA method. All the strains of A. flavus were produced AFB1 on yeast extract sucrose agar media and none of the strains on A. flavus and A. parasiticus agar. Among the 22 strains, two strains from Tamil Nadu (DRAf 009) and Maharashtra (DRAf 015) produced high amount of AFB1 in all the media tested. To assess the genetic variability in A. flavus, the isolates were analyzed by using random amplified polymorphic DNA markers. Isolates showed 17–80% similarity with standard culture of A. flavus (MTCC 2799).