EST-derived genic molecular markers: development and utilization for generating an advanced transcript map of chickpea

Well-saturated linkage maps especially those based on expressed sequence tag (EST)-derived genic molecular markers (GMMs) are a pre-requisite for molecular breeding. This is especially true in important legumes such as chickpea where few simple sequence repeats (SSR) and even fewer GMM-based maps have been developed. Therefore, in this study, 2,496 ESTs were generated from chickpea seeds and utilized for the development of 487 novel EST-derived functional markers which included 125 EST-SSRs, 151 intron targeted primers (ITPs), 109 expressed sequence tag polymorphisms (ESTPs), and 102 single nucleotide polymorphisms (SNPs). Whereas EST-SSRs, ITPs, and ESTPs were developed by in silico analysis of the developed EST sequences, SNPs were identified by allele resequencing and their genotyping was performed using the Illumina GoldenGate Assay. Parental polymorphism was analyzed between C. arietinum ICC4958 and C. reticulatum PI489777, parents of the reference chickpea mapping population, using a total of 872 markers: 487 new gene-based markers developed in this study along with 385 previously published markers, of which 318 (36.5%) were found to be polymorphic and were used for genotyping. The genotypic data were integrated with the previously published data of 108 markers and an advanced linkage map was generated that contained 406 loci distributed on eight linkage groups that spanned 1,497.7 cM. The average marker density was 3.68 cM and the average number of markers per LG was 50.8. Among the mapped markers, 303 new genomic locations were defined that included 177 gene-based and 126 gSSRs (genomic SSRs) thereby producing the most advanced gene-rich map of chickpea solely based on co-dominant markers.     

Evaluation of immunostimulatory and growth promoting effect of seed fractions of Achyranthes aspera in common carp Cyprinus carpio and identification of active constituents

Immunostimulatory and growth promoting properties of Achyranthes aspera seeds were studied with larvae of common carp Cyprinus carpio. Four experimental diets were prepared using raw (D1) and alcohol (D2), petroleum ether (D3) and 50% aqueous alcohol (D4) extracts of A. aspera seeds. Diet without seed served as control (D5). Fish were fed with test/control diet for 30 days and then immunized with 10 μl of c-RBC. Blood samples were collected 7 days after immunization. Survival (93 ± 3%) of fish was significantly (P < 0.05) higher in D1 diet fed group compared to others. Highest specific growth rate was found in fish fed with diet D2. Significantly (P < 0.05) higher levels of serum protein and albumin were found in D1 and D3 compared to others. Highest serum globulin level was found in D1, which was followed by D3, D2, D4 and D5. Hemagglutination titer level was 5-18 folds higher in diet D3 fed fish compared to others. SGOT and SGPT levels were significantly (P < 0.05) higher in control group compared to the treated groups. Myeloperoxidase activity was significantly (P < 0.05) higher in D1 (2.513 ± 0.27 λ 450 nm) and D3 (2.38 ± 0.07 λ 450 nm) diets fed groups compared to others. The best performance of fish was found in raw A. aspera seeds incorporated diet fed group and the active constituents were identified as ecdysterone and two essential fatty acids linolenic acid and oleic acid.     

Neuronal dystonin isoform 2 is a mediator of endoplasmic reticulum structure and function

Dystonin/Bpag1 is a cytoskeletal linker protein whose loss of function in dystonia musculorum (dt) mice results in hereditary sensory neuropathy. Although loss of expression of neuronal dystonin isoforms (dystonin-a1/dystonin-a2) is sufficient to cause dt pathogenesis, the diverging function of each isoform and what pathological mechanisms are activated upon their loss remains unclear. Here we show that dt(27) mice manifest ultrastructural defects at the endoplasmic reticulum (ER) in sensory neurons corresponding to in vivo induction of ER stress proteins. ER stress subsequently leads to sensory neurodegeneration through induction of a proapoptotic caspase cascade. dt sensory neurons display neurodegenerative pathologies, including Ca(2+) dyshomeostasis, unfolded protein response (UPR) induction, caspase activation, and apoptosis. Isoform-specific loss-of-function analysis attributes these neurodegenerative pathologies to specific loss of dystonin-a2. Inhibition of either UPR or caspase signaling promotes the viability of cells deficient in dystonin. This study provides insight into the mechanism of dt neuropathology and proposes a role for dystonin-a2 as a mediator of normal ER structure and function.     

CRIM1 Complexes with ?-catenin and Cadherins,Stabilizes Cell-Cell Junctions and Is Critical for Neural Morphogenesis

In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.     

CRIM1 complexes with ß-catenin and cadherins, stabilizes cell-cell junctions and is critical for neural morphogenesis

In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ?-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ?-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.     

Isolation of biosurfactant-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

An efficient biosurfactant-producing native Pseudomonas aeruginosa RS29 has been isolated from crude oil contaminated soil. Isolation was followed by optimization of different factors to achieve maximum production of biosurfactant in terms of surface tension reduction (STR) and emulsification index (E24). The isolated strain produced highest biosurfactant in the presence of glycerol after 48 h of incubation at 37.5°C, with pH range of 7–8 and at salinity <0.8% (w/v). The extent of STR and the E24 of medium with different nitrogen sources were investigated and found to be maximal for sodium nitrate (26.3 mN/m, E24?=?80%) and potassium nitrate (26.4 mN/m, E24?=?79%). The production of biomass by the designated strain was found to be maximal in ammonium-nitrate-containing medium as compared to the other nitrogen sources. A kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and highest STR was achieved (27.0 mN/m) after 44 h of growth. The biosurfactant was produced as a primary metabolite and 6 g/L crude biosurfactant was extracted by chloroform:methanol (2:1). The critical micelle concentration of the biosurfactant was 90 mg/L. The absorption bands of the FTIR spectra confirmed the rhamnolipid nature of the biosurfactant. The biosurfactant was thermostable (up to 121°C for 15 min) and could withstand a wide range of pH (2–10) and NaCl concentration (2%–10% w/v). The extracted biosurfactant had good foaming and emulsifying activities and was of satisfactory quality in terms of stability (temperature, pH and salinity) and foaming activity.     

Structural basis for the receptor binding specificity of Norwalk virus

Noroviruses are positive-sense, single-stranded RNA viruses that cause acute gastroenteritis. They recognize human histo-blood group antigens as receptors in a strain-specific manner. The structures presented here were analyzed in order to elucidate the structural basis for differences in ligand recognition of noroviruses from different genogroups, the prototypic Norwalk virus (NV; GI-1) and VA387 (GII-4), which recognize the same A antigen but differ in that NV is unable to bind to the B antigen. Two forms of the receptor-binding domain of the norovirus coat protein, the P domain and the P polypeptide, that were previously shown to differ in receptor binding and P-particle formation properties were studied. Comparison of the structures of the NV P domain with and without A trisaccharide and the NV P polypeptide revealed no major ligand-induced changes. The 2.3-A cocrystal structure reveals that the A trisaccharide binds to the NV P domain through interactions with the residues Ser377, Asp327, His329, and Ser380 in a mode distinct from that previously reported for the VA387 P-domain-A-trisaccharide complex. Mutational analyses confirm the importance of these residues in NV P-particle binding to native A antigen. The alpha-GalNAc residue unique to the A trisaccharide is buried deeply in the NV binding pocket, unlike in the structures of A and B trisaccharides bound to VA387 P domain, where the alpha-fucose residue forms the most protein contacts. The A-trisaccharide binding mode seen in the NV P domain complex cannot be sterically accommodated in the VA387 P domain.     

Single particle image reconstruction of the human recombinant Kv2.1 channel

Kv2.1 channels are widely expressed in neuronal and endocrine cells and generate slowly activating K⁺ currents, which contribute to repolarization in these cells. Kv2.1 is expressed at high levels in the mammalian brain and is a major component of the delayed rectifier current in the hippocampus. In addition, Kv2.1 channels have been implicated in the regulation of membrane repolarization, cytoplasmic calcium levels, and insulin secretion in pancreatic β-cells. They are therefore an important drug target for the treatment of Type II diabetes mellitus. We used electron microscopy and single particle image analysis to derive a three-dimensional density map of recombinant human Kv2.1. The tetrameric channel is egg-shaped with a diameter of ∼80 Å and a long axis of ∼120 Å. Comparison to known crystal structures of homologous domains allowed us to infer the location of the cytoplasmic and transmembrane assemblies. There is a very good fit of the Kv1.2 crystal structure to the assigned transmembrane assembly of Kv2.1. In other low-resolution maps of K⁺ channels, the cytoplasmic N-terminal and transmembrane domains form separate rings of density. In contrast, Kv2.1 displays contiguous density that connects the rings, such that there are no large windows between the channel interior and the cytoplasmic space. The crystal structure of KcsA is thought to be in a closed conformation, and the good fit of the KcsA crystal structure to the Kv2.1 map suggests that our preparations of Kv2.1 may also represent a closed conformation. Substantial cytoplasmic density is closely associated with the T1 tetramerization domain and is ascribed to the ∼184 kDa C-terminal regulatory domains within each tetramer.     

Complex I and energy thresholds in the brain

Mitochondrial electron transport chain (ETC) deficiencies are thought to underlie defects in energy metabolism and have been implicated in the neurodegenerative process. In particular, reductions in complex I activities in Parkinson's disease are thought to cause bioenergetic dysfunction with subsequent degeneration of dopaminergic neurons. In terms of bioenergetics and assessing ETC-related problems in the brain, the presence of heterogeneous mitochondria has complicated matters as isolated non-synaptic mitochondria have different energy thresholds and flux control coefficients compared to isolated mitochondria of synaptic origin. The molecular mechanisms that underlie complex I deficiencies in the parkinsonian brain are unknown and are the source of intensive research. This review explores the relationship between complex I activity and energy metabolism in the brain as well as the nature of the complex I defect.