Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C

Spectroscopic methods such as circular dichroism and F?rster resonance energy transfer are current approaches for monitoring protein conformational changes. Those analyses require special equipment and expertise. The need for fluorescence labeling of the protein may interfere with the native structure. We have developed a microtiter plate-based monoclonal antibody (mAb) epitope analysis to detect protein conformational changes in a high throughput manner. This method is based on the concept that the affinity of the antigen-binding site of an antibody for the specific antigenic epitope will change when the 3-D structure of the epitope changes. The effectiveness of this approach was demonstrated in the present study on troponin C (TnC), an allosteric protein in the Ca(2+) regulatory system of striated muscle. Using TnC purified by a highly effective rapid procedure and mAbs developed against epitopes in the N- and C-domains of TnC enzyme-linked immunosorbant assay (ELISA) clearly detected Ca(2+)-induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. On the other hand, Mg(2+)-binding to the C-domain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca(2+)-Mg(2+) exchange at the C-domain metal ion-binding sites. In addition to further understanding of the structure-function relationship of TnC, the data demonstrate that the mAb epitope analysis provides a simple high throughput method for monitoring 3-D structural changes in native proteins under physiological condition and has broad applications in protein structure-function relationship studies.     

Design, synthesis, and evaluation of potential inhibitors of brassinin glucosyltransferase, a phytoalexin detoxifying enzyme from Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a fungal pathogen, which causes stem rot in crucifer crops and in several other plant families resulting in enormous yield losses all over the world. Brassinin is a phytoalexin produced by crucifer plants as part of a general defense mechanism against pathogens and other forms of stress. To the great detriment of crucifers, some fungal pathogens, as for example S. sclerotiorum, can detoxify brassinin. Detoxification of brassinin via glucosylation of the indole nitrogen is carried out by an inducible glucosyltransferase produced in S. sclerotiorum. Because brassinin is a precursor of several phytoalexins active against S. sclerotiorum, brassinin glucosyltransferase (BGT) is a potentially useful metabolic target to control S. sclerotiorum. Toward this end, we have designed, synthesized, and screened several brassinin analogues using both mycelial cultures and cell-free homogenates of S. sclerotiorum. A noticeable decrease in the rate of brassinin detoxification in cell cultures was observed in the presence of methyl (benzofuran-3-yl)methyldithiocarbamate, methyl (benzofuran-2-yl)methyldithiocarbamate, methyl (indol-2-yl)methyldithiocarbamate, 3-phenylindole, 6-fluoro-3-phenylindole, and 5-fluorocamalexin. In addition, these compounds caused substantial inhibition of BGT activity (ca. 80%) in cell-free homogenates of S. sclerotiorum, while only brassinin and 3-phenylindole were transformed to the corresponding beta-d-1-glucopyranosyl products. These results indicate that, although many other glucosyltransferases appear to be produced by S. sclerotiorum in cell cultures, BGT is substrate specific. Overall these results show that selective and potent inhibitors of BGT can be developed.     

Synthesis of O-prenylated and O-geranylated derivatives of 5-benzylidene2,4-thiazolidinediones and evaluation of their free radical scavenging activity as well as effect on some phase II antioxidant/detoxifying enzymes

A series of 5-arylidene-2,4-thiazolidinediones and its geranyloxy or prenyloxy derivative were synthesized and studied for their radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Their comparable scavenging activities were expressed as IC50 value. Compounds 2c, 2d, 4d, and 6a showed appreciable radical scavenging activities. The vanillin based thiazolidinedione compound 2c displayed highest activity comparable to that of alpha-tocopherol. But in vivo, compound 6a showed better results in inducing phase II detoxifying/antioxidative enzyme.     

Breeding Potential of Some Exotic Tomato Lines: A Combined Study of Morphological Variability,Genetic Divergence,and Association of Traits

Tomato (Solanum lycopersicum L.) is called ‘the poor man’s orange’ due to its low price and improved nutritional values. An experiment was conducted to study the breeding potential of some exotic tomato lines by assessing various qualitative and quantitative traits conferring yield and quality attributes. Among the qualitative traits, greater variability was observed for growth type, stem hairiness, and fruit shape and size. A determinate growth habit was observed in the genotype AVTO9802 while the genotype AVTO0102 produced yellow color fruits. A significant (p ≤ 0.01) variation was also observed for the studied quantitative traits. Based on yield and traits attributed to yield, the genotypes AVTO0314, GPB0107, GPB0120 and AVTO9802 were selected as promising genotypes. The differences between the genotypic and phenotypic coefficients of variation (GCV and PCV) of the studied quantitative traits were very low. This suggests that the apparent variation was mainly due to the genotypes. The higher GCV and PCV values were observed for the number of primary branches plant⁻¹ (NPB), number of fruits cluster⁻¹ (NFC), individual fruit weight (IFW) and total soluble solids (TSS). High heritability was recorded for all quantitative traits in a broad sense. However, the individual fruit diameter showed the highest heritability (99.56). The highest (102.75) genetic advance (GA) was observed for the number of fruits plant⁻¹ (NFP). High heritability coupled with high GA as percentage of mean were recorded for the traits NFP, NFC, fruit yield plant⁻¹ (FYP) and IFW. FYP showed a significant positive correlation with NFC (0.714***) and a negative correlation with days to the first harvest (−0.539***) and plant height (−0.492**). Principal component analysis revealed that the first four components explained 78.5% of the total variation among the genotypes. Thus, the promising genotypes (AVTO0314, GPB0107, GPB0120, AVTO9802 and AVTO0102) isolated from this study can be used for developing high-yielding and high-quality tomato varieties.     

Identification of QTLs for Yield and Associated Traits in F₂ Population of Rice

Identification of quantitative trait loci (QTLs) controlling yield and yield-related traits in rice was performed in the F₂ mapping population derived from parental rice genotypes DHMAS and K343. A total of 30 QTLs governing nine different traits were identified using the composite interval mapping (CIM) method. Four QTLs were mapped for number of tillers per plant on chromosomes 1 (2 QTLs), 2 and 3; three QTLs for panicle number per plant on chromosomes 1 (2 QTLs) and 3; four QTLs for plant height on chromosomes 2, 4, 5 and 6; one QTL for spikelet density on chromosome 5; four QTLs for spikelet fertility percentage (SFP) on chromosomes 2, 3 and 5 (2 QTLs); two QTLs for grain length on chromosomes 1 and 8; three QTLs for grain width on chromosomes1, 3 and 8; three QTLs for 1000-grain weight (TGW) on chromosomes 1, 4 and 8 and six QTLs for yield per plant (YPP) on chromosomes 2 (3 QTLs), 4, 6 and 8. Most of the QTLs were detected on chromosome 2, so further studies on chromosome 2 could help unlock some new chapters of QTL for this cross of rice variety. Identified QTLs elucidating high phenotypic variance can be used for marker-assisted selection (MAS) breeding. Further, the exploitation of information regarding molecular markers tightly linked to QTLs governing these traits will facilitate future crop improvement strategies in rice.     

Sources of Nitrogen in Combination with Systems of Irrigation Influence the Productivity of Modern Rice (<i>Oryza sativa</i> L.) Cultivars during Dry Season in Sub-Tropical Environment

In irrigated agricultural systems, nitrogen (N) and water are the vital resources for sustainability of the crop production in the modern era of climate change. The current study aimed to assess the impact of water and N management on the productivity of irrigated rice cultivars. In the context, a field observation was done at the research farm of Bangladesh Agricultural University, Mymensingh, during dry seasons in consecutive two years (2018–2019 and 2019–2020). The experiments were set up following split-plot design assigning water management in the main plots, nitrogen management in the sub-plots, and the cultivars were approved in the split-split plot with three replications. After two years observation, it was revealed that rice cultivar Binadhan-8 gave the maximum value of leaf area index, number effective tillers hill^-1 and grains panicle^-1 which lead to the higher grain yield (GY). Substantial relationships were observed among the concentration of N, growth, total dry matter (TDM) and N content, N uptake, N utilization effectiveness, and GY. However, with little exception, the Combined effect of water and N, cultivars and water management were varied significantly for all parameters. Finally, the results of the current study concluded that application of irrigation at 8 days after the disappearance of ponded water and source of 105 kg N ha^-1 from PU + Poultry manure are the best management approach for the excellent performance of rice cultivar Binadhan-8.     

SHANK3 genetic polymorphism and susceptibility to ASD: evidence from molecular,in silico,and meta-analysis approaches

Molecular Biology Reports - The SHANK3 gene encodes a master synaptic scaffolding protein at the excitatory synapse’s postsynaptic density, which is predominantly responsible for constructing...