Characterization of crystals of an Fab fragment of a murine monoclonal antibody.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.     

Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients

To examine the identity of binding sites for thyrotropin (TSH) and thyroid stimulating antibodies (TSAbs) associated with Graves' disease, we constructed eight human TSH receptor/rat LH-CG receptor chimeras. Substitution of amino acid residues 8-165 of the TSH receptor with the corresponding LH-CG receptor segment (Mc1 + 2) results in a chimera which retains high affinity TSH binding and the cAMP response to TSH but loses both the cAMP response to Graves' IgG and Graves' IgG inhibition of TSH binding. Two of three IgGs from idiopathic myxedema patients which contain thyroid stimulation blocking antibodies (TSBAbs) still, however, react with this chimera. Chimeras which substitute residues 90-165 (Mc2) and 261-370 (Mc4) retain the ability to interact with TSH, Graves' IgG, and idiopathic myxedema IgG. The data thus suggest that residues 8-165 contain an epitope specific for TSAbs and that TSH receptor determinants important for the activities of TSAbs and TSH are not identical. Further, binding sites for TSBAbs in idiopathic myxedema may be different from receptor binding sites for both Graves' IgG TSAb as well as TSH and may be different in individual patients.     

Structural basis of successive adenosine modifications by the conserved ribosomal methyltransferase KsgA

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.     

Natural variation in the promoter of OsHMA3 contributes to differential grain cadmium accumulation between Indica and Japonica rice

Rice is a major source of cadmium(Cd) intake for Asian people. Indica rice usually accumulates more Cd in shoots and grains than Japonica rice. However, underlying genetic bases for differential Cd accumulation between Indica and Japonica rice are still unknown. In this study, we cloned a quantitative trait locus(QTL) grain Cd concentration on chromosome 7(GCC7) responsible for differential grain Cd accumulation between two rice varieties by performing QTL analysis and map-based cloning. We found that the two GCC7 alleles, GCC7~(PA64s) and GCC7~(93-11), had different promoter activity of OsHMA3,leading to different OsHMA3 expression and different shoot and grain Cd concentrations. By analyzing the distribution of different haplotypes of GCC7 among diverse rice accessions, we discovered that the high and low Cd accumulation alleles, namely GCC7~(93-11) and GCC7~(PA64s), were preferentially distributed in Indica and Japonica rice,respectively. We further showed that the GCC7~(PA64s)allele can be used to replace the GCC7~(93-11) allele in the super cultivar 93-11 to reduce grain Cd concentration without adverse effect on agronomic traits. Our results thus reveal that the QTL GCC7 with sequence variation in the OsHMA3 promoter is an important determinant controlling differential grain Cd accumulation between Indica and Japonica rice.     

Nutrients and antinutrient constituents of Amaranthus caudatus L. Cultivated on different soils

This study investigated variations in the concentration of nutrients, antinutrients and mineral content of Amaranthus caudatus harvested from different soil types at various stages of maturity. Four out the five soils namely; sandy clay loam, silty clay loam, clayey loam and loam were experimentally formulated from primary particles of silt, clay and sand in line with the United State Department of Agriculture’s (USDA) soil triangle protocol. The unfractionated soil was used as the control. After harvesting at pre-flowering (61 days after planting), flowering (71 days after planting) and post-flowering (91 days after planting) stages, nutrient and antinutrient analyses were carried out following Association of Official Analytical Chemists (AOAC) and other referenced methods while the Inductively Coupled Plasma- Optical Emission Spectrometer was used to determine mineral compositions of the plant samples. The results of the study revealed that particle size and physicochemical properties of the soil influenced the number of minerals deposited in plant tissues. It was further observed that the nutritional properties of the plant change as plant ages. For an optimal yield of vitamins A and E, clayey loam proved to be the best soil particularly when A. caudatus is harvested before flowering but for vitamin C, sandy clayey loam yielded the highest output at the same stage. Similarly, clayey loam and loam soils yielded the highest proximate compositions at flowering and pre-flowering; however, mineral elements (micro and macro) were highest in control and loam soils.     

Development of Capsicum EST–SSR markers for species identification and in silico mapping onto the tomato genome sequence

Capsicum spp. are widely cultivated for use as vegetables and spices. The Kihara Institute for Biological Research, Yokohama City University, Japan, has stocks of approximately 800 lines of Capsicum spp. collected from various regions of Central and South America, the regions of origin for Capsicum spp. In this study, 5,751 primer pairs for simple sequence repeat markers, based on 118,060 publicly available sequences of expressed sequence tags of Capsicum annuum, were designed and subjected to a similarity search against the genomic sequence of tomato, a model Solanaceae species. Nucleotide sequences spanning 2,245 C. annuum markers were successfully mapped onto the tomato genome, and 96 of these, which spanned the entire tomato genome, were selected for further analysis. In genotyping analysis, 60 out of the 77 markers that produced specific DNA amplicons showed polymorphism among the Capsicum lines examined. On the basis of the resulting data, the 192 tested lines were grouped into five main clusters. The additional sequencing analysis of the plastid genes, matK and rbcL, divided the resources into three groups. As a result, 19 marker loci exhibited genotypes specific to species and cluster, suggesting that the DNA markers are useful for species identification. Information on the DNA markers will contribute to Capsicum genetics, genomics, and breeding.     

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent.     

Extracts of Adipose Derived Stem Cells Slows Progression in the R6/2 Model of Huntington's Disease

Stem cell therapy is a promising treatment for incurable disorders including Huntington''s disease (HD). Adipose-derived stem cell (ASC) is an easily available source of stem cells. Since ASCs can be differentiated into nervous stem cells, it has clinically feasible potential for neurodegenerative disease. In addition, ASCs secrete various anti-apoptotic growth factors, which improve the symptoms of disease from transplanted ASCs. Thus, cell-free extracts of ASCs (ASCs-E) could be a potential candidate for treatment of HD. Here, we investigated effects of ASCs-E on R6/2 HD mouse model and neuronal cells. In R6/2 HD model, injection of ASCs-E improved the performance in Rotarod test. ASCs-E also ameliorated striatal atrophy and mutant huntingtin aggregation in the striatum. In Western blot increased expressions of p-Akt, p-CREB and PGC1α were noted by injection of ASCs-E, when comparing to the R6/2 HD model. Neuro2A neuroblastoma cells treated with ASCs-E showed increased expression of p-CREB and PGC1α. In conclusion, ASCs-E delayed disease progression in animal model of HD by restoring of CREB-PGC1α pathway and could be a potential resource for treatment of HD.     

Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17

Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogs possessing unnatural sugars replacing native d-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as d-quinovosyl-10-deoxymethynolide, l-rhamnosyl-10-deoxymethynolide, l-olivosyl-10-deoxymethynolide, and d-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with l-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar d-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.