Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of β-Barrel Outer Membrane Proteins, Including That of BamA Itself

Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis.     

Novel Genomic Tools and Modern Genetic and Breeding Approaches for Crop Improvement

In recent past, genomic tools especially molecular markers have been extensively used for understanding genome dynamics as well for applied aspects in crop breeding. Several new genomics technologies such as next generation sequencing (NGS), high-throughput marker genotyping, -omics technologies have emerged as powerful tools for understanding genome variation in crop species at DNA, RNA as well as protein level. These technologies promise to provide an insight into the way gene(s) are expressed and regulated in cell and to unveil metabolic pathways involved in trait(s) of interest for breeders not only in model-/major- but even for under-resourced crop species which were once considered “orphan” crops. In parallel, genetic variation for a species present not only in cultivated genepool but even in landraces and wild species can be harnessed by using new genetic approaches such as advanced-backcross QTL (AB-QTL) analysis, introgression libraries (ILs), multi-parent advanced generation intercross (MAGIC) population and association genetics. The gene(s) or genomic regions, responsible for trait(s) of interest, identified either through conventional linkage mapping or above mentioned approaches can be introgressed or pyramided to develop superior genotypes through molecular breeding approaches such as marker-assisted back crossing (MABC), marker assisted recurrent selection (MARS) and genome wide selection (GWS). This article provides an overview on some recent genomic tools and novel genetic and breeding approaches as mentioned above with a final aim of crop improvement.     

Hypoglycemic activity of the antioxidant saponarin, characterized as alpha-glucosidase inhibitor present in Tinospora cordifolia

Tinospora cordifolia, used in anti-diabetic herbal drug preparations, was reported [12] to contain an alpha-glucosidase inhibitor, characterized as saponarin (apigenin-6-C-glucosyl-7-O-glucoside). The leaf extract had appreciable antioxidant and hydroxyl radical scavenging activities and contained the flavonoid in the range of 32.1 +/- 1.5-45.5 +/- 3.5 mg/g of dry solid. Saponarin showed mixed competitive inhibition on activities of alpha-glucosidase and sucrase of different origins. IC(50), Ki and ki' values determined were 48 muM, 8 muM and 19.5 microM respectively for intestinal maltase and 35 microM, 6 microM and 13 microM respectively for intestinal sucrase. When given orally to maltose-fed rat, saponarin showed hypoglycemic activity in the range of 20-80 mg/kg compared to 100-200 mg/kg for acarbose as reported.     

Comparative Functional Analysis of Rat <Emphasis Type="Italic">TGF-β1</Emphasis> and <Emphasis Type="Italic">Xenopus laevis</Emphasis><Emphasis Type="Italic">TGF-β5</Emphasis> Promoters Suggest Differential Regulations

We have carried out a comparative functional analysis of the rat TGF-1 and Xenopus laevis TGF-5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-1 is low as compared to TGF-5 promoter in XTC cells but comparable in A6 cells, while TGF-5 promoter shows nearly negligible activity as compared to TGF-5 promoter in all the tested mammalian cell lines. Moreover, TGF-5 promoter is found to be repressed in XTC cells on treatment with TGF-5 protein. Thus, the regulation of TGF-1 and TGF-5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-1 and TGF-5 proteins, TGF-1 and TGF-5 genes have distinct functions in their respective species. Present address (Kartiki V. Desai): Laboratory of Cell Regulation and Carcinogenesis, NCI, NIH Bldg 41, Room C619, Bethesda, MD 20892, USA     

Regulation of MAP kinase by the BMP-4/TAK1 pathway in Xenopus ectoderm

Bone morphogenetic protein-4 (BMP-4) induces epidermis and represses neural fate in Xenopus ectoderm. Our previous findings implicate p42 Erk MAP kinase (MAPK) in the response to neural induction. We have examined the effects of BMP-4 on MAPK activity in gastrula ectoderm. Expression of a dominant negative BMP-4 receptor resulted in a 4.5-fold elevation in MAPK activity in midgastrula ectoderm. MAPK activity was reduced in ectoderm expressing a constitutively active BMP-4 receptor, or ectoderm treated with BMP-4 protein in the presence or absence of cycloheximide. Overexpression of TAK1 led to a reduction in MAPK activity in early gastrula ectoderm. The inhibitory effects of TAK1 could be reversed by 1 microM SB 203580, a p38 inhibitor. Treatment of isolated ectoderm with SB 203580 led to expression of otx2, NCAM, and noggin. Western blot analyses indicated that the BMP-4 pathway does not activate JNKs in ectoderm. Our findings indicate that BMP-4 inhibits ectodermal MAPK activity through a TAK1/p38-type pathway. MAPK has been shown to inactivate Smad1. Thus, our results suggest that BMP-4 and MAPK pathways are mutually antagonistic in Xenopus ectoderm, and that interactions between these pathways may govern the choice between epidermal and neural fate.     

Cell volume changes affect gluconeogenesis in the perfused liver of the catfish<Emphasis Type="Italic">Clarias batrachus</Emphasis>

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver ofClarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role inC. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.     

Isolation of a library of target-sites for sequence specific DNA binding proteins from chick embryonic heart: a potential tool for identifying novel transcriptional regulators involved in embryonic development

Enormity of the metazoan genomes and divergence in their regulation impose a serious constraint on the comprehensive understanding of context specific gene regulation. DNA elements located in the promoter, enhancer, and other regulatory regions of the genome dictate the temporal and spatial patterns of gene activities. However, owing to the diminutive and variable nature of the regulatory DNA elements, their identification and location remains a major challenge. We have developed an efficient strategy for isolating a repertoire of target sites for sequence specific DNA binding proteins from embryonic chick heart. A comprehensive library of such sequences was constructed and authenticated using various parameters including in silico determination of functional binding sites. This approach, therefore, for the first time, established an experimental and conceptual framework for defining the entire repertoire of functional DNA elements in any cellular context.