Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

Overexpression of extracellular superoxide dismutase has a protective role against hyperoxia-induced brain injury in neonatal mice

There is increasing evidence that hyperoxia, particularly at the time of birth, may result in neurological injury, in particular to the susceptible vasculature of these tissues. This study was aimed at determining whether overexpression of extracellular superoxide dismutase (EC-SOD) is protective against brain injury induced by hyperoxia. Transgenic (TG) mice (with an extra copy of the human extracellular superoxide dismutase gene) and wild-type (WT) neonate mice were exposed to hyperoxia (95% of F(i) o(2) ) for 7 days after birth versus the control group in room air. Brain positron emission tomography (PET) scanning with fludeoxyglucose (FDG) isotope uptake was performed after exposure. To assess apoptosis induced by hyperoxia exposure, caspase 3 ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. Quantitative western blot for the following inflammatory markers was performed: glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, macrophage-inhibiting factor, and phospho-AMP-activated protein kinase. PET scanning with FDG isotope uptake showed significantly higher uptake in the WT hyperoxia neonate brain group (0.14 ± 0.03) than in both the TG group (0.09 ± 0.01) and the control group (0.08 ± 0.02) (P< 0.05). Histopathological investigation showed more apoptosis and dead neurons in hippocampus and cerebellum brain sections of WT neonate mice after exposure to hyperoxia than in TG mice; this finding was also confirmed by TUNEL staining. The caspase 3 assay confirmed the finding of more apoptosis in WT hyperoxia neonates (0.814 ± 0.112) than in the TG hyperoxic group (0.579 ± 0.144) (P < 0.05); this finding was also confirmed by TUNEL staining. Quantitative western blotting for the inflammatory and metabolic markers showed significantly higher expression in the WT group than in the TG and control groups. Thus, overexpression of EC-SOD in the neonate brain offers significant protection against hyperoxia-induced brain damage.     

5-(1H-Benzimidazol-1-yl)-3-alkoxy-2-thiophenecarbonitriles as potent, selective, inhibitors of IKK-epsilon kinase

The identification and hit-to-lead exploration of a novel, potent and selective series of substituted benzimidazole–thiophene carbonitrile inhibitors of IKK-ε kinase is described. Compound 12e was identified with an IKK-ε enzyme potency of pIC₅₀ 7.4, and has a highly encouraging wider selectivity profile, including selectivity within the IKK kinase family.     

Expression of host genes during root nodule development in soybeans

Summary Nine unique nodulin cDNA clones from soybean have been characterized with regard to the size of the RNA and the corresponding protein products. Based on the sequence homology between clones C51 and E27 and the multiple RNA species corresponding to clones D41 and E41, it is suggested that some of the nodulin genes represent members of small gene families. The amino acid sequence deduced from the nucleotide sequence of clones C51 and E27 revealed the presence of a signal peptide and no stop transfer signal, typical of membrane proteins, suggesting that the proteins encoded by these clones are localized in organelles and as such probably involved in ureide biosynthesis (Boland et al. 1982; Schubert and Boland 1984). Based on the timing of appearance of RNA corresponding to the nodulin clones and the pattern of their accumulation, at least three sets of nodulin genes are being represented here. Al1 the nodulin RNAs examined were made in Fix^- nodules formed by strain Ag168 (which does not make Cl component of nitrogenase) at a level comparable to that in Fix⁺ nodules and at a very reduced level in Fix^- nodules formed by strain HS124 (which show very few infected cells). It is concluded that all the nodulin genes examined here are induced independent of nitrogenase activity.     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

Targeting transgene expression in research,agricultural, and environmental applications: Promoters used in plant transformation

Summary Plant genetic engineering has contributed substantially to the understanding of gene regulation and plant development, in the generation of transgenic organisms for widespread usage in agriculture, and has increased the potential uses of crops for industrial and pharmaceutical purposes. As the application of geneticallly engineered plants has widened, so has the need to develop methods to fine-tune control of transgene expression. The availability of a broad spectrum of promoters that differ in their ability to regulate the temporal and spatial expression patterns of the transgene can dramatically increase the successful application of transgenic technology. Indeed, a variety of promoters in necessary at all levels of genetic engineering in plants, from basic research discoveries, concepts and question to development of economically viable crops and plant commodities, to addressing legitimate concerns raised about the safety and containment of transgenic plants in the environment. This review covers the characterization and usage of a broad range of promoters employed in plant genetic engineering, including the widespread use of plant promoters with viral and plant origin that drive constitutive expression. Also covered are selected tissue-specific promoters from fruit, seed and grain, tubers, flowers, pistils, anther and pollen, roots and root nodules, and leaves and green tissue. Topics also include organellar promoters, and those found in specific cell types, as well as the development and evaluation of inducible (endogenous and exogenous origin) and synthetic plant promoter systems. Discussions on the relevance and potential pitfalls within specific applications are included.