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.     

Differential expression of phenylalanine ammonia-lyase and chalcone synthase during soybean nodule development.

We have used conserved and nonconserved regions of cDNA clones for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) isolated from a soybean-nodule cDNA library to monitor the expression of members of the two gene families during the early stages of the soybean-Bradyrhizobium japonicum symbiosis. Our results demonstrate that subsets of the PAL and CHS gene families are specifically induced in soybean roots after infection with B. japonicum. Furthermore, by analyzing a supernodulating mutant line of soybean that differs from the wild-type parent in the number of successful infections, we show that the induction of PAL and CHS is related to postinfection events. Nodulated roots formed by a Nod+ Fix- strain of B. japonicum, resembling a pathogenic association, display induction of another distinct set of PAL and CHS genes. Our results suggest that the symbiosis-specific PAL and CHS genes in soybean are not induced by stress or pathogen interaction.     

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.     

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.     

Accumulation of 15-Kilodalton Zein in Novel Protein Bodies in Transgenic Tobacco

Zeins, the seed storage proteins of maize, are a group of alcohol-soluble polypeptides of different molecular masses that share a similar amino acid composition but vary in their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal is to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whether this protein would be stable in nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, [beta]-phaseolin, in nonseed tissues and to determine the basis for its stability/instability. We have introduced the 15-kD zein and bean [beta]-phaseolin-coding sequences behind the 35S cauliflower mosaic virus promoter into tobacco (Nicotiana tabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein is stable not only in seeds but in all nonseed tissues tested, whereas the [beta]-phaseolin protein accumulated only in mid- and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the [beta]-phaseolin protein in nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention in the ER or to the protease-resistant nature of the protein.