Expression and characterization of isoform 1 of human mitochondrial elongation factor G

Elongation factor G (EF-G) catalyzes the translocation step of protein biosynthesis. Genomic analysis suggests that two isoforms of this protein occur in mitochondria. The region of the cDNA coding for the mature sequence of isoform 1 of human mitochondrial EF-G (EF-G1(mt)) has been cloned and expressed in Escherichia coli. The recombinant protein has been purified to near homogeneity by chromatography on Ni-NTA resins and cation exchange high performance liquid chromatography. EF-G1(mt) is active on both bacterial and mitochondrial ribosomes. Human EF-G1(mt) is considerably more resistant to fusidic acid than many bacterial translocases. A molecular model for EF-G1(mt) has been created and analyzed in the context of its relationship to the translocases from other systems.     

Inorganic nutrient manipulation for highly improved <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration in finger millet—<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.

Summary Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5x the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regnnerated shoots. For long-term callus cultures, 2x KNO₃ or 4x Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.     

Positive and negative regulation of the gamma-secretase activity by nicastrin in a murine model

Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.     

A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.     

Functionalization of pine needles by carboxymethylation and network formation for use as supports in the adsorption of Cr

Pine needles and their carboxymethyl forms were functionalized by network formation with 2-acrylamido-2-methylpropanesulphonic acid (AAmPSA) in the presence of N,N-methylene bisacrylamide. N-Tetramethylethylene diamine and ammonium persulfate were used as accelerator-initiator systems to prepare these hydrogels. The hydrogels were characterized by FTIR, SEM, and nitrogen analysis and for water uptake capacities before and after metal ion sorption with a view to evaluating their use in the removal of toxic ionic species from waste water. A detailed study of Cr⁶⁺ adsorption was carried out as a function of time, temperature, pH, and ionic strength. The thermodynamic parameters of adsorption such as ΔH⁰, ΔS⁰, and ΔG⁰ have been evaluated to understand the underlying mechanism of adsorption. In order to understand their reusability in possible technological applications, biodegradability of these hydrogels and their precursors was studied.     

Evidence for enhanced eNOS function in coronary microvessels during the second window of protection

Nitric oxide (NO) derived from endothelial NO synthase (NOS) (eNOS) has been identified as a trigger for the second window of protection (SWOP), but its role as a mediator during the SWOP is a matter of debate. Eighteen mongrel dogs were chronically instrumented to measure left ventricular function, coronary blood flow, and wall thickening. Myocardial preconditioning was induced by 10 min coronary artery occlusion. After 24 h of reperfusion (during the SWOP), the hearts were excised. Coronary microvessels were isolated and incubated in presence of 1) the endothelium-dependent agonists carbachol and bradykinin, 2) the calcium ionophore A23187, and 3) the angiotensin-converting enzyme (ACE) inhibitors enalaprilat and ramiprilat. Nitrite, a metabolite of NO, was measured. Under baseline conditions, nitrite production in microvessels from SWOP was 30% higher than that from normal (96 +/- 4 vs. 74 +/- 3 pmol/mg, P < 0.01, respectively). Nitrite production in response to carbachol, bradykinin, and A23187 was also enhanced in microvessels from SWOP (P < 0.05). These enhanced responses were abolished by N(G)-nitro-l-arginine methyl ester (l-NAME) or the endothelial receptor-specific antagonists atropine and HOE-140. The level of eNOS protein in the SWOP myocardium was twofold higher than that in the non-SWOP myocardium. Nitrite production in response to the ACE inhibitors was greater in microvessels from SWOP. These effects were blocked by l-NAME, HOE-140, or dichloroisocoumarin (which inhibits kinin formation). We found that a brief ischemic episode induced delayed, enhanced NO production in coronary microvessels and an upregulation of eNOS protein. These findings suggest that eNOS is a mediator during the SWOP. The ability of ACE inhibitors to enhance NO release during the SWOP points to an additional clinical application for these drugs.     

Mineral proximity influences mechanical response of proteins in biological mineral-protein hybrid systems

The organic phase of nacre, which is composed primarily of proteins, has an extremely high elastic modulus as compared to that of bulk proteins, and also undergoes large deformation before failure. One reason for this unusually high modulus could be the mineral-organic interactions. In this work, we elucidate the specific role of mineral proximity on the structural response of proteins in biological structural composites such as nacre through molecular modeling. The "glycine-serine" domain of a nacre protein Lustrin A has been used as a model system. It is found that the amount of work needed to unfold is significantly higher when the GS domain is pulled in the proximity of aragonite. These results indicate that the proximity of aragonite has a significant effect on the unfolding mechanisms of proteins when pulled. These results will provide very useful information in designing synthetic biocomposites, as well as further our understanding of mechanical response in structural composites in nature.