GTPase activity analysis of eRF3 in Euplotes octocarinatus

In eukaryotes, eRF3 participates translation termination and belongs to the superfamily of GTPase. In this work, dissociation constants for E. octocarinatus eRF3 binding to nucleosides in presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, the GTP hydrolyzing assay of Eo-eRF3 was carried out by HPLC methods and the kinetic parameter for GTP hydrolysis by eRF3 was determined. The results showed eRF1a could promote GTP binding to eRF3 and hydrolyzing GTP activity of eRF3. The observation is consistent with the data from human. Whereas E. octocarinatus eRF3 alone can bind GTP in contrast to no GTP binding observed in the absence of eRF1 in human eRF3. The affinity for Eo-eRF3 binding nucleotides is different from that in human. Structure model and amino acids sequence alignment of potential G domains indicated these different may be due to Valine 317 and Glutamate 452 displacing conserved Glycine and Lysine, which were involved in GTP binding.     

Body reserves affect the reproductive endocrine responses to an acute change in nutrition in mature male sheep

Metabolic status is a powerful regulator of reproductive activity, but the metabolic mediators involved and the relationships between fat reserves, food intake and the systems that control reproduction are not fully understood. In this study with mature male Merino sheep, we tested whether the effect of an acute nutritional stimulus on pulsatile LH secretion depended on body condition. Two groups of rams ("Fat" and "Lean") were fed differentially for 4 months to achieve high or low levels of body mass and body condition score. Half of each group was then assigned to be fed either their maintenance requirement or twice their maintenance requirement and, 7 days later, plasma samples were collected every 20 min for 24 h. All samples were used for the analysis of LH pulses and pooled samples were used for the measurement of metabolic hormone concentrations. In the rams that were fed the maintenance diet, the frequency of LH pulses was similar for the Fat and Lean groups, but plasma concentrations of leptin and insulin were significantly higher in the Fat group than in the Lean group. Following an acute increase in food intake, plasma concentrations of insulin were significantly increased in both Fat and Lean rams, but plasma leptin concentrations were increased only in Fat rams and LH pulse frequency was increased only in Lean rams. We concluded that the secretion of LH and leptin, but not insulin, is differentially influenced by nutritional status and body condition and that the role of leptin in the central regulation of the GnRH-LH system is probably permissive.     

Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain,human blood,and glial cells. Autacoids in anti-inflammation

Docosahexaenoic acid (DHA, C22:6) is highly enriched in brain, synapses, and retina and is a major omega-3 fatty acid. Deficiencies in this essential fatty acid are reportedly associated with neuronal function, cancer, and inflammation. Here, using new lipidomic analyses employing high performance liquid chromatography coupled with a photodiode-array detector and a tandem mass spectrometer, a novel series of endogenous mediators was identified in blood, leukocytes, brain, and glial cells as 17S-hydroxy-containing docosanoids denoted as docosatrienes (the main bioactive member of the series was 10,17S-docosatriene) and 17S series resolvins. These novel mediators were biosynthesized via epoxide-containing intermediates and proved potent (pico- to nanomolar range) regulators of both leukocytes reducing infiltration in vivo and glial cells blocking their cytokine production. These results indicate that DHA is the precursor to potent protective mediators generated via enzymatic oxygenations to novel docosatrienes and 17S series resolvins that each regulate events of interest in inflammation and resolution.     

Protein structure and import into the peroxisomal matrix

Proteins destined for the peroxisomal matrix are synthesized in the cytosol, and imported post-translationally. It has been previously demonstrated that stably folded proteins are substrates for peroxisomal import. Mammalian peroxisomes do not contain endogenous chaperone molecules. Therefore, it is possible that proteins are required to fold into their stable, tertiary conformation in order to be imported into the peroxisome. These investigations were undertaken to determine whether proteins rendered incapable of folding were also substrates for import into peroxisomes. Reduction of albumin resulted in a less compact tertiary structure as measured by analytical centrifugation. Microinjection of unfolded albumin molecules bearing the PTS1 targeting signal resulted in their import into peroxisomes. Kinetic analysis indicated that native and unfolded molecules were imported into peroxisomes at comparable rates. While import was unaffected by treatment with cycloheximide, hsc70 molecules were observed to be imported along with the unfolded albumin molecules. These results indicate that proteins, which are incapable of assuming their native conformation, are substrates for peroxisomal import. When combined with previous observations demonstrating the import of stably folded proteins, these results support the model that tertiary structure has no effect on protein import into the peroxisomal matrix .     

Soluble production and function of vascular endothelial growth factor/basic fibroblast growth factor complex peptide

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important proangiogenic factors in tumor procession. The autocrine and paracrine bFGF and the VEGF in tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis. A VEGF/bFGF Complex Peptide (VBP3) was designed on the basis of epitope peptides from both VEGF and bFGF to elicit in vivo production of anti‐bFGF and anti‐VEGF antibodies. In this study, we reported on the production of recombinant VBP3 using high cell density fermentation. Fed‐batch fermentation for recombinant VBP3 production was conducted, and the production procedure was optimized in a 10‐L fermentor. The fraction of soluble VBP3 protein obtained reached 78% of total recombinant protein output under fed‐batch fermentation. Purified recombinant VBP3 could inhibit tumor cell proliferation in vitro and stimulate C57BL/6 mice to produce high titer anti‐VEGF and anti‐bFGF antibodies in vivo. A melanoma‐grafted mouse model and an immunohistochemistry assay showed that tumor growth and tumor angiogenesis were significantly inhibited in VBP3‐vaccinated mice. These results demonstrated that soluble recombinant VBP3 could be produced by large‐scale fermentation, and the product, with good immunogenicity, elicited production of high‐titer anti‐bFGF and anti‐VEGF antibodies, which could be used as a therapeutic tumor vaccine to inhibit tumor angiogenesis and tumor growth. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:194–203, 2015     

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO)

The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogaster by measurement of two different genetic endpoints. We used the wing-spot test and the comet assay. The comet assay was conducted with larval haemocytes. The results from the wing-spot test showed that neither lead chloride, PbCl(2), nor lead nitrate, Pb(NO(3))(2), were able to induce significant increases in the frequency of mutant spots. In addition, the combined treatments with gamma-radiation and PbCl(2) or Pb(NO(3))(2) did not show significant variations in the frequency of the three categories of mutant spots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indicate that the lead compounds tested do not interact with the repair of the genetic damage induced by ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay with haemocytes, Pb(NO(3))(2) was effective in inducing significant increases of DNA damage with a direct dose-response pattern. These results confirm the usefulness of the comet assay with haemocytes as an in vivo model and support the assumption that there is a genotoxic risk associated with lead exposure.     

Synergistic interaction between leptin and cholecystokinin in the rat nodose ganglia is mediated by PI3K and STAT3 signaling pathways: implications for leptin as a regulator of short term satiety

Research has shown that the synergistic interaction between vagal cholecystokinin-A receptors (CCKARs) and leptin receptors (LRbs) mediates short term satiety. We hypothesize that this synergistic interaction is mediated by cross-talk between signaling cascades used by CCKARs and LRbs, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. Whole cell patch clamp recordings were performed on isolated rat nodose ganglia neurons. Western immunoblots elucidated the intracellular signaling pathways that modulate leptin/CCK synergism. In addition, STAT3, PI3K, Src, and MAPK genes were silenced by lentiviral infection and transient Lipofectamine transfection of cultured rat nodose ganglia to determine the effect of these molecules on leptin/CCK synergism. Patch clamp studies showed that a combination of leptin and CCK-8 caused a significant increase in membrane input resistance compared with leptin or CCK-8 alone. Silencing the STAT3 gene abolished the synergistic action of leptin/CCK-8 on neuronal firing. Leptin/CCK-8 synergistically stimulated a 7.7-fold increase in phosphorylated STAT3 (pSTAT3), which was inhibited by AG490, C3 transferase, PP2, LY294002, and wortmannin, but not PD98059. Silencing the Src and PI3K genes resulted in a loss of leptin/CCK-stimulated pSTAT3. We conclude that the synergistic interaction between vagal CCKARs and LRbs is mediated by the phosphorylation of STAT3, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/Src/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways. Malfunctioning of these signaling molecules may result in eating disorders.     

Crystal structure of ubiquitin-like small archaeal modifier protein 1 (SAMP1) from Haloferax volcanii

The ubiquitin-like (Ubl) system has been shown to be ubiquitous in all three kingdoms of life following the very recent characterization of ubiquitin-like small archaeal modifier proteins (SAMP1 and 2) from Haloferax volcanii. The ubiquitin (Ub) and Ubl molecules in eukaryotes have been studied extensively and their cellular functions are well established. Biochemical and structural data pertaining to prokaryotic Ubl protein (Pup) continue to be reported. In contrast to eukaryotes and prokaryotes, no structural information on the archaeal Ubl molecule is available. Here we determined the crystal structure of SAMP1 at 1.55 Å resolution and generated a model of SAMP2. These were then compared with other Ubl molecules from eukaryotes as well as prokaryotes. The structure of SAMP1 shows a β-grasp fold of Ub, suggesting that the archaeal Ubl molecule is more closely related to eukaryotic Ub and Ubls than to its prokaryotic counterpart. The current structure identifies the location of critical elements such a single lysine residue (Lys4), C-terminal di-glycine motif, hydrophobic patches near leucine 60, and uniquely inserted α-helical segments (α1 and α3) in SAMP1. Based on the structure of SAMP1, several Ub-like features of SAMPs such as poly-SAMPylation and non-covalent interactions have been proposed, which should provide the basis for further investigations concerning the molecular function of archaeal Ubls and the large super-family of β-grasp fold proteins in the archaeal kingdom.