The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity

The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance. The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3). It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth. Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa. When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min. In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously. Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats.     

Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.     

Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endothelium-denuded arteries after restoration of the response by exposure to 10-10 M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.     

Genetic traits of the mosquito Anopheles gambiae: red stripe,frizzled, and homochromy1

The expression, inheritance, and linkage relationships of three genetic traits were studied in the malaria vector Anopheles gambiae. Red stripe (Rs) is a common phenotypic polymorphism in numerous A. gambiae populations, whereas frizzled (f) and homochromy1 (hom1) were isolated from (60)Co-irradiated mosquitoes. Red stripe appears as a diffuse stripe of pigment on the dorsum of larvae and pupae and is variable in expressivity and penetrance. Our data demonstrate that Red stripe results from a heterozygous collarless genotype (i.e., c+ c, chromosome 2) and is essentially sex-limited to females. frizzled is a sex-linked recessive semi-lethal identified by deformed lateral larval setae; its lethality manifests as low rates of adult emergence and brief adult survival. frizzled is located on the X chromosome between pink eye and Mosaic, 3 cM from Mosaic and approximately 12 cM from pink eye. Finally, the mutation homochromy1 (hom1) is on chromosome 2 and causes a recessive phenotype that prevents normal darkening of larvae when reared in a black container. Unlike mutants with this characteristic described thus far, the eye color of hom1 mutants is normal. We determined that hom1 is located between Dieldrin resistance and collarless, approximately 3 cM from the latter. We discuss the possibility of differences in male and female recombination values and the range of values that have been observed in test crosses for chromosome 2 markers.     

A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8(+) T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8(+) T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8(+) T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.     

Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons

The establishment and refinement of neuronal connections depend on dynamic modification of the morphology and physiology of developing axons in response to extrinsic factors. In embryonic cultures of Xenopus spinal neurons, acute application of brain-derived neurotrophic factor (BDNF) induced rapid collateral protrusion of filopodium-like microspikes and lamellipodia along the neurite processes, leading to a morphologic alternation of the neuron. Both types of membrane protrusions contained high concentrations of actin filaments and depended on the polymerization of the actin cytoskeleton. Immunofluorescent staining, however, revealed the presence of microtubules (MTs) in lamellipodia induced by BDNF. These MTs appeared to have arisen from debundling of MTs in the neurite shaft at the protrusion sites, splaying and extending in the rapidly protruding lamellipodia. Inhibition of microtubule polymerization by nocodazole largely abolished the formation of lamellipodia but not of microspikes. Taken together, our results suggest that collateral sprouting of microspikes and lamellipodia involve distinctly different cytoskeletal mechanisms. Although the actin cytoskeleton is solely responsible for microspike formation, cooperative efforts by microtubules and actin filaments are essential for lamellipodial protrusion in response to extrinsic factors.     

Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro

Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.     

A study of prey-predator relations for mammals

In this paper, we present a prey-predator nonlinear model for mammals, consisting of large- and small-size prey species with group defence, in a partially protected habitat. If the prey size is small, then it is more prone to the predator at higher densities. Conversely, large prey size at higher densities tend to develop group defence. Therefore, the predator will be attracted towards that area where prey are less in number. A new physical constant has been introduced into the radiation-type condition on that part of the boundary where interaction between prey and predator takes place. This constant allows us to efficiently model group defence capabilities of the herds and its numerical values have to be determined for different pairs of prey-predator species from field observations. A way of measuring the constants involved in the model is suggested. Numerical results are provided and thoroughly discussed for a habitat of circular shape. The obtained results show that in the region away from the protected area, the density of large-size prey species is higher than that of small-size prey species, a fact that is in accordance with observations.