Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

Mucor indicus: Biology and industrial application perspectives: A review

Mucor indicus, one of the most important strains of zygomycetes fungi, has been the subject of several studies since a couple of hundred years ago. This fungus, regarded as a non-pathogenic dimorphic microorganism, is used for production of several beers and foods. Morphology of the fungus can be manipulated and well controlled by changing a number of parameters. Furthermore, M. indicus can grow on a variety of substrates including lignocellulosic hydrolysates which are mixtures of hexoses, pentoses, and different severe fermentation inhibitors. Indeed, high yield ethanol production is among the most important features of this strain. Presence of considerable amounts of chitosan in the cell wall is another important aspect of the fungus. Besides production of ethanol and chitosan, the biomass of this fungus has shown a great potential to be used as a rich nutritional source, e.g. fish feed. The fungus is also among the oleaginous fungi and produces high amounts of polyunsaturated fatty acids particularly γ-linolenic acid. Furthermore, the biomass autolysate has a high potential for yeast extract replacement in fermentation by the fungus. Additionally, the strain has shown promising results in heavy metal removal from wastewaters. This review discusses different aspects of biology and industrial application perspectives of M. indicus. Furthermore, open areas for the future basic and applied levels of research are also presented.     

Pathogenicity of Two Species of Entomopathogenic Nematodes Against the Greenhouse Whitefly,Trialeurodes vaporariorum (Hemiptera: Aleyrodidae), in Laboratory and Greenhouse Experiments

The greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) is a polyphagous pest in greenhouse crops. The efficacy of two entomopathogenic nematodes (EPN), Steinernema feltiae and Heterorhabditis bacteriophora, as biological control agents against T. vaporariorum was evaluated using two model crops typical of vegetable greenhouse productions: cucumber and pepper. Laboratory tests evaluated adults and second nymphal instars for pest susceptibility to different EPN species at different concentrations of infective juveniles (IJ; 0, 25, 50, 100, 150, 200, and 250 IJ per cm²); subsequent greenhouse trials against second nymphal instars on cucumber and pepper plants evaluated more natural conditions. Concentrations were applied in combination with Triton X-100 (0.1% v/v), an adjuvant for increasing nematode activity. In laboratory studies, both life stages were susceptible to infection by the two nematode species, but S. feltiae recorded a lower LC₅₀ than H. bacteriophora for both insect stages. Similarly, in greenhouse experiments, S. feltiae required lower concentrations of IJ than H. bacteriophora to reach the same mortality in nymphs. In greenhouse trials, a significant difference was observed in the triple interaction among nematode species × concentration × plant. Furthermore, the highest mortality rate of the second nymphal instars of the T. vaporariorum was obtained from the application of S. feltiae concentrated to 250 IJ/cm² on cucumber (49 ± 1.23%). The general mortality caused by nematodes was significantly higher in cucumber than in pepper. These promising results support further investigation for the optimization of the best EPN species/concentration in combination with insecticides or adjuvants to reach a profitable control of this greenhouse pest.     

Phytotoxicity of green synthesized silver nanoparticles on Camelina sativa L

Due to the increased production and release of silver nanoparticles (AgNPs) in the environment, the concerns about the possibility of toxicity and oxidative damage to plant ecosystems should be considered. In the present study, the effects of different concentrations of AgNPs (0, 0.5, 1, 2, 3 and 4 g/L) synthesized using the extract of camelina (Camelina sativa) leaves on the growth and the biochemical traits of camelina seedlings were investigated. The results showed that AgNPs significantly increased Ag accumulation in the roots and shoots which decreased the growth and photosynthetic pigments of camelina seedlings. The highest decrease in the height and total dry weight was observed by 53.1 and 61.8% under 4 g/L AgNPs, respectively over control plants. AgNPs application over 2 g/L enhanced the accumulation of proline, malondialdehyde, hydrogen peroxide and methylglyoxal, and up-regulated the activity of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) and glyoxalase (glyoxalase I and II) system which indicates oxidative stress induction in camelina seedlings. Moreover, AgNPs reduced ASA and GSH contents and increased DHA and GSSG contents, hence disrupting the redox balance. These results showed that AgNPs at 4 g/L had the most toxic effects on the camelina growth. Therefore, increasing oxidative stress markers and the activity of antioxidant enzymes and enzymes involved in glyoxalase system indicated the oxidative stress induced by AgNPs treatments over 2 g/L as well as the induction of antioxidant defense systems to combat AgNPs-induced oxidative stress.     

Aberrant cardiolipin metabolism is associated with cognitive deficiency and hippocampal alteration in tafazzin knockdown mice

Cardiolipin (CL) is a key mitochondrial phospholipid essential for mitochondrial energy production. CL is remodeled from monolysocardiolipin (MLCL) by the enzyme tafazzin (TAZ). Loss-of-function mutations in the gene which encodes TAZ results in a rare X-linked disorder called Barth Syndrome (BTHS). The mutated TAZ is unable to maintain the physiological CL:MLCL ratio, thus reducing CL levels and affecting mitochondrial function. BTHS is best known as a cardiac disease, but has been acknowledged as a multi-syndrome disorder, including cognitive deficits. Since reduced CL levels has also been reported in numerous neurodegenerative disorders, we examined how TAZ-deficiency impacts cognitive abilities, brain mitochondrial respiration and the function of hippocampal neurons and glia in TAZ knockdown (TAZ kd) mice. We have identified for the first time the profile of changes that occur in brain phospholipid content and composition of TAZ kd mice. The brain of TAZ kd mice exhibited reduced TAZ protein expression, reduced total CL levels and a 19-fold accumulation of MLCL compared to wild-type littermate controls. TAZ kd brain exhibited a markedly distinct profile of CL and MLCL molecular species. In mitochondria, the activity of complex I was significantly elevated in the monomeric and supercomplex forms with TAZ-deficiency. This corresponded with elevated mitochondrial state I respiration and attenuated spare capacity. Furthermore, the production of reactive oxygen species was significantly elevated in TAZ kd brain mitochondria. While motor function remained normal in TAZ kd mice, they showed significant memory deficiency based on novel object recognition test. These results correlated with reduced synaptophysin protein levels and derangement of the neuronal CA1 layer in hippocampus. Finally, TAZ kd mice had elevated activation of brain immune cells, microglia compared to littermate controls. Collectively, our findings demonstrate that TAZ-mediated remodeling of CL contributes significantly to the expansive distribution of CL molecular species in the brain, plays a key role in mitochondria respiratory activity, maintains normal cognitive function, and identifies the hippocampus as a potential therapeutic target for BTHS.     

Pulmonary hypertension and ascites as affected by dietary protein source in broiler chickens reared in cool temperature at high altitudes

As canola meal protein (CMP) has a lower arginine (Arg) content than soybean meal protein (SBMP), the main objective of this study was to investigate if replacing SBMP by CMP affected the incidence of pulmonary hypertension and ascites. Two hundred and seventy broiler chicks (Ross 308) were used to evaluate the effect of replacing CMP at proportionally 0, 0.5, and 1 for SBMP in a complete randomized block design, as mentioned in the Statistical analysis section (dietary treatments designated as CMP0, CMP0.5, and CMP1, respectively). The experiment was carried out on litter flooring from 6 to 42 days of age. The results indicate that broiler performance (body weight gain, feed intake, and feed conversion ratio) was significantly (P<0.05) impaired in the CMP1 group at 6–21 days but not at 21–42 days. Inclusion of canola meal (CM) in the diet significantly increased ascites and total mortality (P<0.05) and caused a decrease in the serum nitric oxide (NO) concentration with a concomitant increase (P<0.05) in the right ventricular weight ratio (RV/TV). Haematocrit, circulatory triiodothyronine and thyroxine thyroid hormone concentrations and serum alkaline phosphatase activity were unaffected by dietary treatments. In conclusion, the results of this study indicate that substitution of CMP for SBMP was associated with pulmonary hypertension (i.e., higher RV/TV) and ascites mortality in broiler chickens.     

Age and parasitism status of Tuta absoluta eggs alter the foraging responses of the predator Nabis pseudoferus

We studied the effect of Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) parasitism of Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) eggs on the foraging behavior of Nabis pseudoferus Remane (Hemiptera: Nabidae) feeding on T. absoluta eggs of different ages. Older parasitized eggs (48–72 h old) were rejected after probing with the probosis. A separate experiment showed that probing did not harm developing parasitoids. Bugs greatly reduced their feeding times on older parasitized eggs and spent more time grooming and probing eggs and leaves. Predators exhibited a type III functional response to 24 h-old eggs, but a type II response to older eggs, whether parasitized or not. 24 h-old eggs suffered higher attack rates, and required less handling time than older eggs. Although the predatory efficiency of N. pseudoferus was reduced in the presence of parasitized eggs, the partial refuge enjoyed by T. brassicae in older eggs may permit an additive impact on the pest, and should facilitate the joint augmentation of both species against T. absoluta.

     

Elucidation of metabolism in hybridoma cells grown in fed‐batch culture by genome‐scale modeling

Genome‐scale modeling of mouse hybridoma cells producing monoclonal antibodies (mAb) was performed to elucidate their physiological and metabolic states during fed‐batch cell culture. Initially, feed media nutrients were monitored to identify key components among carbon sources and amino acids with significant impact on the desired outcome, for example, cell growth and antibody production. The monitored profiles indicated rapid assimilation of glucose and glutamine during the exponential growth phase. Significant increase in mAb concentration was also observed when glutamine concentration was controlled at 0.5 mM as a feeding strategy. Based on the reconstructed genome‐scale metabolic network of mouse hybridoma cells and fed‐batch profiles, flux analysis was then implemented to investigate the cellular behavior and changes in internal fluxes during the cell culture. The simulated profile of the cell growth was consistent with experimentally measured specific growth rate. The in silico simulation results indicated (i) predominant utilization of glycolytic pathway for ATP production, (ii) importance of pyruvate node in metabolic shifting, and (iii) characteristic pattern in lactate to glucose ratio during the exponential phase. In future, experimental and in silico analyses can serve as a promising approach to identifying optimal feeding strategies and potential cell engineering targets as well as facilitate media optimization for the enhanced production of mAb or recombinant proteins in mammalian cells. Biotechnol. Bioeng. 2009;102: 1494–1504. © 2008 Wiley Periodicals, Inc.