Effects of acylation on the structure,lipid binding,and transfer activity of wheat lipid transfer protein

Study of the effect of protein chemical acylation on their functional properties or activity often brings valuable information regarding structure-function relationships. We performed such work on wheat lipid transfer protein, LTP1, to investigate the role of grafted acyl chains on the lipid binding and transfer properties. LTP1 was acylated by using anhydride derivatives of various chain lengths from C2 to C6. Only the chemical modifications with hexanoic acid yielded a marked effect on the tertiary structure and a slight change in the secondary structure. The affinity of the modified proteins for myristoyl-lysophosphatidylcholine was similar to that of the native protein accompanied by a slight decrease in stoichiometry. Interestingly, the acylation of LTP1 enhanced the lipid transfer activity by at least a factor of 10 for hexanoic chain length. Finally, the grafting of acyl chains was investigated by means of molecular modelling, and an attempt is made to correlate with our experimental data.     

Flow Visualization of Rhinoceros Beetle (Trypoxylus dichotomus) in Free Flight

Aerodynamic characteristics of the beetle,Trypoxylus dichotomus,which has a pair of elytra (forewings) and flexible hind wings,are investigated.Visualization experiments were conducted for various flight conditions of a beetle,Trypoxylus dichotomus:free,tethered,hovering,forward and climbing flights.Leading edge,trailing edge and tip vortices on both wings were observed clearly.The leading edge vortex was stable and remained on the top surface of the elytron for a wide interval during the downstroke of free forward flight.Hence,the elytron may have a considerable role in lift force generation of the beetle.In addition,we reveal a suction phenomenon between the gaps of the hind wing and the elytron in upstroke that may improve the positive lift force on the hind wing.We also found the reverse clap-fling mechanism of the T.dichotomus beetle in hovering flight.The hind wings touch together at the beginning of the upstroke.The vortex generation,shedding and interaction give a better understanding of the detailed aerodynamic mechanism of beetle flight.     

Properties and biosynthetic connection of the nucleotide pyrophosphatases of rat liver plasma membrane and endoplasmic reticulum

The detergent-solubilized nucleotide pyrophosphatases of the rat liver plasma membrane and endoplasmic reticulum fractions were purified by lectin affinity chromatography. They have the same molecular mass of 148 000 dalton; their catalytic properties are also very similar and correspond to those of the trypsin-solubilized activities from the same membrane preparations. Pulse-chase experiments on isolated perfused livers using [3H]leucine indicated different labelling kinetics of the proteins isolated from plasma membrane and endoplasmic reticulum. The plasma membrane enzyme became only slightly labelled in the presence of 100 microM vinblastine. The data support a precursor-product relationship of the nucleotide pyrophosphatases from endoplasmic reticulum and plasma membrane.     

Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase

The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.     

Nutrition and Physical Activity Program to Attenuate Obesity and Promote Physical and Metabolic Fitness in Elementary School Children

Obesity and low levels of physical and metabolic fitness are risk factors for cardiovascular disease and diabetes. The purpose of this investigation was to attenuate obesity and improve physical and metabolic fitness in elementary school children. Schools have the opportunity, mechanisms, and personnel in place to deliver nutrition education, fitness activities, and a school food service that is nutritious and healthy. Cohorts from grades 3 to 5 in two school districts in rural Nebraska (Intervention/Control) participated in a 2-year study of physical activity and modified school lunch program. Data collection for aerobic capacity, body composition, blood chemistry, nutrition knowledge, energy intake, and physical activity was at the beginning and end of each year. Int received enhanced physical activity, grade specific nutrition education, and a lower fat and sodium school lunch program. Con continued with a regular school lunch and team sports activity program. At year 2, Int lunches had significantly less energy (9%), fat (25%), sodium (21%), and more fiber (17%). However, measures of 24-hour energy intake for Int and Con showed significant differences for sodium only. Physical activity in the classroom was 6% greater for Int compared to Con (p < 0.05) but physical activity outside of school was ?16 % less for Int compared to Con (p < 0.05). Body weight and body fat were not different between schools for normal weight or obese children. No differences were found for cholesterol, insulin, and glucose; however, HDL cholesterol was significantly greater and cholesterol/HDL was significantly less for Int compared to Con (p < 0.05). It appears that compensation in both energy intake and physical activity outside of school may be responsible for the lack of differences between Int and Con.     

Operon prediction in Pyrococcus furiosus

Identification of operons in the hyperthermophilic archaeon Pyrococcus furiosus represents an important step to understanding the regulatory mechanisms that enable the organism to adapt and thrive in extreme environments. We have predicted operons in P.furiosus by combining the results from three existing algorithms using a neural network (NN). These algorithms use intergenic distances, phylogenetic profiles, functional categories and gene-order conservation in their operon prediction. Our method takes as inputs the confidence scores of the three programs, and outputs a prediction of whether adjacent genes on the same strand belong to the same operon. In addition, we have applied Gene Ontology (GO) and KEGG pathway information to improve the accuracy of our algorithm. The parameters of this NN predictor are trained on a subset of all experimentally verified operon gene pairs of Bacillus subtilis. It subsequently achieved 86.5% prediction accuracy when applied to a subset of gene pairs for Escherichia coli, which is substantially better than any of the three prediction programs. Using this new algorithm, we predicted 470 operons in the P.furiosus genome. Of these, 349 were validated using DNA microarray data.     

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.     

DAB1 and Reelin effects on amyloid precursor protein and ApoE receptor 2 trafficking and processing

Numerous cytoplasmic adaptor proteins, including JIP1, FE65, and X11alpha, affect amyloid precursor protein (APP) processing and Abeta production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP beta-C-terminal fragment and secreted Abeta. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the beta-C-terminal fragment of APP and Abeta. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands.     

General mutagenesis of F plasmid TraI reveals its role in conjugative regulation

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies approximately 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.