Altered nitrogen balance and decreased urea excretion in male rats fed cafeteria diet are related to arginine availability

Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids.We analyzed whether reduced urea excretion was a consequence of higherNO ; (nitrite,nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion.There were no differences in plasma nitrate or nitrite. NO and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithinewhen comparedwith controls,whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability.

Comparative performance of the stable isotope signatures of carbon, nitrogen and oxygen in assessing early vigour and grain yield in durum wheat

The present paper studied the performance of the stable isotope signatures of carbon (δ13C), nitrogen (δ15N) and oxygen (δ18O) in plants when used to assess early vigour and grain yield (GY) in durum wheat growing under mild and moderate Mediterranean stress conditions. A collection of 114 recombinant inbred lines was grown under rainfed (RF) and supplementary irrigation (IR) conditions. Broad sense heritabilities (H2) for GY and harvest index (HI) were higher under RF conditions than under IR. Broad sense heritabilities for δ13C were always above 0·60, regardless of the plant part studied, with similar values for IR and RF trials. Some of the largest genetic correlations with GY were those shown by the δ13C content of the flag leaf blade and mature grains. Under both water treatments, mature grains showed the highest negative correlations between δ13C and GY across genotypes. Flag leaf δ13C was negatively correlated with GY only under RF conditions. The δ13C in seedlings was negatively correlated, under IR conditions only, with GY but also with early vigour. The sources of variation in early vigour were studied by stepwise analysis using the stable isotope signatures measured in seedlings. The δ13C was able to explain almost 0·20 of this variation under RF, but up to 0·30 under IR. In addition, nitrogen concentration in seedlings accounted for another 0·05 of variation, increasing the amount explained to 0·35. The sources of variation in GY were also studied through stable isotope signatures and biomass of different plant parts: δ13C was always the first parameter to appear in the models for both water conditions, explaining c. 0·20 of the variation. The second parameter (δ15N or N concentration of grain, or biomass at maturity) depended on the water conditions and the plant tissue being analysed. Oxygen isotope composition (δ18O) was only able to explain a small amount of the variation in GY. In this regard, despite the known and previously described value of δ13C as a tool in breeding, δ15N is confirmed as an additional tool in the present study. Oxygen isotope composition does not seem to offer any potential, at least under the conditions of the present study.

Laboratory projects using inquiry-based learning: an application to a practical inorganic course

This paper reports how laboratory projects (LP) coupled to inquiry-based learning (IBL) were implemented in a practical inorganic chemistry course. Several coordination compounds have been successfully synthesised by students according to the proposed topics by the LP-IBL junction, and the chemistry of a number of metals has been studied. Qualitative data were collected from written reports, oral presentations, lab-notebook reviews and personal discussions with the students through an experimental course with undergraduate second-year students at the Universidad Nacional de Colombia during the last 5 years. Positive skills production was observed by combining LP and IBL. Conceptual, practical, interpretational, constructional (questions, explanations, hypotheses), communicational, environmental and application abilities were revealed by the students throughout the experimental course.

Dft theoretical study of energetic nitrogen-rich C4N6H8-n(NO2)n derivatives

Density functional theory (DFT) calculations at the B3LYP/6-31G** theoretical level were performed for a series of guanidine-fused bicyclic skeleton derivatives C4N6H8-n(NO2)n (n = 1 - 6). The heats of formation (HOFs) were calculated by isodesmic reactions, and the detonation properties were evaluated using the Kamlet - Jacobs equations. The bond dissociation energies were also analyzed to investigate the thermal stability and sensitivity of the compounds. The results show that all of the derivatives have high positive HOFs, compound G has the highest theoretical density, and compound F1 has the highest detonation velocity and detonation pressure. Considering both the detonation properties and thermal stabilities, compounds D1 and D4 (3 nitro substituents), E1 - E6 (4 nitro substituents), and G (6 nitro substituents) can be regarded as potential candidates for high-energy density materials.

Uptake and translocation of nitrogen, phosphorus and calcium in soybean infected with Meloidogyne incognita and M. javanica

Two soybean (Glycine max) cultivars were used in this study, Ocepar 4, rated as moderately resistant to Meloidogyne incognita race 3 but susceptible to M. javanica, and 'BR 16', susceptible to both nematodes. The effect of nematodes infection on the uptake and transport of N, P and Ca to the shoot was studied in plants growing in a split root system. The upper half was inoculated with 0, 3,000, 9,000 or 27,000 eggs/plant while the lower half received 15N, 32P or 45Ca. Infected plants showed an increase of root but a decrease of shoot mass with increasing inoculum levels. In general, total endogenous nutrients increased in the roots and tended to decrease in the shoots with increasing inoculum levels. When concentrations were calculated, there was an increase in the three nutrients in the roots, and an increase of Ca but no significant variation of N and P was observed in the shoots. The total amount of 15N in the roots increased at the highest inoculum levels but 32P and 45Ca decreased. In the shoots there was a reduction of 32P and 45Ca. The specific concentrations of the labelled nutrients (abundance or radioactivity/tissue mass) also showed a decrease of 32P and 45Ca in the shoots and roots of infected plants and an increase of 15N in the shoots. Considering that overall nutrient concentrations reflect cumulative nutrient uptake and the data from labelled elements gave information at a specific moment of the infection, thus nematodes do interfere with nutrient uptake and translocation.

Basicities of primary arylamines and calculated amine nitrogen electronic charges

Mülliken charges on nitrogen atoms were calculated for several arylamines, utilizing the AM1 Quantum Chemistry method, relating their values to experimental amine pKa . Direct relation between pKa and nitrogen charges was found. The amines energies of protonation, calculated by the same method, also correlate directly with these charges.

Physiological cost of induced resistance in cotton plants at different nitrogen levels

Resistance induction through the use of chemical inducers often results in physiological costs to the plant. In this study, induced resistance in cotton plants was evaluated with regard to physiological costs in a cultivar susceptible to Colletotrichum gossypii var. cephalosporioides (CNPA GO 2002 - 7997). Plants were cultivated in substrates with two levels of nitrogen and received two applications of acibenzolar-S-methyl (ASM), jasmonic acid (JA) and Agro-Mos® (AM) disease resistance inducers. Plant height (H), internodal length (IL), shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW) and root dry weight (RDW) were evaluated. The activity of the phenylalanine ammonia lyase (PAL) and peroxidase (POX) was also determined. The plants treated with ASM presented high physiological costs with an accentuated reduction in H, SFW and SDW, whereas those treated with JA exhibited a significant increase in SDW, and did not significantly differ from H and IL. In the potting mix supplemented with nitrogen, all inducers differed from the control treatment regarding to internodal length, whereas only ASM and AM presented a significant difference between one another in the potting mix without the addition of nitrogen. Significant correlations (P=0.05) were found for most of the variables analyzed, with greater correlations observed between SFW and SDW (0.94); IL and H (0.74); SFW and H (0.70); and SDW and H (0.70). ASM induced the least amount of PAL activity, significantly differing from the remaining treatments. Greater POX activity was observed in ASM, which significantly differed from the control. AM and JA, however, presented lower activity than the control with regard to these enzymes, and it was not possible to confirm induction resistance in these two treatments.

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.