On the relationship between C and N fixation in nodulated alfalfa (Medicago sativa)

Legumes such as alfalfa (Medicago sativa L.) are vital N2-fixing crops accounting for a global N2 fixation of ~35 MtNyear-1. Although enzymatic and molecular mechanisms of nodule N2 fixation are now well documented, some uncertainty remains as to whether N2 fixation is strictly coupled with photosynthetic carbon fixation. That is, the metabolic origin and redistribution of carbon skeletons used to incorporate nitrogen are still relatively undefined. Here, we conducted isotopic labelling with both 15N2 and 13C-depleted CO2 on alfalfa plants grown under controlled conditions and took advantage of isotope ratio mass spectrometry to investigate the relationship between carbon and nitrogen turn-over in respired CO2, total organic matter and amino acids. Our results indicate that CO2 evolved by respiration had an isotopic composition similar to that in organic matter regardless of the organ considered, suggesting that the turn-over of respiratory pools strictly followed photosynthetic input. However, carbon turn-over was nearly three times greater than N turn-over in total organic matter, suggesting that new organic material synthesised was less N-rich than pre-existing organic material (due to progressive nitrogen elemental dilution) or that N remobilisation occurred to sustain growth. This pattern was not consistent with the total commitment into free amino acids where the input of new C and N appeared to be stoichiometric. The labelling pattern in Asn was complex, with contrasted C and N commitments in different organs, suggesting that neosynthesis and redistribution of new Asn molecules required metabolic remobilisation. We conclude that the production of new organic material during alfalfa growth depends on both C and N remobilisation in different organs. At the plant level, this remobilisation is complicated by allocation and metabolism in the different organs. Additional keywords: carbon exchange, carbon isotopes, nitrogen fixation, nitrogen 15 isotope

Study of selenocompounds from selenium-enriched culture of edible sprouts

Selenium is recognised as an essential micronutrient for humans and animals. One of the main sources of selenocompounds in the human diet is vegetables. Therefore, this study deals with the Se species present in different edible sprouts grown in Se-enriched media. We grew alfalfa, lentil and soy in a hydroponic system amended with soluble salts, containing the same proportion of Se, in the form of Se(VI) and Se(IV). Total Se in the sprouts was determined by acidic digestion in a microwave system and by ICP/MS. Se speciation was carried out by enzymatic extraction (Protease XIV) and measured by LC-ICP/MS. The study shows that the Se content of plants depends on the content in the growth culture, and that part of the inorganic Se was biotransformed mainly into SeMet. These results contribute to our understanding of the uptake of inorganic Se and its biotransformation by edible plants.

Protected maleimide building blocks for the decoration of peptides, peptoids and peptide nucleic acids

Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides into polyamides such as peptides, peptide nucleic acids, and peptoids were prepared, as well as the corresponding oligomers. Suitable maleimide deprotection conditions were established in each case. The stability of the adducts generated by Michael-type maleimide-thiol reaction and Diels-Alder cycloaddition to maleimide deprotection conditions was exploited to prepare a variety of conjugates from peptide and PNA scaffolds incorporating one free and one protected maleimide. The target molecules were synthesized by using two subsequent maleimide-involving click reactions separated by a maleimide deprotection step. Carrying out maleimide deprotection and conjugation simultaneously gave better results than performing the two reactions subsequently.

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

Effect of diet composition and ration size on key enzyme activities of glycolysis-gluconeogenesis, pentose phosphate pathway and amino acid metabolism in liver of Sparus aurata

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). Highcarbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

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.

Brazilian soybean Glycine max (L.) Merr. cultivars adapted to low latitude regions: seed composition and content of bioactive proteins

Among the goals of the Brazilian soybean improvement programmes, the breeding strategies for cultivars adapted to low latitudes have been included to extend crop areas and to increase production. Seeds of nine Brazilian soybean cultivars adapted to low latitudes were investigated regarding to their composition, and amino acid and antinutritional/toxic protein contents. Protein (394.5 ± 13.1 to 445.3 ± 8.0 g kg-1 dry matter) and oil (200.6 ± 1.2 to 232.3 ± 4.7 g kg-1 dry matter) contents showed low correlation to each other (r = -0.06). The total carbohydrate (141.7 ± 6.1 to 211.1 ± 15.0 g kg-1 dry matter) and ash contents (48.2 ± 4.2 to 52.2 ± 0.5 g kg-1 dry matter) were similar to data available for other soybean cultivars. All soybean cultivars presented low levels of tryptophan and sulphur amino acids. The lectin (1,152 to 147,456 HU kg-1 flour), trypsin inhibitor (34.45 ± 2.28 to 77.62 ± 2.63 g trypsin inhibited kg-1 flour), toxin (6,210 ± 134 to 34,650 ± 110 LD50 kg-1 flour) and urease (0.74 ± 0.02 to 1.22 ± 0.10 g kg¹ flour) presented variations in their contents amongst the cultivars. Compared to other soybean cultivars, urease was higher, the acute toxicity lower and the lectin and trypsin inhibitor contents similar to data available. In general, soybean cultivars showed similar biochemical composition to those developed in different geographic regions. The relevance of these findings to the agronomic features and to choice of soybean cultivars to be used as food or feed is discussed.

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8 µM, kcat = 5.3 min-1, kcat/Km = 2 min-1 µM-1 and Km = 5.0 µM, kcat = 7.0 min-1, kcat/Km = 1.4 min-1 µM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), a-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by a-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations

Effect of a leucine-supplemented diet on body composition changes in pregnant rats bearing Walker 256 tumor

Cancer patients present high mobilization of host protein, with a decrease in lean body mass and body fat depletion occurring in parallel to neoplastic growth. Since leucine is one of the principal amino acids used by skeletal muscle for energy, we investigated the changes in body composition of pregnant tumor-bearing rats after a leucine-supplemented diet. Sixty pregnant Wistar rats divided into six groups were fed a normal protein diet (18%, N) or a leucine-supplemented diet (3% L-leucine, L). The pregnant groups were: control (CN), Walker 256 carcinoma-bearing rats (WN), control rats pair-fed with tumor-bearing rats (pfN), leucine-supplemented (CL), leucine-supplemented tumor-bearing (WL), and leucine-supplemented rats pair-fed with tumor-bearing rats (pfL). At the end of pregnancy, all animals were sacrificed and body weight and tumor and fetal weight were determined. The carcasses were then analyzed for water, fat and total, collagen and non-collagen nitrogen content. Carcass weight was reduced in the WN, WL, pfN and pfL groups compared to control. The lean body mass and total carcass nitrogen were reduced in both tumor-bearing groups. Despite tumor growth and a decrease in fetal weight, there was a slight decrease in collagen (7%) and non-collagen nitrogen (8%) in the WL group compared with the WN group which showed a decrease of 8 and 12%, respectively. Although the WL group presented severe tumor growth effects, total carcass nitrogen and non-collagen nitrogen were particularly higher in this leucine-supplemented group compared to the WN group. These data suggest that the leucine-supplemented diet had a beneficial effect, probably attenuating body wasting.

Amino acid composition of parturient plasma, the intervillous space of the placenta and the umbilical vein of term newborn infants

The objective of the present study was to determine the levels of amino acids in maternal plasma, placental intervillous space and fetal umbilical vein in order to identify the similarities and differences in amino acid levels in these compartments of 15 term newborns from normal pregnancies and deliveries. All amino acids, except tryptophan, were present in at least 186% higher concentrations in the intervillous space than in maternal venous blood, with the difference being statistically significant. This result contradicted the initial hypothesis of the study that the plasma amino acid levels in the placental intervillous space should be similar to those of maternal plasma. When the maternal venous compartment was compared with the umbilical vein, we observed values 103% higher on the fetal side which is compatible with currently accepted mechanisms of active amino acid transport. Amino acid levels of the placental intervillous space were similar to the values of the umbilical vein except for proline, glycine and aspartic acid, whose levels were significantly higher than fetal umbilical vein levels (average 107% higher). The elevated levels of the intervillous space are compatible with syncytiotrophoblast activity, which maintain high concentrations of free amino acids inside syncytiotrophoblast cells, permitting asymmetric efflux or active transport from the trophoblast cells to the blood in the intervillous space. The plasma amino acid levels in the umbilical vein of term newborns probably may be used as a standard of local normality for clinical studies of amino acid profiles.

Elevated amniotic fluid amino acid levels in fetuses with gastroschisis

Our objective was to measure maternal plasma and amniotic fluid amino acid concentrations in pregnant women diagnosed as having fetuses with gastroschisis in the second trimester of pregnancy. Twenty-one pregnant women who had fetuses with gastroschisis detected by ultrasonography (gastroschisis group) in the second trimester and 32 women who had abnormal triple screenings indicating an increased risk for Down syndrome but had healthy fetuses (control group) were enrolled in the study. Amniotic fluid was obtained by amniocentesis, and maternal plasma samples were taken simultaneously. The chromosomal analysis of the study and control groups was normal. Levels of free amino acids and non-essential amino acids were measured in plasma and amniotic fluid samples using EZ:fast kits (EZ:fast GC/FID free (physiological) amino acid kit) by gas chromatography (Focus GC AI 3000 Thermo Finnigan analyzer). The mean levels of essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) and non-essential amino acids (alanine, glycine, proline, and tyrosine) in amniotic fluid were found to be significantly higher in fetuses with gastroschisis than in the control group (P < 0.05). A significant positive correlation between maternal plasma and amniotic fluid concentrations of essential and nonessential amino acids was found only in the gastroschisis group (P < 0.05). The detection of significantly higher amino acid concentrations in the amniotic fluid of fetuses with a gastroschisis defect than in healthy fetuses suggests the occurrence of amino acid malabsorption or of amino acid leakage from the fetus into amniotic fluid.

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice - 15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

Amniotic fluid amino acid levels in non-immune hydrops fetalis: a case-control study

In a prospective case-control study, we compared the amniotic fluid amino acid levels in non-immune hydrops fetalis (NIHF) and normal fetuses. Eighty fetuses underwent amniocentesis for different reasons at the prenatal diagnosis unit of the Department of Obstetrics and Gynecology, Faculty of Medicine, Dicle University. Forty of these fetuses were diagnosed with NIHF. The study included 40 women each in the NIHF (mean age: 27.69 ± 4.56 years) and control (27.52 ± 5.49 years) groups, who had abnormal double- or triple-screening test values with normal fetuses with gestational ages of 23.26 ± 1.98 and 23.68 ± 1.49 weeks at the time of sample collection, respectively. Amniotic fluid amino acid concentrations (intra-assay variation: 2.26-7.85%; interassay variation: 3.45-8.22%) were measured using EZ:faast kits (EZ:faast GC/FID free (physiological) amino acid kit; Phenomenex, USA) by gas chromatography. The standard for quantitation was a mixture of free amino acids from Phenomenex. The levels of 21 amino acids were measured. The mean phosphoserine and serine levels were significantly lower in the NIHF group, while the taurine, α-aminoadipic acid (aaa), glycine, cysteine, NH4, and arginine (Arg) levels were significantly higher compared to control. Significant risk variables for the NIHF group and odds coefficients were obtained using a binary logistic regression method. The respective odds ratios and 95% confidence intervals for the risk variables phosphoserine, taurine, aaa, Arg, and NH4 were 3.31 (1.84-5.97), 2.45 (1.56-3.86), 1.78 (1.18-2.68), 2.18 (1.56-3.04), and 2.41 (1.66-3.49), respectively. The significant difference between NIHF and control fetuses suggests that the amniotic fluid levels of some amino acids may be useful for the diagnosis of NIHF.