Effect of substituent structure in fluorene based compounds: Experimental and theoretical study.

Fluorene-based low molar weight derivatives were synthesized in Suzuki reactions by using key starting materials 9-benzylidene-2,7-dibromofluorene or 3-(2,7-dibromofluoren-9-ylmethylen)-9-ethylcarbazole and various aryl boronic acids. Photophysical properties of the compounds were investigated in different solutions as well as in solid state. The thermal investigations showed that the obtained compounds are highly thermally stable with temperatures of 5% mass loss (T5%) in the range of 311-432 °C. Some of the compounds also exhibited very high glass transition temperatures exceeding 125 °C. The presented molecules were electrochemically active and showed the energy band gap below 2.97 eV. The investigations were supported by DFT calculations and the photovoltaic ability of the presented compounds was tested in the organic-inorganic solar cells.

The use of the ERAS protocol in malnourished and properly nourished patients undergoing elective surgery: a questionnaire study.

INTRODUCTION: Enhanced recovery after surgery (ERAS) is a modern approach to perioperative management. This study aimed to evaluate compliance with certain aspects of the ERAS protocol in malnourished and properly nourished patients undergoing elective surgery. MATERIAL AND METHODS: A questionnaire study was conducted among 197 patients undergoing elective surgery at the university hospital. We divided patients into two groups according to nutritional status. RESULTS: The study's results showed that 67 patients (34%) lost weight before admission (the weight-loss group). Twenty-five participants (37%) in the weight-loss group and 15 patients (12%) in the preserved-weight group underwent surgery due to cancer ( P < 0.001). More patients in the weight loss group (45 of 67) than in the preserved-weight group (40 of 129, P < 0.001) limited their food intake a week before the surgery. The preserved-weight group participants were mobilized earlier than the weight-loss group ( P = 0.04). The median number of hours since drinking their last fluids and eating their last meals before the surgery were 12.2 hours and 25.4 hours for both groups, respectively. Only eight patients received preoperative carbohydrate loading. We found higher serum protein concentrations in the preserved-weight group (7.10 [0.5] vs. 6.92 [0.71], P = 0.023); however, white blood cell count was higher in the weight-loss group (7.85 (2.28) vs.7.10 (0.50), P = 0.04). Both groups were highly satisfied with their hospital treatments. CONCLUSIONS: Our study revealed relatively high malnutrition in patients undergoing elective surgery. As a standard of perioperative care in the studied centre, the ERAS protocol implementation level is low.

Synthesis and Characterization of New Conjugated Azomethines End-Capped with Amino-thiophene-3,4-dicarboxylic Acid Diethyl Ester.

A new series of thiophene-based azomethines differing in the core structure was synthesized. The effect of the central core structure in azomethines on the thermal, optical and electrochemical properties was investigated. The obtained compounds exhibited the ability to form a stable amorphous phase with a high glass transition temperature above 100 °C. They were electrochemically active and undergo oxidation and reduction processes. The highest occupied (HOMO) and the lowest unoccupied molecular (LUMO) orbitals were in the range of -3.86--3.60 eV and -5.46--5.17 eV, respectively, resulting in a very low energy band gap below 1.7 eV. Optical investigations were performed in the solvents with various polarity and in the solid state as a thin film deposited on a glass substrate. The synthesized imines absorbed radiation from 350 to 600 nm, depending on its structure and showed weak emission with a photoluminescence quantum yield below 2.5%. The photophysical investigations were supported by theoretical calculations using the density functional theory. The synthesized imines doped with lithium bis-(trifluoromethanesulfonyl)imide were examined as hole transporting materials (HTM) in hybrid inorganic-organic perovskite solar cells. It was found that both a volume of lithium salt and core imine structure significantly impact device performance. The best power conversion efficiency (PCE), being about 35-63% higher compared to other devices, exhibited cells based on the imine containing a core tiphenylamine unit.

Polymers in High-Efficiency Solar Cells: The Latest Reports.

Third-generation solar cells, including dye-sensitized solar cells, bulk-heterojunction solar cells, and perovskite solar cells, are being intensively researched to obtain high efficiencies in converting solar energy into electricity. However, it is also important to note their stability over time and the devices' thermal or operating temperature range. Today's widely used polymeric materials are also used at various stages of the preparation of the complete device-it is worth mentioning that in dye-sensitized solar cells, suitable polymers can be used as flexible substrates counter-electrodes, gel electrolytes, and even dyes. In the case of bulk-heterojunction solar cells, they are used primarily as donor materials; however, there are reports in the literature of their use as acceptors. In perovskite devices, they are used as additives to improve the morphology of the perovskite, mainly as hole transport materials and also as additives to electron transport layers. Polymers, thanks to their numerous advantages, such as the possibility of practically any modification of their chemical structure and thus their physical and chemical properties, are increasingly used in devices that convert solar radiation into electrical energy, which is presented in this paper.

Synthesis and Thermal, Photophysical, Electrochemical Properties of 3,3-di[3-Arylcarbazol-9-ylmethyl]oxetane Derivatives.

Novel oxetane-functionalized derivatives were synthesized to find the impact of carbazole substituents, such as 1-naphtyl, 9-ethylcarbazole and 4-(diphenylamino)phenyl, on their thermal, photophysical and electrochemical properties. Additionally, to obtain the optimized ground-state geometry and distribution of the frontier molecular orbital energy levels, density functional theory (DFT) calculations were used. Thermal investigations showed that the obtained compounds are highly thermally stable up to 360 °C, as molecular glasses with glass transition temperatures in the range of 142-165 °C. UV-Vis and photoluminescence studies were performed in solvents of differing in polarity, in the solid state as a thin film on glass substrate, and in powders, and were supported by DFT calculations. They emitted radiation both in solution and in film with photoluminescence quantum yield from 4% to 87%. Cyclic voltammetry measurements revealed that the materials undergo an oxidation process. Next, the synthesized molecules were tested as hole transporting materials (HTM) in perovskite solar cells with the structure FTO/b-TiO2/m-TiO2/perovskite/HTM/Au, and photovoltaic parameters were compared with the reference device without the oxetane derivatives.

New Acceptor-Donor-Acceptor Systems Based on Bis-(Imino-1,8-Naphthalimide).

In this paper, six novel symmetrical bis-(imino-1,8-naphthalimides) differing in core and N-substituent structure were synthesized, and their thermal (TGA, DSC), optical (UV-Vis, PL), electrochemical (DPV, CV) properties were evaluated. The compounds were stable to 280 °C and could be transferred into amorphous materials. Electrochemical investigations showed their ability to occur reductions and oxidations processes. They exhibited deep LUMO levels of about -3.22 eV and HOMO levels above -5.80 eV. The optical investigations were carried out in the solutions (polar and non-polar) and in films and blends with PVK:PBD. Bis-(imino-1,8-naphthalimides) absorbed electromagnetic radiation in the range of 243-415 nm and emitted light from blue to yellow. Their capacity for light emission under voltage was preliminarily tested in devices with an active layer consisting of a neat compound and a blend with PVK:PBD. The diodes emitted green or red light.

Oligomeric S100A4 Is Associated With Monocyte Innate Immune Memory and Bypass of Tolerance to Subsequent Stimulation With Lipopolysaccharides.

Objectives: Most DAMPs in inflammatory diseases are TLR2- and TLR4-ligands and according to the current concept, repeated stimuli would result in tolerance. Aims of the study were to verify this assumption, to investigate whether epigenetic effectors are involved and to explore the situation in rheumatoid arthritis (RA). Methods: A trained immunity (TI) and tolerance protocol was established using peripheral blood monocytes from healthy donors, ß-glucan and lipopolysaccharide (LPS). The training or tolerance capacities of RA-relevant DAMPs were tested. Results: ß-Glucan-, oS100A4-, HMBG1-, and HSP90-pretreated monocytes showed increased IL-6 responses to LPS re-stimulation. ß-Glucan, oS100A and tenascin C induced training of monocytes to release more TNFα. In comparison to ß-glucan, most DAMPs tested induced less TI, with exception of oS100A4. Monocytes exposed to oS100A4 showed increased IL-1ß, IL-6, and TNFα in response to LPS, in spite that both stimulate TLR4. RNASEq upon ß-glucan or oS100A4 revealed similar changes in chemokines/cytokines and epigenetic effectors; 17 epigenetic effectors correlated with chemokine/cytokine gene expression; PRDM8 was associated with more chemokine and cytokine transcripts. Knockdown of PRDM8 abolished TI induced by oS100A4. In RA, plasma S100A4 correlated with increased CSF2, and increased PRDM8 transcription in RA monocytes was associated with increased plasma CCL5 and IL-6, as well as therapy-resistance. Conclusion: Bypass of tolerance by DAMPs might be a phenomenon as important as TI, since it could explain how chronic inflammation can be maintained in spite of an environment with multiple TLR2/TLR4-ligands. In RA monocytes, a PRDM8-dependent TI mechanism could be responsible for sustained chemokine/cytokines levels.

Structural basis of potassium activation in plant asparaginases.

l-asparaginases (EC 3.5.1.1) play an important role in nitrogen mobilization in plants. Here, we investigated the biochemical and biophysical properties of potassium-dependent (PvAspG1) and potassium-independent (PvAspG-T2) l-asparaginases from Phaseolus vulgaris. Our previous studies revealed that PvAspG1 requires potassium for catalytic activation and its crystal structure suggested that Ser-118 in the activation loop plays a critical role in coordinating the metal cation. This amino acid residue is replaced by isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, allosteric effector binding, secondary structure conformation, and pH profile were studied. Introduction of the serine residue conferred potassium activation in PvAspG-T2. Conversely, the PvAspG1-S118I mutant could no longer be activated by potassium. PvAspG1 and the PvAspG-T2-I117S mutant had a similar half-maximal effective concentration (EC50 ) value for potassium activation, between 0.1 and 0.3 mm. Potassium binding elicited a similar conformational change in PvAspG1 and PvAspG-T2-I117S, as studied by circular dichroism. However, no change in conformation was observed for PvAspG-T2 and PvAspG1-S118I. Analysis of kinetic parameters in function of pH indicated that potassium activation mediated by Ser-118 influences the ionization of specific functional groups in the enzyme-substrate complex. Together, the results indicate that Ser-118 of PvAspG1 is essential and sufficient for potassium activation in plant l-asparaginases. ENZYME: l-Asparaginase (EC 3.5.1.1).

Expression of matrix metalloproteinases and components of the endocannabinoid system in the knee joint are associated with biphasic pain progression in a rat model of osteoarthritis.

Matrix metalloproteinases (MMPs) are considered important in articular cartilage breakdown during osteoarthritis (OA). Similarly, the endocannabinoid system (ECS) is implicated in joint function and modulation of nociceptive processing. Functional interplay between ECS and MMPs has been recently indicated. Here, we tested if changes in the expression of selected MMPs and major ECS elements temporally correlate with the intensity of OA-related pain. Knee OA was induced in male Wistar rats by intra-articular sodium monoiodoacetate injection. OA-like pain behavior was tested using the dynamic weight bearing. Joint tissue samples at different time points after OA induction were subjected to gene (quantitative polymerase chain reaction) and protein (Western blot) expression analyses. Monoiodoacetate-induced nocifensive responses in rats showed a biphasic progression pattern. The alterations in expression of selected MMPs elegantly corresponded to the two-stage development of OA pain. The most substantial changes in the expression of the ECS system were revealed at a later stage of OA progression. Alterations within ECS are involved in the process of adaptation to persistent painful stimuli. The accumulation of MMPs in osteoarthritic cartilage may have a role in the biphasic progression of OA-related pain. Temporal association of changes in ECS and MMPs expression shows a potential therapeutic approach that utilizes the concept of combining indirect ECS-mediated MMP inhibition and ECS modulation of pain transduction.

The multiplicity of spinal AA-5-HT anti-nociceptive action in a rat model of neuropathic pain.

There is considerable evidence to support the role of anandamide (AEA), an endogenous ligand of cannabinoid receptors, in neuropathic pain modulation. AEA also produces effects mediated by other biological targets, of which the transient receptor potential vanilloid type 1 (TRPV1) has been the most investigated. Both, inhibition of AEA breakdown by fatty acid amide hydrolase (FAAH) and blockage of TRPV1 have been shown to produce anti-nociceptive effects. Recent research suggests the usefulness of dual-action compounds, which may afford greater anti-allodynic efficacy. Therefore, in the present study, we examined the effect of N-arachidonoyl-serotonin (AA-5-HT), a blocker of FAAH and TRPV1, in a rat model of neuropathic pain after intrathecal administration. We found that treatment with AA-5-HT increased the pain threshold to mechanical and thermal stimuli, with highest effect at the dose of 500nM, which was most strongly attenuated by AM-630, CB2 antagonist, administration. The single action blockers PF-3845 (1000nM, for FAAH) and I-RTX (1nM, for TRPV1) showed lower efficacy than AA-5-HT. Moreover AA-5-HT (500nM) elevated AEA and palmitoylethanolamide (PEA) levels. Among the possible targets of these mediators, only the mRNA levels of CB2, GPR18 and GPR55, which are believed to be novel cannabinoid receptors, were upregulated in the spinal cord and/or DRG of CCI rats. It was previously reported that AA-5-HT acts in CB1 and TRPV1-dependent manner after systemic administration, but here for the first time we show that AA-5-HT action at the spinal level involves CB2, with potential contributions from GRP18 and/or GPR55 receptors.

Characterization of aromatic aminotransferases from Ephedra sinica Stapf.

Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using L-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.

Higher endogenous methionine in transgenic Arabidopsis seeds affects the composition of storage proteins and lipids.

Previous in vitro studies demonstrate that exogenous application of the sulfur-containing amino acid methionine into cultured soybean cotyledons and seedlings reduces the level of methionine-poor storage proteins and elevates those that are methionine-rich. However, the effect of higher endogenous methionine in seeds on the composition of storage products in vivo is not studied yet. We have recently produced transgenic Arabidopsis seeds having significantly higher levels of methionine. In the present work we used these seeds as a model system and profiled them for changes in the abundances of 12S-globulins and 2S-albumins, the two major groups of storage proteins, using 2D-gels and MALDI-MS detection. The findings suggest that higher methionine affects from a certain threshold the accumulation of several subunits of 12S-globulins and 2S-albumins, regardless of their methionine contents, resulting in higher total protein contents. The mRNA abundances of most of the genes encoding these proteins were either correlated or not correlated with the abundances of these proteins, implying that methionine may regulate storage proteins at both transcriptional and post-transcriptional levels. The elevations in total protein contents resulted in reduction of total lipids and altered the fatty acid composition. Altogether, the data provide new insights into the regulatory roles of elevated methionine levels on seed composition.

Soybean seeds overexpressing asparaginase exhibit reduced nitrogen concentration.

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K+ -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the ß-subunit of ß-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing ß-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11-18%, and an increase in free aspartic acid levels, by 25-60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9-13%. Their levels of extractible globulins were reduced by 11-30%. This was accompanied by an increase in oil concentration, by 5-8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation.

The importance of TRPV1-sensitisation factors for the development of neuropathic pain.

Transient receptor potential vanilloid type 1 (TRPV1), classically associated with transduction of high-temperature and low-pH pain, underlies pain hypersensitivity in neuropathic pain. The molecular regulation of TRPV1 channel activity is not yet fully understood. Therefore, we investigated factors regulating sensitisation of this receptor during development of neuropathic pain in a rat model of chronic construction injury (CCI) in the dorsal root ganglia (DRG). In the rat CCI model, elevated levels of pro-inflammatory cytokines (TNFα, IL-1ß and IL-6) in DRG corresponded to development of neuropathic pain. We assessed the expression of known kinases influencing TRPV1 sensitisation at the mRNA and/or protein level. Protein kinase C ε (PKCε) showed the strongest upregulation at the mRNA and protein levels among all tested kinases. Co-expression of PKCε and TRPV1 in L5 DRG of CCI animals was high during the development of neuropathic pain. The number of neurons expressing PKCε increased throughout the experiment. We provide complex data on the expression of a variety of factors involved in TRPV1 sensitisation in a CCI model of neuropathic pain. Our study supports evidence for involvement of TRPV1 in the development of neuropathic pain, by showing increased expression of interleukins and kinases responsible for the channel sensitisation. TNFα and NGF seem to play a role in the transition from acute to neuropathic pain, while PKCε in its maintenance. Further studies might confirm their significance as novel targets for the treatment of neuropathic pain.

Na⁺/K⁺ exchange switches the catalytic apparatus of potassium-dependent plant L-asparaginase.

Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type L-asparaginase from Phaseolus vulgaris (PvAspG1) differing in the type of associated alkali metal ions (K(+), Na(+) or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type L-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to α and ß subunits, thus creating the mature heterotetramer or dimer of heterodimers (αß)2. The αß subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two ß-sheets flanked on each side by a layer of helices. In addition to the `sodium loop' (here referred to as the `stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the `activation loop') in subunit α (residues Val111-Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type L-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K(+) is coordinated) or OFF (when Na(+) is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent L-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.

Characterization of a cruciferin deficient mutant of Arabidopsis and its utility for overexpression of foreign proteins in plants.

Plant seeds naturally accumulate storage reserves (proteins, carbohydrates, lipids) that are mobilized during germination to provide energy and raw materials to support early seedling growth. Seeds have been exploited as bioreactors for the production to foreign materials, but stable, high level expression has been elusive, in part due to the intrinsic bias for producing the natural reserves in their typical proportions. To identify mutants governing seed filling, we screened a population of mutagenized Arabidopsis plants for a mutant that failed to fill its seeds. Here we report the identification of ssp1, a recessive, viable mutant that accumulates approximately 15% less protein than wildtype seeds. Molecular analyses revealed that ssp1 is due to the introduction of a premature stop codon in CRU3, one of the major cruciferin genes. Unlike many other reserve mutants or transgenic lines in which seed storage protein levels are reduced by antisense/RNAi technologies, ssp1 exhibits low level compensation by other reserves, and represents a mutant background that might prove useful for high level expression of foreign proteins. To test this hypothesis, we used a bean phytohemagglutinin (PHA) gene as a reporter and compared PHA expression levels in single copy insertion lines in ssp1 vs. wildtype. These near isogenic lines allow reporter protein levels to be compared without the confounding and sometimes unknown influences of transgene copy number and position effects on gene expression. The ssp1 lines consistently accumulated more PHA than the backcrossed counterparts, with increases ranging from 12% to 126%. This proof of principle study suggests that similar strategies in crop plants may improve the yield of foreign proteins of agronomic and economic interest.

Characterization of Arabidopsis serine:glyoxylate aminotransferase, AGT1, as an asparagine aminotransferase.

Asparagine (Asn) is a major form of nitrogen transported to sink tissues. Results from a previous study have shown that an Arabidopsis mutant lacking asparaginase activity develops relatively normally, highlighting a possible compensation by other types of asparagine metabolic enzymes. Prior studies with barley and tobacco mutants have associated Asn aminotransferase activity with the photorespiratory enzyme, serine (Ser):glyoxylate aminotransferase. This enzyme is encoded by AGT1 in Arabidopsis thaliana. Recombinant N-terminal His-tagged AGT1 purified from Escherichia coli was characterized with Ser, alanine (Ala) and Asn as amino acid donors and glyoxylate, pyruvate and hydroxypyruvate as organic acid acceptors. The V(max) of AGT1 with Asn was higher than with Ser or Ala by ca. 5- to 20-fold. As a result, the catalytic efficiency (V(max)/K(m)) was slightly higher with Asn than with the two other amino acids. In the roots of 10-day-old seedlings treated for 2h with 20mM Asn, the AGT1 transcript levels were raised by 2-fold. During this treatment, the concentration of Asn in root was raised by ca. 5-fold. These results suggest that AGT1 is involved in Asn metabolism in Arabidopsis.

Transcripts of sulphur metabolic genes are co-ordinately regulated in developing seeds of common bean lacking phaseolin and major lectins.

The lack of phaseolin and phytohaemagglutinin in common bean (dry bean, Phaseolus vulgaris) is associated with an increase in total cysteine and methionine concentrations by 70% and 10%, respectively, mainly at the expense of an abundant non-protein amino acid, S-methyl-cysteine. Transcripts were profiled between two genetically related lines differing for this trait at four stages of seed development using a high density microarray designed for common bean. Transcripts of multiple sulphur-rich proteins were elevated, several previously identified by proteomics, including legumin, basic 7S globulin, albumin-2, defensin, albumin-1, the Bowman-Birk type proteinase inhibitor, the double-headed trypsin inhibitor, and the Kunitz trypsin inhibitor. A co-ordinated regulation of transcripts coding for sulphate transporters, sulphate assimilatory enzymes, serine acetyltransferases, cystathionine ß-lyase, homocysteine S-methyltransferase and methionine gamma-lyase was associated with changes in cysteine and methionine concentrations. Differential gene expression of sulphur-rich proteins preceded that of sulphur metabolic enzymes, suggesting a regulation by demand from the protein sink. Up-regulation of SERAT1;1 and -1;2 expression revealed an activation of cytosolic O-acetylserine biosynthesis. Down-regulation of SERAT2;1 suggested that cysteine and S-methyl-cysteine biosynthesis may be spatially separated in different subcellular compartments. Analysis of free amino acid profiles indicated that enhanced cysteine biosynthesis was correlated with a depletion of O-acetylserine. These results contribute to our understanding of the regulation of sulphur metabolism in developing seed in response to a change in the composition of endogenous proteins.

Relationship between asparagine metabolism and protein concentration in soybean seed.

The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine.

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.