Determination of Carbon and Nitrogen Isotope Fractions in Asparagine, Aspartic Acid, Threonine and Methionine

The nomenclature for compounds that are modified with isotopes is growing every day. Compounds can be modified with isotopes either individually, in a functional group or groups, or completely with all atomic centers of the element. This diversity of isotope-modified compounds increases the range of researches that can be studied using them. Compounds modified with isotopes of carbon-13 or nitrogen-15 can be converted into carbon monoxide, carbon dioxide and molecular nitrogen. Currently, only the average value of carbon-13 or nitrogen-15 isotopes can be determined. However, by directly determining the atomic share of these isotopes in organic compounds modified with isotopes, information about the isotopic centers of the element can be obtained. The atomic fraction of an element is defined as a single carbon or nitrogen isotope-modified center or centers, or all centers that are isotope-modified with that element at the same time. Carbon-13 or nitrogen-15 isotopes’ atomic fraction can be determined molecularly or with fragment ions of different elemental content, or both. This makes the method self-verifying, increasing the accuracy and reliability of the results obtained. Amino acids, such as asparagine, aspartic acid, methionine, and threonine, are essential for the human body. This proposed method of isotopic analysis will increase the possibilities for scientific research using these compounds.

Oral administration of asparagine and 3-indolepropionic acid prolongs survival time of rats with traumatic colon injury

Background: Traumatic colon injury(TCI) is a common disease during wartime. Prolongation of posttraumatic survival time is an effective approach to patient outcome improvement. However, there is a lack of basic research in this field.This study aimed to elucidate the mechanisms underlying TCI progression and to develop novel regimens to buy time for TCI patients on the battlefield.Methods: A total of 669 Sprague–Dawley rats were used in this study. Surgical colon incision was performed to generate the TCI rat model. The landscape of colon microbiota compositions was depicted using 16S rRNA sequencing and metabolites in the intestinal contents were detected by metabolomics profiling. The signaling transduction in the intestinal epithelium was investigated using antibody microarrays and Western blotting. The enzyme-linked immunosorbent assay(ELISA) was conducted to measure the levels of interleukin-6 and tumor necrosis factor-α in intestines and plasma for the detection of inflammatory responses. Diamine oxidase, D-lactate and endotoxin in plasma and protein expression of zonula occludens 1 and occludin were selected as the indicators of intestinal barrier permeability. To investigate alterations of microbiota symbiosis, the relative abundances of specific bacterial genera were detected using quantitative real-time PCR(qRT-PCR).Results: As a type of lethal injury, TCI induced acute disruption of intestinal homeostasis, characterized by inflammatory responses, intestinal barrier hyperpermeability and microbiota dysbiosis(P<0.05). Significant alterations in bacterial metabolic patterns were detected with decreases in many metabolites. After a series of screenings,we found that oral administration of asparagine(Asn) and 3-indolepropionic acid(IPA) effectively prolonged posttraumatic survival time [Asn plus IPA vs. Vehicle: hazard ratio(HR)=0.105, 95%CI 0.031–0.356, P=0.0003] and restored intestinal homeostasis in TCI rats(P<0.05). Mechanistically, this combinational strategy protected the rats against TCI through synergistic activation of Akt signaling in the intestinal epithelium(P<0.05).Conclusions: Abrupt dysregulation of intestinal homeostasis plays a critical role in the progression toward TCI induced death. Oral administration of Asn plus IPA may serve as an effective regimen to restore intestinal functions and prolong the posttraumatic survival time.

Genome-wide identification and expression analysis of asparagine synthetase family in apple

Asparagine is an efficient nitrogen transport and storage carrier. Asparagine synthesis occurs by the amination of aspartate which is catalyzed by asparagine synthetase(ASN) in plants. Complete genome-wide analysis and classifications of the ASN gene family have recently been reported in different plants. However, systematic analysis and expression profiles of these genes have not been performed in apple(Malus domestica). Here, a comprehensive bioinformatics approach was applied to identify MdASNs in apple. Then, plant phylogenetic tree, chromosome location, conserved protein motif, gene structure, and expression pattern of MdASNs were analyzed. Five members were identified and distributed on 4 chromosomes with conserved GATase-7 and ASN domains. Expression analysis indicated that all MdASNs mRNA accumulated at the highest level in reproductive organs, namely flowers or fruits, which may be associated with the redistribution of free amino acids in plant metabolic organs and reservoirs. Additionally, most of Md ASNs were dramatically up-regulated under various nitrogen supplies, especially in the aboveground part. Taken together, MdASNs may be assigned to be responsible for the nitrogen metabolism and asparagine synthesis in apple.

Asparagine Synthetase Is Partially Localized to the Plasma Membrane and Upregulated by L-asparaginase in U937 Cells

This study investigated the intracellular localization of asparagine synthetase （ASNS） in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively. Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively. U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining. Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface. Moreover, Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells. Immunofluorescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells, except for its distribution in the cytosol. In addition, ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells. It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.

An Interspecies Conserved Antigen of Plasmodium falciparum Containing Clusters of Asparagine Residues

An interspecies conserved Plasmodium asparagine rich antigen, designated as ARK26, was isolated by immunoscreening P.falciparum genomic DNA expression library with mouse convalescent anti P.yeolii serum. Partial DNA sequence analysis reveals that ARK26 contains clusters of asparagines and no randomly repeated amino acid sequence motifs are observed. A 65 ×10 3 GST fusion protein is expressed by recombinant plasmid PGEX 5X 1(ARK26) in E.coli C strain ABLE K. Computer programs predict that two asparagine rich regions are among the possible antigenic epitopes of p37 encoded by ARK26. Interestingly, the sequence of ARK26 displays significant similarity to yeast and several other species' mitochondrial genes, and its possible function is discussed.

Kinetics of the thermal disappearance of radicals formed during the radiolysis of L-α-anhydrous asparagine

An EPR study of paramagnetic centers formed by irradiation of polycrystalline L-α-anhydrous asparagine (L-Asn) was performed. The EPR spectra of gamma irradiated samples at room temperature, shown the presence of three types of paramagnetic centers. A possible mechanisms of formation for the three radical species is suggested, based also on literature data. The kinetics of the disappearance of radicals during thermal annealing indicated a complex mechanism.

EXPERIMENTAL STUDIES OF YOUNG G1NKGO EMBRYOS——THE EFFECT OF CASEIN HYDROLYSATE, GLUTAMINE, ASPARAGINE, 2,4-D, AND ADENINE SULPHATE ON EMBRYOS GROWN IN VITRO

In the investigations of young embryos and other reproductive organs cultured in vitro, casein hydrolysate, glutamine, and asparagine have been used as supplementing growth substances. 2,4-D has been used to induce callus from rye embryos cultured in vitro. Adenine sulphate also has been used in embryo culture. The present report deals with the effects of casein hydrolysate, glutamine, asparagine, 2,4-D, and adenine sulphate on young Ginkgo embryos. In the dissection and disinfection of the material and the preparation and sterilization of basic medium the methods used this

Concomitant NH_4^+ Secretion During Astaxanthin Synthesis in Haematococcus pluvialis Under High Irradiance and Nitrogen Defi-cient Conditions

The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin synthesis in the cultures of high light and nitrogen-free （HF）, high light and nitrogen-repletion （HR）, and low light and nitrogen-free （LF）, （1） endopeptidase （EP） activities increased along with decrease in protein content, （2） asparagine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, （3） ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, （LR）, the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas （02 and N2）, astaxan- thin content increased as the protein level decreased. These results strongly suggest that （1） the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, （2） endopeptidase was involved in the degradative process, （3） for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.

Novel synthesis of a strained para-cyclophane derivative

A [P. Rajakumar, A.M.A. Rasheed, Tetrahedron 61（22） （2005） 5351] para-cyclophane derivative was synthesized via intramolecular esterification of a dipeptide surrogate containing asparagine to form tetrahydropyrimidinone ring. The structures of the product and intermediates were characterized by 1H NMR, 13C NMR and mass spectrum.

Influence of Amino Acids on the Secondary Somatic Embryogenesis Proliferation Process of Moroccan Cork Oak (<i>Quercus suber</i>L.)

The present study aims to explore the regeneration potential of Moroccan cork oak through the secondary somatic embryogenesis process. Particularly, we focus on the analysis of amino acids influence on the quantity and quality of the regenerated secondary embryos. The amino acids tested are: Glutamine, asparagine, arginine, tryptophane, methionine, casein hydrolysate and urea. Each amino acid is added in the Margara (N30K) medium at different concentrations ranged between 10 and 500 mg/l. The results are collected after 2 months of culture. First analysis shows that the glutamine and the casein hydrolysate gives a maximum number of somatic embryos, clusters and pre-embryos newly formed on the clusters. By comparison to the control medium, the increase of the secondary embryos number directly formed exceed 36% in the case of casein hydrolysate and 35% of in the case of glutamine both at the concentration of 30 mg/l. However, the test of the combination of these amino acids did not have any significant results. In terms of quality, the influence of amino acids on the morphology of secondary embryos was analyzed.

Cloning and Analysis of TasA Gene in Bacillus subtilis HAS

In order to clarify the mechanism for the inhibitory action of the Bacillus subtilis strain HAS on Sporisorium scitamineum （ Syd. ）, which can cause sugarcane smut, the full-length TasA gene which encodes a protein with broad-spectrum antimicrobial activity, was PCR-amplified from HAS, and cloned into pMD18-T vector. Sequence analysis indicated that the full-length TasA cloned from HAS consisted of 786 nucleotides, and shared 99% homology in nucleotide sequence with the TasA gene sequence published in Genbank （AJ871386.1）. It was predicted that the molecular weight of TasA protein was about 28 kD. Base transitions or transversions ~curred at positions 104, 164, 169,250, 399,623 and 627, at the 2nd, 2nd, 3rd , 1st , 3rd, 2nd and 3rd bases of TasA codons. The mutations in the seven bases may cause the missense mutations of the polypeptide chain. Compared with the amino acid sequences of the TasA protein encoded by Bacillus aubtilis subsp, subtilis str. 168, mutations in two amino acids at positions 150 and 209 of the protein encoded by the cloned TasA gene were found, and as a result, an ala- nine was replaced with a threonine.

Hydrothermally Synthesized NanobioMOFs, Evaluated by Photocatalytic Hydrogen Generation

Three new materials, nanobioMOFs (cobalt argeninate, cobalt asparaginate and cobalt glutaminate) have been hydrothermally synthesized. Nano sized morphology of all these materials have been obtained by scanning electron microscopic technique. Mass spectrometric studies of all these materials have been conducted for determination of their molar masses. All these nanobioMOFs have been found to exhibit photocatalytic hydrogen generation in pure water upon irradiation at wavelengths longer than 650 nm. The amounts of quantum yield of hydrogen generation at 650 nm in water was 4.5%, 4.0% and 3.5% for cobalt argeninate, cobalt asparaginate and cobalt glutaminate respectively. The apparently higher yield of hydrogen generation from these amine functionalized nanobioMOFs can direct to the development of more nano sized functionalized MOFs for water splitting.

C/EBPβ/AEP Signaling Drives Alzheimer's Disease Pathogenesis

Alzheimer’s disease(AD)is the most common type of dementia.Almost two-thirds of patients with AD are female.The reason for the higher susceptibility to AD onset in women is unclear.However,hormone changes during the menopausal transition are known to be associated with AD.Most recently,we reported that follicle-stimulating hormone(FSH)promotes AD pathology and enhances cognitive dysfunctions via activating the CCAAT-enhancer-binding protein(C/EBPβ)/asparagine endopeptidase(AEP)pathway.This review summarizes our current understanding of the crucial role of the C/EBPβ/AEP pathway in driving AD pathogenesis by cleaving multiple critical AD players,including APP and Tau,explaining the roles and the mechanisms of FSH in increasing the susceptibility to AD in postmenopausal females.The FSH-C/EBPβ/AEP pathway may serve as a novel therapeutic target for the treatment of AD.

Identification of important precursors and theoretical toxicity evaluation of byproducts driving cytotoxicity and genotoxicity in chlorination

Chlorination,the most widely used disinfection process for water treatment,is unfortunately always accompanied with the formation of hazardous disinfection byproducts(DBPs).Various organic matter species,like natural organic matter(NOM)and amino acids,can serve as precursors of DBPs during chlorination but it is not clear what types of organic matter have higher potential risks.Although regulation of DBPs such as trihalomethanes has received much attention,further investigation of the DBPs driving toxicity is required.This study aimed to identify the important precursors of chlorination by measuring DBP formation from NOM and amino acids,and to determine the main DBPs driving toxicity using a theoretical toxicity evaluation of contributions to the cytotoxicity index(CTI)and genotoxicity index(GTI).The results showed that NOM mainly formed carbonaceous DBPs(CDBPs),such as trichloromethane,while amino acids mainly formed nitrogenous DBPs(N-DBPs),such as dichloroacetonitrile(DCAN).Among the DBPs,DCAN had the largest contribution to the toxicity index and might be the main driver of toxicity.Among the precursors,aspartic acid and asparagine gave the highest DCAN concentration(200 g/L)and the highest CTI and GTI.Therefore,aspartic acid and asparagine are important precursors for toxicity and their concentrations should be reduced as much as possible before chlorination to minimize the formation of DBPs.During chlorination of NOM,tryptophan,and asparagine solutions with different chlorine doses and reaction times,changes in the CTI and GTI were consistent with changes in the DCAN concentration.

Increased β-Cyanoalanine Nitrilase Activity Improves Cyanide Tolerance and Assimilation in Arabidopsis

Plants naturally produce cyanide （CN） which is maintained at low levels in their cells by a process of rapid assimilation. However, high concentrations of environmental CN associated with activities such as industrial pollution are toxic to plants. There is thus an interest in increasing the CN detoxification capacity of plants as a potential route to phytoremediation. Here, Arabidopsis seedlings overexpressing the Pseudomonas fluorescens β-cyanoalanine nitrilase pinA were compared with wild-type and a β-cyanoalanine nitrilase knockout line （△Atnit4） for growth in the presence of exogenous CN. After incubation with CN, ＋PfpinA seedlings had increased root length, increased fresh weight, and decreased leaf bleaching compared with wild-type, indicating increased CN tolerance. The increased tolerance was achieved without an increase in β-cyanoalanine synthase activity, the other enzyme in the cyanide assimilation pathway, suggesting that nitrilase activity is the limiting factor for cyanide detoxification. Labeling experiments with [^13C] KCN demonstrated that the altered CN tolerance could be explained by differences in flux from CN to Asn caused by altered β-cyanoalanine nitrilase activity. Metabolite profiling after CN treatment provided new insight into downstream metabolism, revealing onward metabolism of Asn by the photorespiratory nitrogen cycle and accumulation of aromatic amino acids.

Source of inorganic N affects the cost of growth in a legume tree species(Virgilia divaricata)from the Mediterrean-type Fynbos ecosystem

Aims In Mediterranean-type ecosystem,the Cape Fynbos,legumes may be able to switch between soil N and atmospheric N_(2) sources during growth to adjust the carbon costs of N acquisition.This study investigated the utilization of different inorganic N sources by Virgilia divaricata,a native legume from the Mediterranean-type ecosystem of the Cape Floristic Region.Methods Plants were cultivated in sterile quartz sand,supplied with 25%strength Long Ashton nutrient solution,modified to contain 500μM Phosphate.At the phosphate level(500μM),plants were treated with 500μM NH_(4)NO_(3)(treatment named N),or grown in N-free nutrient solution and inoculated with effective Burkholderia sp.(Bact.)or treated with combined N sources(500μM NH_(4)NO_(3))and inoculated with effective Burkholderia sp.(N+Bact.).Important Findings The application of NH_(4)NO_(3) to the legumes resulted in a greater increase in plant dry matter.Carbon construction costs were higher in plants that were supplied with mineral and symbiotic N sources.Maximum photosynthetic rates per leaf area was maintained,irrespective of the N sources.Although the plant roots were nodulated,the plant dependence on N_(2) fixation decreased with addition of N.Roots and nodules of the plants solely reliant on N_(2) fixation showed an increase in glutamine content.These results show that V.divaricata is highly adapted for growth at the forest margin.Fynbos and possibly anthropic soils by utilizing both atmospheric and soil N sources.