Effects of Agricultural Practices on Carbon Emission and Soil Health

The agricultural sector is a source of greenhouse gas emissions that directly affect the global problem of climate change and contribute approximately 11% in total greenhouse gas emissions in the world and in Croatia too. Irregular and irresponsible agricultural practices, such as excessive tillage and improper fertilization often lead to soil carbon loss and increased carbon dioxide emissions to the atmosphere. This field study provides results how agricultural practices affect carbon dioxide emissions from soil, carbon sequestration and soil quality during the cultivation of winter wheat. The field experiment was conducted in a temperate continental climate on distric Stagnosol. Four investigated treatments were: organic fertilization, mineral fertilization, control treatment and black fallow. The lowest carbon dioxide emission was recorded on bare soil and the highest on organic fertilization treatment. The application of manure, mineral fertilizers and calcification rendered significant effect on some soil chemical characteristics and daily carbon dioxide flux

Rolle der N-terminalen Domänen (NTDs) bei der Assemblierung und Funktion von N-Methyl-D-Aspartat (NMDA)-Rezeptoren

N-Methyl-D-Aspartat-Rezeptoren (NMDARs) gehören zusammen mit den non-NMDA-Rezeptoren (AMPA- und Kainat-Rezeptoren) zu der Familie der ionotropen Glutamatrezeptoren (iGluRs). NMDARs sind wichtig für die Weiterleitung von exzitatorischen Signalen zwischen Neuronen, Prozesse wie Lernen und Gedächtnisbildung sowie die Pathophysiologie von neurologischen Erkrankungen. Das gemeinsame Merkmal der iGluRs ist ihr modularer Aufbau, wobei die jeweiligen Domänen eine vergleichbare Struktur sowie Funktion zu besitzen scheinen. Für die extrazellulär angeordneten N-terminalen Domänen (NTDs) wird angenommen, dass sie bei allen iGluRs die Determinanten der Assemblierung sind und bei den NMDARs zusätzlich die Modulation der Rezeptorfunktion bedingen. Der tetramere NMDAR ist aus homologen NR1-, NR2A-D- und NR3A-B-Untereinheiten aufgebaut. Der sogenannte ´konventionelle´ NMDAR setzt sich aus den Glyzin-bindenden NR1- und Glutamat-bindenden NR2-Untereinheiten zusammen. Die Ligandenbindungsdomänen (LBDs) dieser Untereinheiten bilden zweiblättrige Strukturen aus, welche in einer ´Rücken-an-Rücken´-Konformation angeordnet sind. Dadurch entstehen intermolekulare Wechselwirkungen im NR1/NR2-Hetero-Dimer, deren Stabilität das Ausmaß der NMDAR-Aktivierung und der Desensitisierung bestimmt. Aufgrunddessen wird das Hetero-Dimer als die funktionelle Einheit im Rezeptor angesehen. Beim NR1/NR3-Rezeptor, welcher durch Gylzin allein aktivierbar ist, bewirkt die Ligandierung der NR3-LBD die Aktivierung des Rezeptors. Die anschließende Ligandierung der NR1-LBD führt dagegen zur Inaktivierung, d.h. zur Auto-Inhibition des NR1/NR3-Rezeptors, deren Determinanten noch unbekannt sind. Das Ziel der vorliegenden Arbeit war es die Rolle der NTDs bei der Assemblierung, Stöchiometrie und Funktion von NMDARs zu untersuchen. Für die biochemische Analyse dieser Fragestellung wurden die NMDARs rekombinant in Xenopus laevis Oozyten exprimiert und mit [35S]-Methionin bzw. Cy5 markiert. Anschließend folgte die affinitätschromatographische Aufreinigung über die Hexahistidyl-Markierung von einer der exprimierten Untereinheiten sowie die Analyse anhand der blau-nativen- und der SDS-Polyacrylamid-Gelelektophorese. In der vorliegenden Arbeit konnte gezeigt werden, dass bei NMDARs die NTD-Deletion keine Auswirkung auf die Assemblierung, die Stöchiometrie oder die Zelloberflächen-Expression hatte. Zusammenfassend sind, im Unterschied zu den non-NMDARs, bei NMDARs die NTDs nicht für die Ausbildung funktioneller Rezeptoren notwendig. Die funktionelle Analyse der Glyzin-vermittelten Rezeptorströme von Wildtyp- und NTD-deletierten NR1/NR3A- und NR1/NR3B-Rezeptoren anhand der Zwei-Elektroden Spannungsklemme zeigte, dass die NTD-Deletion zur Aufhebung der NR1-LBD-vermittelten Auto-Inhibition führt. Somit bestimmen die NTDs das Ausmaß der Rezeptoreffizienz. Dieser Befund korreliert mit der Verringerung der Rezeptordesensitisierung bei NR1/NR3A-Rezeptoren. Für das Auftreten beider Effekte war schon die selektive NR3-NTD-Deletion ausreichend. NR1/NR3A/NR3B-Rezeptoren zeichnen sich durch große Glyzin-induzierte Rezeptorströme auf, die nicht auto-inhibiert werden. Im Einklang damit, hatte die NTD-Deletion keine Auswirkung auf die Rezeptoreffizienz. Die Aufhebung der Auto-Inhibition bei NR1/NR3A/NR3B-Rezeptoren und das Vorhandensein dieser bei NR1/NR3A- bzw. NR1/NR3B-Rezeptoren können auf Unterschiede in den Wechselwirkungen zwischen den funktionellen Einheiten zurückgeführt werden. Anders als bei NR1/NR3A- und NR1/NR3B-Rezeptoren, agiert bei NR1/NR3A/NR3B-Rezeptoren Zn2+ als ein hoch-affiner allosterischer Inhibitor von Glyzin-induzierten Strömen. Die Mutation der NR1-Glyzinbindetasche bedingt zwar ein geringeres Ausmaß der Zn2+-Inhibition, jedoch nicht deren Aufhebung. Somit besitzt Zn2+ mindestens eine weitere Bindestelle im NR1/NR3A/NR3B-Rezeptor. Die hier erhaltenen Daten zeigen auf, dass es andere Determinanten für die NMDAR-Assemblierung als die NTDs geben muss und diese sich somit in den verbleibenden NMDAR-Domänen befinden. Die Erkenntnisse über die NTD-bedingte Modulation der NR1/NR3-NMDA-Rezeptorfunktion können bei zukünftigen Studien als ein Mittel dienen, diese Rezeptoren in vivo, z.B. durch den Einsatz von spezifischen Proteasen, zu detektieren. Das Wissen der Eigenschaften von funktionellen Einheiten kann für die Aufklärung der vermeintlich konträr agierenden Hetero-Dimere in NR1/NR2/NR3-Rezeptoren, die bereits in vivo detektiert wurden, von großer Bedeutung sein

The N-terminal domains of both NR1 and NR2 subunits determine allosteric Zn2+ inhibition and glycine affinity of N-methyl-D-aspartate receptors.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric protein composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking, and function. Here, we investigated how deletion of the NR1- and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/NR2 receptors and coexpression of the NTD-deleted NR1 subunit with wild-type or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn2+ and ifenprodil, consistent with the idea that interdomain interactions between these domains are important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly

SOIL SAMPLING WITH NEW SOIL SAMPLING PROBE

Abstract: Conventional soil sampling usually considers sample weight of 2 kg per 4-5 ha area, which means that representative sample in relation to soil mass up to 30 cm depth, is presented through the ratio 1:10,000,000. In order to increase the volume of sampled soil and thus to increase the accuracy of sampling, we have designed new circular soil probe apparatus that can take 16 soil samples in one run (PCT/HR2011/000021 Rotary Soil Sampling Assembly). Samples are distributed in a 50 cm radius from the center of the probe. After sampling all 16 samples are mixed and homogenized in one composite sample, which is used for chemical soil analyses. We have tested new soil sampling probe at the field of 84 ha. Sampling was performed with grid sampling at intersections in a grid of 50 x 50 m. Results of soil pH and plant available phosphorus are based on 330 soil samples and they provide sound basis for recommendations for variable liming and phosphorus fertilization rates. Results were processed with ordinary kriging

Formation of NR1/NR2 and NR1/NR3 heterodimers constitutes the initial step in N-methyl-D-aspartate receptor assembly.

N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine- and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo- and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry

The N-terminal domain of the GluN3A subunit determines the efficacy of glycine-activated NMDA receptors.

N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1(ΔNTD)) and GluN3 (GluN3(ΔNTD)) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3A(ΔNTD) receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, whereas the glycine-induced currents of GluN1(ΔNTD)/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3A(R319C) receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function

Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function.

Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity.

N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A-D) and two GluN3 (A-B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics