Active nuclear import and cytoplasmic retention of activation-induced deaminase

The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.

Effect of environmental conditions on the N uptake route of soil microorganisms and adaption of a method to determine amino acid oxidase in soil

Soil microorganisms have evolved two possible mechanisms for their uptake of organic N: the direct route and the mobilization-immobilization-turnover (MIT) route. In the direct route, simple organic molecules are taken up via various mechanisms directly into the cell. In the MIT route, the deamination occurs outside the cell and all N is mineralized to NH4+ before assimilation. A better understanding of the mechanisms controlling the different uptake routes of soil microorganisms under different environmental conditions is crucial for understanding mineralization processes of organic material in soil. For the first experiment we incubated soil samples from the long term trial in Bad Lauchstädt with corn residues with different C to N ratios and inorganic N for 21 days at 20 °C. Under the assumption that all added amino acids were taken up or mineralized, the direct uptake route was more important in soil amended with corn residues with a wide C to N ratio. After 21 days of incubation the direct uptake of added amino acids increased in the order addition of corn residue with a: “C to N ratio of 40 & (NH4)2SO4 and no addition (control)” (69% and 68%, respectively) < “C to N ratio of 20” (73%) < “C to N ratio of 40” (95%). In all treatments the proportion of the added amino acids that were mineralized increased with time, indicating that the MIT route became more important over time. To investigate the effects of soil depth on the N uptake route of soil microorganisms (experiment II), soil samples in two soil depths (0-5 cm; 30-40 cm) were incubated with corn residues with different C to N ratios and inorganic N for 21 days at 20 °C and 60% (WHC). The addition of corn residue resulted in a marked increase of protease activity in both depths due to the induction from the added substrate. Addition of corn residue with a wide C to N ratio resulted in a significantly greater part of the direct uptake (97% and 94%) than without the addition of residues (85% and 80%) or addition of residue with a small C to N ratio (90% and 84%) or inorganic N (91% and 79% in the surface soil and subsoil, respectively), suggesting that under conditions of sufficient mineralizable N (C to N ratio of 20) or increased concentrations of NH4+, the enzyme system involved in the direct uptake is slightly repressed. Substrate additions resulted in an initially significantly higher increase of the direct uptake in the surface soil than in the subsoil. As a large proportion of the organic N input into soil is in form of proteinaceous material, the deamination of amino acids is a key reaction of the MIT route. Therefore the enzyme amino acid oxidase contribute to the extracellular N mineralization in soil. The objective of experiment III was to adapt a method to determine amino acid oxidase in soil. The detection via synthetic fluorescent Lucifer Yellow derivatives of the amino acid lysine is possible in soil. However, it was not possible to find the substrate concentration at which the reaction rate is independent of substrate concentration and therefore we were not able to develop a valid soil enzyme assay.

Effects of essential oils on ruminal microorganisms and their protein metabolism

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.

Strategies for optimizing nitrogen use by ruminants

The efficiency of N utilization in ruminants is typically low (around 25%) and highly variable (10% to 40%) compared with the higher efficiency of other production animals. The low efficiency has implications for the production performance and environment. Many efforts have been devoted to improving the efficiency of N utilization in ruminants, and while major improvements in our understanding of N requirements and metabolism have been achieved, the overall efficiency remains low. In general, maximal efficiency of N utilization will only occur at the expense of some losses in production performance. However, optimal production and N utilization may be achieved through the understanding of the key mechanisms involved in the control of N metabolism. Key factors in the rumen include the efficiency of N capture in the rumen (grams of bacterial N per grams of rumen available N) and the modification of protein degradation. Traditionally, protein degradation has been modulated by modifying the feed (physical and chemical treatments). Modifying the rumen microflora involved in peptide degradation and amino acid deamination offers an alternative approach that needs to be addressed. Current evidence indicates that in typical feeding conditions there is limited net recycling of N into the rumen (blood urea-N uptake minus ammonia-N absorption), but understanding the factors controlling urea transport across the rumen wall may reverse the balance to take advantage of the recycling capabilities of ruminants. Finally, there is considerable metabolism of amino acids (AA) in the portal-drained viscera (PDV) and liver. However, most of this process occurs through the uptake of AA from the arterial blood and not during the ‘absorptive’ process. Therefore, AA are available to the peripheral circulation and to the mammary gland before being used by PDV and the liver. In these conditions, the mammary gland plays a key role in determining the efficiency of N utilization because the PDV and liver will use AA in excess of those required by the mammary gland. Protein synthesis in the mammary gland appears to be tightly regulated by local and systemic signals. The understanding of factors regulating AA supply and absorption in the mammary gland, and the synthesis of milk protein should allow the formulation of diets that increase total AA uptake by the mammary gland and thus reduce AA utilization by PDV and the liver. A better understanding of these key processes should allow the development of strategies to improve the efficiency of N utilization in ruminants.

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4+ ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O-2(center dot-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 mu M) and Fe3+ ion (100 mu M) addition to the cell cultures had no effect, whereas Cu2+ (5.0-100 mu M) significantly increased cell death. Supplementation of the AA- and Cu2+-containing culture medium with antioxidants, such as catalase (5.0 mu M), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu2+ ions (10-50 mu M) relative to control cultures. This may imply higher activity of antioxidant enzymes C, in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) Plus Cu2+ (100 mu M) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

SYNTHESIS AND RETRO AZA DIELS-ALDER REACTION OF SOME NEW ISOQUINUCLIDINE DERIVATIVES

N-Benzyl- and N-(alpha-methoxycarbonylethyl)-2,4,6-triphenyl-1,2-dihydropyridines were submitted to Diels-Alder reactions with maleic anhydride or N-phenylmaleimide yielding, diastereoselectively, the corresponding endo-anti adducts. These novel isoquinuclidines showed to be resistant to N-alkylation or N-protonation, undergoing an unexpected fragmentation via a retro aza Diels-Alder process.

Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Enzimas termoestáveis: fontes, produção e aplicação industrial

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil

Objectives: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined.Methods: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Lowenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 by upstream from the start codon was determined by DNA sequencing of purified PCR products.Results: of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region: Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity.Conclusions: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Conformation and lytic activity of eumenine mastoparan: A new antimicrobial peptide from wasp venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Suplementação da dieta de vacas leiteiras mantidas em pastagens com virginiamicina e soja crua ou tostada

Pós-graduação em Zootecnia - FCAV

Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) and thereby potentially alter the information content and structure of cellular RNAs. Notably, although the overwhelming majority of such editing events occur in transcripts derived from Alu repeat elements, the biological function of non-coding RNA editing remains uncertain. Here, we show that mutations in ADAR1 (also known as ADAR) cause the autoimmune disorder Aicardi-Goutieres syndrome (AGS). As in Adar1-null mice, the human disease state is associated with upregulation of interferon-stimulated genes, indicating a possible role for ADAR1 as a suppressor of type I interferon signaling. Considering recent insights derived from the study of other AGS-related proteins, we speculate that ADAR1 may limit the cytoplasmic accumulation of the dsRNA generated from genomic repetitive elements.

Staphylococcus aureus thiaminase II: oligomerization warrants proteolytic protection against serine proteases

Staphylococcus aureus TenA (SaTenA) is a thiaminase type II enzyme that catalyzes the deamination of aminopyrimidine, as well as the cleavage of thiamine into 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 5-(2-hydroxyethyl)-4-methylthiazole (THZ), within thiamine (vitamin B1) metabolism. Further, by analogy with studies of Bacillus subtilis TenA, SaTenA may act as a regulator controlling the secretion of extracellular proteases such as the subtilisin type of enzymes in bacteria. Thiamine biosynthesis has been identified as a potential drug target of the multi-resistant pathogen S. aureus and therefore all enzymes involved in the S. aureus thiamine pathway are presently being investigated in detail. Here, the structure of SaTenA, determined by molecular replacement and refined at 2.7 A ° resolution to an R factor of 21.6% with one homotetramer in the asymmetric unit in the orthorhombic space group P212121, is presented. The tetrameric state of wild-type (WT) SaTenA was postulated to be the functional biological unit and was confirmed by small-angle X-ray scattering (SAXS) experiments in solution. To obtain insights into structural and functional features of the oligomeric SaTenA, comparative kinetic investigations as well as experiments analyzing the structural stability of the WT SaTenA tetramer versus a monomeric SaTenA mutant were performed.

GDH activity and ammonium excretion during growth in the marine mysid, "Leptomysis lingvura"

[EN] Ammonium (NH4+) and nitrate (NO3-) are the main constituents of the inorganic nitrogen pool that supports primary production in marine systems. NH4+ release via glutamate deamination in heterotrophic organisms represents the largest recycled nitrogen source in the euphotic zone, supporting around the 80 % of the primary producers requirements (Harrison, 1992). Glutamate dehydrogenase (GDH) is the enzyme that catalyzes this process. This fact has lead to the use of GDH activity as an index, a proxy, for physiological NH4+ formation. The result is a measure of potential excretion that avoids incubation artefacts due to manipulation of the organisms. The relationship between GDH activity and NH4+ excretion in cultures of the marine mysid Leptomysis lingvura is analyzed here. With interspecific and environmental interferences minimized, the study shows that the relationship between GDH activity and NH4+ excretion in L. lingvura is similar to equivalent results measured on mixed assemblages of zooplankton.