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Catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase-amidase containing microbial whole-cell catalyst under mild conditions, enantioselective desymmetrizations of meso-cyclopentane-1,3-dicarbonitriles and cyclopentane-1,3-dicarboxamides were studied. Although the nitrile hydratase was found to exhibit high enzymatic activity, but low 1 R enantioselectivity toward dinitriles, a number of 2,2-unsymmetrically substituted meso-cyclopentane-1,3-dicarboxamide substrates were converted by the 1 S enantioselective amidase into quaternary carbon-bearing enantiopure (1 S,2 R,3 R)-3-carbamoylcyclopentanecarboxylic acids in yields up to 94 %. The application of the method was demonstrated by convenient and practical transformations of the resulting (1 S,2 R,3 R)-2-allyl-3-carbamoylcyclopentanecarboxylic acid derivatives into functionalized cyclopentane-fused δ-lactam and δ-lactone compounds. [ABSTRACT FROM AUTHOR]

Objectives To identify β-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals. Methods A deletion mutant of the putative class A β-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth. Results The B. thetaiotaomicron BT_4507 gene encodes a β-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated β-lactamases that could degrade cefotaxime. β-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime. Conclusions The production of membrane vesicles carrying surface-associated β-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against β-lactam antibiotics. [ABSTRACT FROM PUBLISHER]

Histone deacetylase inhibitors have gained a great deal of attention recently for the treatment of cancers and inflammatory diseases. So design of new inhibitors is of great importance in pharmaceutical industries and labs. Creating pharmacophor models in order to design new molecules or search a library for finding lead compounds is of great interest. This approach reduces the overall cost associated with the discovery and development of a new drug. Here we elaborated an exact pharmacophore model for histone deacetylase inhibitors by using pharmacophore query and docking study. The data set used for the modelling exercise comprised of 383 molecules collated from the original literature. These molecules were used to crating the model and docking study was held with Zolinza, the recently FDA approved drug as potent histone deacetylase inhibitor. Our model consists of 5 features: Hydrogen bond donors, Hydrogen bond acceptors, H-bond donor/acceptors, Aromatic ring centers, and hydrophobic centers. With the aid of this pharmacophore model and docking result, 3D searches in large databases can be performed, leading to a significant enrichment of active analogs. [ABSTRACT FROM AUTHOR]

Purpose: To describe clinical and CT features of L-asparaginase-associated pancreatitis (L-AP) and to correlate CT grades with clinical parameters. Methods: A total of 16 children (M:F = 9:7; mean age, 8.1 years) who developed L-AP after L-asparaginase (L-asp) treatment and underwent abdominal CT scan were included. We retrospectively reviewed clinical data (age, sex, signs, and symptoms related to pancreatic toxicity and its complications, the number of L-asp doses receiving before L-AP); laboratory test results (serum amylase, lipase, C-reactive protein (CRP), calcium, blood urea nitrogen (BUN), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), glucose, and serum albumin); and clinical course (the number of days of hospitalization, number of NPO days, use of nasogastric tube, intravenous (IV) narcotics, total parenteral nutrition (TPN) or any surgical intervention). We also reviewed CT images and modified CT severity index (MCSI) for grading the severity of AP and classified to three groups (mild, moderate, and severe) or two groups (low and high score) according to MCSI. Results: L-AP typically occurred early in the course of therapy. Use of IV narcotics ( P = .014) and peak amylase ( P = .009) showed a significant difference between mild and severe L-AP groups according to MCSI. Between the low and high score groups, Use of IV narcotics ( P = .046), BUN ( P = .039), and peak amylase level ( P = .013) was significantly different. However, the L-asp dose, hospital day, and other clinical date associated with prognosis did not show any significant difference. Conclusion: In L-AP with pediatric ALL patients, MCSI may correlate with usage of IV narcotics, BUN, and peak amylase levels. [ABSTRACT FROM AUTHOR]

The expression of a new acylamidase gene from R. erythropolis TA37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4′-nitroacetanilide, N-isopropylacrylamide, N′N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrile hydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system. [ABSTRACT FROM AUTHOR]

Allantoate degradation is an essential step for recycling purine-ring nitrogen in all plants, but especially in tropical legumes where the ureides allantoate and allantoin are the main compounds used to store and transport the nitrogen fixed in nodules. Two enzymes, allantoate amidohydrolase (AAH) and allantoate amidinohydrolase (allantoicase), could catalyze allantoate breakdown, although only AAH-coding sequences have been found in plant genomes, whereas allantoicase-related sequences are restricted to animals and some microorganisms. A cDNA for AAH was cloned from Phaseolus vulgaris leaves. PvAAH is a single-copy gene encoding a polypeptide of 483 amino acids that conserves all putative AAH active-site domains. Expression and purification of the cDNA in Nicotiana benthamiana showed that the cloned sequence is a true AAH protein that yields ureidoglycine and ammonia, with a Km of 0.46 m M for allantoate. Optimized in vitro assay, quantitative RT-PCR and antibodies raised to the PvAAH protein were used to study AAH under physiological conditions. PvAAH is ubiquitously expressed in common bean tissues, although the highest transcript levels were found in leaves. In accordance with the mRNA expression levels, the highest PvAAH activity and allantoate concentration also occurred in the leaves. Comparison of transcript levels, protein amounts and enzymatic activity in plants grown with different nitrogen sources and upon drought stress conditions showed that PvAAH is regulated at posttranscriptional level. Moreover, RNAi silencing of AAH expression increases allantoate levels in the transgenic hairy roots, indicating that AAH should be the main enzyme involved in allantoate degradation in common bean. [ABSTRACT FROM AUTHOR]

High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5- f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy- N′-phenyloctandiamide. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of L-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. L-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of L-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from L-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L L-lysine in 12 h. Because L-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate. [ABSTRACT FROM AUTHOR]

Background: Dihydropyrimidinase Related Proteins (DRPs) have known homologous to the Collapsing Response Mediator Proteins (CRMPs). The DRP gene family has comprised four members, DRP 1, 2, 3, and 4, all out of which have considered to be involved in axonal outgrowth and path-finding. Methods: The protein has extracted from tumor, normal brain tissues, and then the protein purity has evaluated by Bradford test and spectrophotometric methods. In this study, proteins has separated by Two-Dimensional Gel (2DG) electrophoresis method and then spots have analyzed and compared using statistical data and specific software (Progenesis Same Spots). Spots have identified by pH isoelectric, molecular weights and data banks. Results: The 2D gel has shown 800 spots totally. Two spots have reported for DRP2, and one spot has reported for DRP3 in the human brain proteome, that have differed in pH isoelectric, and Molecular Weights values. Conclusion: This protein family has involved in neuronal differentiation and axonal guidance, and abundantly influenced in the developing brain, but their expression persisted into adulthood. DRP2 has regulated by phosphorylation, Glycogen synthase kinase 3, regulate phosphorylation of DRP2 an inactive from, and induced neuronal polarity. [ABSTRACT FROM AUTHOR]

A novel soft-hard cooperative approach was developed to synthesize bioactive mesoporous composite by pre-wrapping Penicillin G amidase with poly(acrylaimde) nanogel skin and subsequently incorporating such Penicillin G amidase nanocapsules into hierarchically mesoporous silica. The as-received bioactive mesoporous composite exhibited comparable activity and extraordinarily high stability in comparison with native Penicillin G amidase and could be used repetitively in the water-medium hydrolysis of penicillin G potassium salt. Furthermore, this strategy could be extended to the synthesis of multifunctional bioactive mesoporous composite by simultaneously introducing glucose oxidase nanocapsules and horseradish peroxidase nanocapsules into hierarchically mesoporous silica, which demonstrated a synergic effect in one-pot tandem oxidation reaction. Improvements in the catalytic performances were attributed to the combinational unique structure from soft polymer skin and hard inorganic mesoporous silica shell, which cooperatively helped enzyme molecules to retain their appropriate geometry and simultaneously decreased the enzyme-support negative interaction and mass transfer limitation under heterogeneous conditions. [ABSTRACT FROM AUTHOR]

Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaHPO-NaHPO) with acetamide as the substrate and showed activity over the pH range of 4-8 and the temperature range of 4-95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and l-glutamine. Enzyme activity was induced by 1 mM Ca, 1 mM Al, and 1-10 mM Mg, but strongly inhibited by Zn, Cu, Ni, and Fe. The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5 % methanol nor 5 % isopropanol had any significant effect on AmiE activity (99 and 96 % retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78 % identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site. [ABSTRACT FROM AUTHOR]

Polyamidase was able to hydrolyze the amide bond of insoluble polymer. In the present study, a polyamidase from Nocardia farcinica CGMCCC4.1166 was cloned and expressed in E. coli BL21(DE3). The recombinant polyamidase was purified to homogeneity, through a combination of chromatography of anion exchange, and hydrophobic interaction. The purified enzyme was characterized in detail. The optimum temperature of the enzyme was 50°C, and it was stable below 40°C. The enzyme had an optimum pH of 8.0, with pH stability between pH 7.0 and 9.0. The enzyme does not need metal ion as cofactor. In addition, when the enzyme was utilized to hydrolyze polyamide, the monomeric product of adipic acid was verified by HPLC analysis; as well, the wettability and dyeability of polyamide fabric after enzyme treatment were significantly improved, which differed from those of its inactive S173A mutant, and the amidase from Rhodococcus pyridinivorans. Furthermore, the structural features near the active site of polyamidase, different from other amidases, were explored. [ABSTRACT FROM AUTHOR]

Species of the genus Variovorax are often isolated from nitrile or amide-containing organic compound-contaminated soil. However, there have been few biological characterizations of Variovorax and their contaminant-degrading enzymes. Previously, we reported a new soil isolate, Variovorax boronicumulans CGMCC 4969, and its nitrile hydratase that transforms the neonicotinoid insecticide thiacloprid into an amide metabolite. In this study, we showed that CGMCC 4969 is able to degrade acrylamide, a neurotoxicant and carcinogen in animals, during cell growth in a mineral salt medium as well as in its resting state. Resting cells rapidly hydrolyzed 600 mg/L acrylamide to acrylic acid with a half-life of 2.5 min. In in vitro tests, CGMCC 4969 showed plant growth-promoting properties; it produced a siderophore, ammonia, hydrogen cyanide, and the phytohormone salicylic acid. Interestingly, in soil inoculated with this strain, 200 mg/L acrylamide was completely degraded in 4 days. Gene cloning and overexpression in the Escherichia coli strain Rosetta (DE3) pLysS resulted in the production of an aliphatic amidase of 345 amino acids that hydrolyzed acrylamide into acrylic acid. The amidase contained a conserved catalytic triad, Glu59, Lys 134, and Cys166, and an 'MRHGDISSS' amino acid sequence at the N-terminal region. Variovorax boronicumulans CGMCC 4969, which is able to use acrylamide for cell growth and rapidly degrade acrylamide in soil, shows promising plant growth-promoting properties. As such, it has the potential to be developed into an effective Bioaugmentation strategy to promote growth of field crops in acrylamide-contaminated soil. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal, and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates. A predicted N-acetylmuramoyl- l-alanine amidase or MurNAc-LAA (also known as peptidoglycan aminohydrolase, NAMLA amidase, NAMLAA, amidase 3, and peptidoglycan amidase; EC 3.5.1.28) was identified that would hydrolyze the amide bond between N-acetylmuramoyl and l-amino acids in certain cell wall glycopeptides. The gene encoding this protein was subsequently cloned from genomic DNA of a C. perfringens isolate by polymerase chain reaction, and the gene product (PlyCpAmi) was expressed to determine if it could be utilized as an antimicrobial to control the bacterium. By spot assay, lytic zones were observed for the purified amidase and the E. coli expression host cellular lysate containing the amidase gene. Turbidity reduction and plate counts of C. perfringens cultures were significantly reduced by the expressed protein and observed morphologies for cells treated with the amidase appeared vacuolated, non-intact, and injured compared to the untreated cells. Among a variety of C. perfringens strains, there was little gene sequence heterogeneity that varied from 1 to 21 nucleotide differences. The results further demonstrate that it is possible to discover lytic proteins encoded in the genomes of bacteria that could be utilized to control bacterial pathogens. [ABSTRACT FROM AUTHOR]

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram-negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N-acetylmuramyl-L-alanine amidases AmiA/ B/ C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 Å crystal structure of AmiC which includes the first report of an AMIN domain structure, a β-sandwich of two symmetrical four-stranded β-sheets exposing highly conserved motifs on the two outer faces. We show that this N-terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan-binding domain. The C-terminal catalytic domain shows an auto-inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity. [ABSTRACT FROM AUTHOR]

Clofarabine was the latest new drug to be approved, in 2004, for relapsed or refractory acute lymphoblastic leukaemia ( ALL). To investigate its value in the frontline treatment of ALL we applied clofarabine 5 × 40 mg/m2 in combination with pegylated asparaginase ( PEG- ASP) 1 × 2500 iu/m2 in high risk ALL patients as a novel post-induction element in the German Co-operative Study Group for treatment of ALL (Co ALL) trial 08-09. Newly diagnosed ALL patients, defined by a significant minimal residual disease ( MRD) load at the end of induction (B-progenitor ALL at day 29 ≥ 10−4 and T- ALL at day 43 ≥ 10−3) were eligible for this phase II trial. All other patients received the standard treatment consisting of high-dose cytarabine ( HIDAC) 4 × 3 g/m² in combination with Peg- ASP 2500 iu/m². Forty-two patients (39 B-progenitor; 3 T- ALL) fulfilled the criteria, were stratified and received the clofarabine/ PEG- ASP treatment resulting in 24/39 (61%) MRD-negative B-progenitor patients compared to 18/39 (46%) after HIDAC/ PEG- ASP in Co ALL 07-03. Overall, the toxicity profile of clofarabine/ PEG- ASP was similar to HIDAC/ PEG- ASP without unexpected severe side effects. Clofarabine combined with PEG- ASP is safe and effective in the frontline treatment of ALL. A prospective, randomized trial is warranted to evaluate the antileukaemic efficacy of clofarabine versus HIDAC combined with PEG- ASP. [ABSTRACT FROM AUTHOR]

Development of isoform-selective histone deacetylase (HDAC) inhibitors is important in elucidating the function of individual HDAC enzymes and their potential as therapeutic agents. Among the eleven zinc-dependent HDACs in humans, HDAC6 is structurally and functionally unique. Here, we show that a hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalytic activity in vivo and in vitro. HPOB causes growth inhibition of normal and transformed cells but does not induce cell death. HPOB enhances the effectiveness of DNA-damaging anticancer drugs in transformed cells but not normal cells. HPOB does not block the ubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitor HPOB has therapeutic potential in combination therapy to enhance the potency of anticancer drugs. [ABSTRACT FROM AUTHOR]

The Gram-negative bacteria type VI secretion system (T6SS) has been found to play an important role in interbacterial competition, biofilm formation and many other virulence-related processes. The bacteria harboring T6SS inject the effectors into their recipient’s cytoplasm or periplasm to kill them and meanwhile, to avoid inhibiting itself, the cognate immunity proteins were produced to acts as the effector inhibitor. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity (EI) pairs. We have recently solved the structures of StTae4-Tai4 and EcTae4-Tai4 complexes from the human pathogens Salmonella typhimurium and Enterobacter cloacae, respectively. It is very interesting and important to discover whether there is cross-neutralization between St- and EcTai4 and whether their effector inhibition mechanism is conserved. Here, we determined the crystal structure of StTae4 in complex with EcTai4. The solution conformation study revealed it is a compact heterotetramer that consists of an EcTai4 homodimer binding two StTae4 molecules in solution, different from that in crystal. A remarkable shift can be observed in both the flexible winding loop of StTae4 and protruding loop of EcTai4 and disulfide bonds are formed to stabilize their overall conformations. The in vitro and in vivo interactions studies showed EcTai4 can efficiently rescue the cells from the toxicity of its cognate effectors StTae4, but can not neutralize the toxic activities of the effectors from other families. These findings provide clear structural evidence to support the previous observation of cross-immunity within T6SS families and provide a basis for understanding their important roles in polymicrobial environments. [ABSTRACT FROM AUTHOR]

Dihydroorotases are universal proteins catalyzing the third step of pyrimidine biosynthesis. These zinc metalloenzymes belong to the superfamily of cyclic amidohydrolases, comprising also other enzymes that are involved in degradation of either purines (allantoinases), pyrimidines (dihydropyrimidinases) or hydantoins (hydantoinases). The evolutionary relationships between these mechanistically related enzymes were estimated after designing a method to build an accurate multiple sequence alignment. The amino acid sequences that have been crystallized were used to build a seed alignment. All the remaining homologues were progressively added by aligning their HMM profiles to the seed HMM profile, allowing to obtain a reliable phylogeny of the superfamily. This helped us to propose a new evolutionary classification of dihydroorotases into three major types, while at the same time disentangling an important part of the history of their complex structure-function relationships. Although differing in their substrate specificity, allantoinases, hydantoinases and dihydropyrimidinases are found to be phylogenetically closer to DHOase Type I than the proximity of the three DHOase types to each other. This suggests that the primordial cyclic amidohydrolase was a multifunctional, highly evolvable generalist, with high conformational diversity allowing for promiscuous activities. Then, successive gene duplications allowed resolving the primordial substrate ambiguity in various substrate specificities. The present-day superfamily of cyclic amidohydrolases is the result of the progressive divergence of these ancestral paralogous copies by descent with modification. [ABSTRACT FROM AUTHOR]

Background:Soft tissue sarcomas (STS) are rare tumours for which treatment options are limited in the advanced setting. Histone deacetylase inhibitors have shown activity in preclinical models of STS.Methods:We conducted a single-arm, open-label, multicentre phase II study to assess the efficacy and tolerability of panobinostat given orally, 40 mg thrice weekly in patients with advanced pretreated STS. The primary endpoint was the 3-month progression-free rate.Results:Forty-seven STS patients were enrolled between January 2010 and December 2010. Median age was 59 (range 21-79) years, 22 (47%) patients were males. Panobinostat dose was lowered to 20 mg thrice weekly after nine patients were enrolled, based on the recommendation of an independent safety committee. The most common grade 3/4 adverse events were thrombocytopenia, fatigue, lymphopenia and anaemia. Forty-five patients were evaluable for the primary endpoint. Among them, nine patients (20%, 95% CI (10-35%)) were progression-free at 3 months. No partial response was seen, but 17 patients (36%) had stable disease (SD) as their best response. Six patients were progression-free at 6 months.Conclusion:Panobinostat was poorly tolerated at 40 mg thrice a week. Efficacy in unselected advanced STS was limited, although some patients had prolonged SD. [ABSTRACT FROM AUTHOR]

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5-11.5) and temperature (40-90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co, Hg, Cu, Ni, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K and V of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min mg protein by analyzing Michaelis-Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus. [ABSTRACT FROM AUTHOR]

N-Acyl- d-amino acid amidohydrolases ( d-aminoacylases) are often used as tools for the optical resolution of d-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding d-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-) d-Phe (0.05-6.32 μmol min mg) under conditions without CoCl. Addition of 1 mM CoCl enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the d-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min mg for N-Ac- d-Phe and N-Ac- d-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. d-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0-9.0. It was stable at pH 5.5-9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl- d-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl- d-Phe was 2.1-fold higher than that toward N-Ac- d-Phe, indicating that the structure of N-acylated portion of substrate altered the activity. [ABSTRACT FROM AUTHOR]

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA170-247 and yGPAA170-339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70-247 and 70-339 respectively. The secondary structural content of the stable and monodisperse yGPAA170-247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA170-247 has an Rg (radius of gyration) of 2.72± 0.025 nm and Dmax (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA170-247. The large elliptical shape of yGPAA170-247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA170-247 will be discussed together with the recently determined low-resolution structures of yPIG-K24-337 and yPIG-S38-467 from S. cerevisiae in the GPI transamidase complex. [ABSTRACT FROM AUTHOR]

Disinfectants and fertilisers exert strong impact on soil processes by affecting the structure and the activity of the soil microbial community. Most relevant studies examined these impacts independently, under laboratory conditions and without crop cover. In this study, we have monitored the response of soil chemical, microbial, and biochemical properties to disinfectant and fertiliser treatments in field plots cultivated with beans. The measured properties comprised microbial C and N, asparaginase, gultaminase, urease, and acid phosphomonoesterase activities and contents of organic N, organic C, inorganic N, and inorganic P. We ran four different treatments using different combinations of chemical (metham sodium) and biological disinfectant (a mixture of neem cake and essential oils) and fertilisers (NPK 8-16-24 and cow manure) in plots cultivated with shell beans, while the control soil was neither treated nor cropped with beans. The data were expressed as percentage (%RC) in relation to the control values. The disinfectant and fertiliser treatments had less impact on soil properties compared to bean crop growth (except for microbial C and N, and content of organic C). In comparison to the control, higher activities of urease and asparaginase and content of inorganic N were recorded in bean cropped plots at the stage of seedlings (June), while higher activities of acid phosphomonoesterase and glutaminase and content of organic N were recorded at the stage of plant flowering (August). In October, the values of all properties were higher in the control plots compared to the treated plots. The joint effect of disinfectants x fertilisers affected the response of content of organic C and N and extractable P and glutaminase activity. The %RC of the properties exhibited more negative values in plots treated with chemical disinfectant and chemical fertiliser than in the other treatments. We suggested that the response of soil properties to disinfectants and fertilisers were influenced by the growth of P. vulgaris. [ABSTRACT FROM AUTHOR]

Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6-8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs. [ABSTRACT FROM AUTHOR]

Polyvinylpyrrolidone (PVP) is a frequendy used polymer in the pharmaceutical and foodstuff industries. Because it is not subject to metabolic changes and is virtually nondegradable, trace concentra-tions of PVP are often found in community wastewaters. The literature finds that the partial removal of PVP in wastewater treatment plants probably occurs through sorption. The primary objective of this study was to find an effective method to remove PVP from wastewaters. In this regard, the literature indicates the theoretical potential to use specific enzymes (e.g., 'ϒ-lactamases, amidases) to gradually degrade PVP molecules. Polyvinylpyrrolidone biodegradability tests were conducted using suitable heterogeneous cultures (activated sludge) collected from a conventional wastewater treatment plant, treatment plants connected to a pharmaceutical factory, and using select enzymes. Aerobic biodégra-dation of PVP in a conventional wastewater environment was ineffective, even after adaptation of activated sludge using the nearly identical monomer l-methyl-2-pyrrolidone. Another potential method for PVP removal involves pretreating the polymer prior to biological degradation. Based on the results (approximately 10 to 15% biodégradation), pretreatment was partially effective, realistically, it could only be applied with difficulty at wastewater treatment plants. Sorption of PVP to an active carbon sorbent (Chezacarb S), which corresponded to the Langmuir isotherm, and sorption to activated sludge, which correspond-ed to the Freundlich isotherm, were also evaluated. From these sorption tests, it can be concluded that the considerable adsorption of PVP to activated sludge occurred primarily at low PVP concentrations. Based on the test results, the authors recommend the following methods for PVP removal from wastewater: (1) sorption; (2) application of specific microorganisms; and (3) alkaline hydrolysis, which is the least suitable of the three for use in wastewater treatment plants. [ABSTRACT FROM AUTHOR]

We introduce PFunkel, a versatile method for extensive, researcher-defined DNA mutagenesis using a ssDNA or dsDNA template. Once the template DNA is prepared, the method can be completed in a single day in a single tube, and requires no intermediate DNA purification or sub-cloning. PFunkel can be used for site-directed mutagenesis at an efficiency approaching 100%. More importantly, PFunkel allows researchers the unparalleled ability to efficiently construct userdefined libraries. We demonstrate the creation of a library with site-saturation at four distal sites simultaneously at 70% efficiency. We also employ PFunkel to create a comprehensive codon mutagenesis library of the TEM-1 ß-lactamase gene. We designed this library to contain 18,081 members, one for each possible codon substitution in the gene (287 positions in TEM-1 x 63 possible codon substitutions). Deep sequencing revealed that ∼97% of the designed single codon substitutions are present in the library. From such a library we identified 18 previously unreported adaptive mutations that each confer resistance to the ß-lactamase inhibitor tazobactam. Three of these mutations confer resistance equal to or higher than that of the most resistant reported TEM-1 allele and have the potential to emerge clinically. [ABSTRACT FROM AUTHOR]

Superfamily of alpha-beta hydrolases is one of the largest groups of structurally related enzymes with diverse catalytic functions. Bioinformatic analysis was used to study how lipase and amidase catalytic activities are implemented into the same structural framework. Subfamily-specific positions—conserved within lipases and peptidases but different between them—that were supposed to be responsible for functional discrimination have been identified. Mutations at subfamily-specific positions were used to introduce amidase activity into Candida antarctica lipase B (CALB). Molecular modeling was implemented to evaluate influence of selected residues on binding and catalytic conversion of amide substrate by corresponding library of mutants. In silico screening was applied to select reactive enzyme-substrate complexes that satisfy knowledge-based criteria of amidase catalytic activity. Selected CALB variants with substitutions at subfamily-specific positions Gly39, Thr103, Trp104, and Leu278 were produced and showed significant improvement of experimentally measured amidase activity. Based on these results, we suggest that value of subfamily-specific positions should be further explored in order to develop a systematic tool to study structure-function relationship in enzymes and to use this information for rational enzyme engineering. [ABSTRACT FROM PUBLISHER]

The article discusses research on the use of class IIa histone deacetylases (HDACs) in treating type 2 diabetes. It references the article "Class IIa histone deacetylases are hormoneactivated regulators of FOXO and mammalian glucose homeostasis," by M.M. Mihayloma in the 2011 issue of the periodical "Cell." It reveals that HDACs can regulate liver glucose production, making it feasible for type 2 diabetes treatment.

Beneficial microbe functions range from host dietary supplementation to development and maintenance of host immune system. In mammals, newborn progeny are quickly colonized with a symbiotic fauna that is provisioned in mother's milk and that closely resembles that of the parent. Tsetse fly (Diptera: Glossinidae) also depends on the obligate symbiont Wigglesworthia for nutritional supplementation, optimal fecundity, and immune system development. Tsetse progeny develop one at a time in an intrauterine environment and receive nourishment and symbionts in mother's milk. We show that the host Peptidoglycan Recognition Protein (PGRP-LB) is expressed only in adults and is a major component of the milk that nourishes the developing progeny. The amidase activity associated with PGRP-LB may scavenge the symbiotic peptidoglycan and prevent the induction of tsetse's Immune Deficiency pathway that otherwise can damage the symbionts. Reduction of PGRP-LB experimentally diminishes female fecundity and damages Wigglesworthia in the milk through induction of antimicrobial peptides, including Attacin. Larvae that receive less maternal PGRP-LB give rise to adults with fewer Wigglesworthia and hyperimmune responses. Such adults also suffer dysregulated immunity, as indicated by the presence of higher trypanosome densities in parasitized adults. We show that recPGRP-LB has antimicrobial and antitrypanosomal activities that may regulate symbiosis and impact immunity. Thus, PGRP-LB plays a pivotal role in tsetse's fitness by protecting symbiosis against host-inflicted damage during development and by controlling parasite infections in adults that can otherwise reduce host fecundity. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a Cterminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. Methodology/Principal Findings: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. Conclusions/Significance: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT--rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA--mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer. [ABSTRACT FROM AUTHOR]

The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaHPO/NaHPO buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaHPO/NaHPO buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90-95% (mol mol) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization. [ABSTRACT FROM AUTHOR]

Asparaginase preparations deplete asparagine in acute lymphoblastic leukemia (ALL) blasts. Asparaginase in its various forms is an important component of treatment regimens for pediatric ALL. Recently, interest and use of asparaginase in adult patients with ALL has increased, particularly in young adults. There is much less information on asparaginase use and toxicity in adult compared with pediatric populations. This review surveys prior published studies of the three most commonly used asparagine preparations as used in adult patients, and discusses important toxicities encountered in adult patients who receive asparaginase preparations [ABSTRACT FROM AUTHOR]

There is increasing evidence that regulation of local chromatin structure is a critical mechanism underlying the consolidation of long-term memory (LTM), however considerably less is understood about the specific mechanisms by which these epigenetic effects are mediated. Furthermore, the importance of histone acetylation in Drosophila memory has not been reported. The histone deacetylase (HDAC) Rpd3 is abundant in the adult fly brain, suggesting a post-mitotic function. Here, we investigated the role of Rpd3 in long-term courtship memory in Drosophila. We found that while modulation of Rpd3 levels predominantly in the adult mushroom body had no observed impact on immediate recall or onehour memory, 24-hour LTM was severely impaired. Surprisingly, both overexpression as well as RNAi-mediated knockdown of Rpd3 resulted in impairment of long-term courtship memory, suggesting that the dose of Rpd3 is critical for normal LTM. [ABSTRACT FROM AUTHOR]

ABSTRACT Monascus purpureus grown on rice secretes several polyketide pigments used for coloring foods. The amidase enzymes, L-asparaginase and L-glutaminase activities were identified by rapid plate assay. The pink zone formed around colonies of M. purpureus (Microbial Type Culture Collection [MTCC] 410), hyper pigment (Central Food Research [CFR] 410-11) and albino (CFR 410-22) were 2.0, 2.4 and 1.2 cm, respectively. The L-asparaginase (0.103 IU) and L-glutaminase (0.139 IU) activity was comparatively more in CFR 410-11 than MTCC 410 and CFR 410-22. Higher acid protease activity (5,170 Units) was observed in CFR 410-22. The mutant CFR 410-11 has secreted more red pigment cultured on rice (2.248 optical density [OD] units) and in broth (0.841 OD units). Significant difference in amidase and acid protease activities of MTCC 410 and its mutant CFR 410-11 and CFR 410-22 have revealed the importance of these enzymes in pigment production. PRACTICAL APPLICATION The isolated albino (CFR 410-22) mutant has overproduced acid protease enzyme. This enzyme finds applications in debittering of protein hydrolysate, cheese ripening and preparation of amino acid mixture from protein. The amidase enzymes produced by Monascus sp. may play a major role in tumor inhibition. [ABSTRACT FROM AUTHOR]

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μμL of cell-free extract containing 7.47 mg protein mL−−1, 2 μμL of glutaraldehyde (5%, v/v) and 10 μμL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0--10.0 μμM urea, a detection limit of 2.0 μμM for urea, a response time of 20 s, a sensitivity of 58.245 % per μμM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition. [ABSTRACT FROM AUTHOR]

The article discusses the use of comparative modeling to predict the three-dimensional (3D) structure of cysteine, histidine-dependent amidohydrolase and peptidase domain of the LysK endolysin (CHAP k protein) which is derived from bacteriophage K. It notes the presence of highly conserved amino acid residues in CHAP proteins which include an invariant cystine (Cys), and histidine (His). The author also cites the use of web-based servers, and visualization programs in the comparative analysis.

Background: Histone deacetylases (HDACs) are crucial components of the oestrogen receptor (ER) transcriptional complex. Preclinically, HDAC inhibitors can reverse tamoxifen/aromatase inhibitor resistance in hormone receptor-positive breast cancer. This concept was examined in a phase II combination trial with correlative end points.Methods: Patients with ER-positive metastatic breast cancer progressing on endocrine therapy were treated with 400 mg of vorinostat daily for 3 of 4 weeks and 20 mg tamoxifen daily, continuously. Histone acetylation and HDAC2 expression in peripheral blood mononuclear cells were also evaluated.Results: In all, 43 patients (median age 56 years (31-71)) were treated, 25 (58%) received prior adjuvant tamoxifen, 29 (67%) failed one prior chemotherapy regimen, 42 (98%) progressed after one, and 23 (54%) after two aromatase inhibitors. The objective response rate by Response Evaluation Criteria in Solid Tumours criteria was 19% and the clinical benefit rate (response or stable disease >24 weeks) was 40%. The median response duration was 10.3 months (confidence interval: 8.1-12.4). Histone hyperacetylation and higher baseline HDAC2 levels correlated with response.Conclusion: The combination of vorinostat and tamoxifen is well tolerated and exhibits encouraging activity in reversing hormone resistance. Correlative studies suggest that HDAC2 expression is a predictive marker and histone hyperacetylation is a useful pharmacodynamic marker for the efficacy of this combination. [ABSTRACT FROM AUTHOR]

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the Nacetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA241) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin384) or the shorter (Lysin241) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various Grampositive bacteria and mycobacteria [ABSTRACT FROM AUTHOR]

On seedlings of winter wheat ( Triticum aestivum L.) and cucumber ( Cucumis sativus L.), the dynamics of cysteine and serine trypsin-like proteinases and also trypsin inhibitors at cold hardening (5°C for wheat and 10°C for cucumber) was studied. Activation of proteinases and inhibitors coincided in time or preceded an increased tolerance in wheat and cucumber seedlings in the early period of their hardening. After attaining the highest wheat tolerance, activity amidases reduced, whereas the increased activity levels of cysteine proteinases and trypsin inhibitors was maintained during the entire period of hardening. In cucumber, in these period activities of amidases and trypsin inhibitors reduced, whereas the activity of cysteine proteinases was maintained at the level close to the initial one. It is suggested that cysteine proteinases, amidases, and trypsin inhibitors are involved in plant adaptation to cold. [ABSTRACT FROM AUTHOR]

Little information is available on the effects of urease inhibitor, N-( n-butyl)thiophosphoric triamide (NBPT), and nitrification inhibitor, dicyandiamide (DCD), on nitrous oxide (NO) emissions from fluvo-aquic soil in the North China Plain. A field experiment was conducted at the Fengqiu State Key Agro-Ecological Experimental Station, Henan Province, China, to study the influence of urea added with NBPT, DCD, and combination of both NBPT and DCD on NO emissions during the maize growing season in 2009. Two peaks of NO fluxes occurred during the maize growing season: the small one following irrigation and the big one after nitrogen (N) fertilizer application. There was a significant positive relationship between ln [NO flux] and soil moisture during the maize growing season excluding the 11-day datasets after N fertilizer application, indicating that NO flux was affected by soil moisture. Mean NO flux was the highest in the control with urea alone, while the application of urea together with NBPT, DCD, and NBPT + DCD significantly lowered the mean NO flux. Total NO emission in the NBPT + DCD, DCD, NBPT, and urea alone treatments during the experimental period was 0.41, 0.47, 0.48, and 0.77 kg NO-N ha, respectively. Application of urea with NBPT, DCD, and NBPT + DCD reduced NO emission by 37.7%, 39.0%, and 46.8%, respectively, over urea alone. Based on our findings, the combination of DCD and NBPT together with urea may reduce NO emission and improve the maize yield from fluvo-aquic soil in the North China Plain. [ABSTRACT FROM AUTHOR]

Histone deacetylase inhibitors (HDI) dampen cellular innate immune response by decreasing interferon production and have been shown to increase the growth of vesicular stomatitis virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective vaccinia virus in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet did not increase the replication of vaccinia in normal tissues. The combination of TSA and VV increased survival of mice harbouring human HCT116 colon tumour xenografts as compared to mice treated with either agent alone. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells. [ABSTRACT FROM AUTHOR]

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of b-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms. [ABSTRACT FROM AUTHOR]

Background: Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown. Methodology/Principal Findings: To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression. Conclusions/Significance: The most frequent molecular events associated with ependymoma recurrence were overexpression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors. [ABSTRACT FROM AUTHOR]

The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicate that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters. [ABSTRACT FROM AUTHOR]

In the last decades the idea to use enzymes for environmental bioremediation has been more and more proposed and, in the light of this, new solutions have been suggested and detailed studies on some classes of enzymes have been performed. In particular, our attention in the last few years has been focused on the enzymes belonging to the amidohydrolase superfamily. Several members of this superfamily are endowed with promiscuous activities. The term 'catalytic promiscuity' describes the capability of an enzyme to catalyse different chemical reactions, called secondary activities, at the active site responsible for the main activity. Recently, a new family of microbial lactonases with promiscuous phosphotriesterase activity, dubbed PTE-Like Lactonase (PLL), has been ascribed to the amidohydrolase superfamily. Among members of this family are enzymes found in the archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, which show high thermophilicity and thermal resistance. Enzymes showing phosphotriesterase activity are attractive from a biotechnological point of view because they are capable of hydrolysing the organophosphate phosphotriesters (OPs), a class of synthetic compounds employed worldwide both as insecticides and chemical warfare agents. Furthermore, from a basic point of view, studies of catalytic promiscuity offer clues to understand natural evolution of enzymes and to translate this into in vitro adaptation of enzymes to specific human needs. Thermostable enzymes able to hydrolyse OPs are considered good candidates for the set-up of efficient detoxification tools. [ABSTRACT FROM AUTHOR]

The direct, 100 % atom-economic, and selective synthesis of amines is a challenging task that can be achieved, making use of early transition-metal catalysts. Here we report the synthesis and application of group 4 and 5 high-oxidation-state metal amidate complexes in catalytic C-N (hydroamination) and C-C (hydroaminoalkylation) bond-forming reactions to access substituted amines. [ABSTRACT FROM AUTHOR]