cda1+, encoding chitin deacetylase is required for proper spore formation in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, a major role of chitin is to build up a complete spore. Here, we analyzed the cda1(+) gene (SPAC19G12.03), which encodes a protein homologous to chitin deacetylases, to know whether it is required for spore formation in S. pombe. The homothallic Deltacda1 strain constructed by homologous recombination was found to form a little amount of abnormal spores that contained one, two, or three asci, similar to (but not as strong as) the phenotype observed in a deletion mutant of chs1 encoding chitin synthase 1. This phenotype is reversed by expression of S. cerevisiae chitin deacetylase CDA1 or CDA2, suggesting that cda1 encodes a chitin deacetylase. To support the role of Cda1 in sporulation, the timing of expression of cda1(+) mRNA increased during sporulation process. We also found that the Cda1 protein self-associated when its binding was tested both by two-hybrid system and immunoprecipitation. Thus, these data indicated that cda1(+) is required for proper spore formation in S. pombe.     

CDA: Combinatorial Drug Discovery Using Transcriptional Response Modules

Background

Anticancer therapies that target single signal transduction pathways often fail to prevent proliferation of cancer cells because of overlapping functions and cross-talk between different signaling pathways. Recent research has identified that balanced multi-component therapies might be more efficacious than highly specific single component therapies in certain cases. Ideally, synergistic combinations can provide 1) increased efficacy of the therapeutic effect 2) reduced toxicity as a result of decreased dosage providing equivalent or increased efficacy 3) the avoidance or delayed onset of drug resistance. Therefore, the interest in combinatorial drug discovery based on systems-oriented approaches has been increasing steadily in recent years.

Methodology

Here we describe the development of Combinatorial Drug Assembler (CDA), a genomics and bioinformatics system, whereby using gene expression profiling, multiple signaling pathways are targeted for combinatorial drug discovery. CDA performs expression pattern matching of signaling pathway components to compare genes expressed in an input cell line (or patient sample data), with expression patterns in cell lines treated with different small molecules. Then it detects best pattern matching combinatorial drug pairs across the input gene set-related signaling pathways to detect where gene expression patterns overlap and those predicted drug pairs could likely be applied as combination therapy. We carried out in vitro validations on non-small cell lung cancer cells and triple-negative breast cancer (TNBC) cells. We found two combinatorial drug pairs that showed synergistic effect on lung cancer cells. Furthermore, we also observed that halofantrine and vinblastine were synergistic on TNBC cells.

Conclusions

CDA provides a new way for rational drug combination. Together with phExplorer, CDA also provides functional insights into combinatorial drugs. CDA is freely available at http://cda.i-pharm.org.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability.

We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively. We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C). The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDA(Bsubt). Both enzymes were found to be homotetramers of approximately 58 kDa. CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald). After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated. Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature.     

Synthesis of cellulose dehydroabietate in ionic liquid [bmim]Br

A new type of cellulose derivative, cellulose dehydroabietate (CDA), was synthesized by the O-acylation reaction of cellulose with dehydroabietic acid chloride (DHAC) using ionic liquid 1-butyl-3-methylimidazolium bromide ([bmim]Br) as a solvent and 4-dimethyl-aminopyridine (DMAP) as a catalyst. The resulting CDA was characterized by means of FT-IR, X-ray diffraction (XRD), scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), and elemental analysis. Also, some properties of CDA were determined. These results showed that CDA has better solubility, water-repellency, and resistance to acids and bases than raw cellulose, and these properties increase with the DS of CDA.     

Microbioluminescent study of the general toxicity and mutagenicity of pollutants

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37 degrees C and pH 6.5. The mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn2+, and Cd2+ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.     

Quantification of carbamylated dehydroascorbate derivative produced from cyanate and dehydroascorbate

We established a high-performance liquid chromatographic method for separating and quantifying carbamylated dehydroascorbate derivative (CDA), a reaction product of cyanate with dehydroascorbate. The separation of CDA from interfering substances was achieved by anion-exchange HPLC using a TSK gel SAX (250×4.6 mm I.D.) column and 0.12 M NaCl eluent. The detection of CDA was achieved through two steps: (1) degradation of CDA to cyanate and amino compounds in alkaline solution, and (2) detection of these products by an indophenol reaction. For the processing of plasma and urine samples, anion-exchange solid-phase extraction was used. The detection limit for quantitative determination was 0.1 μM CDA (S/N=3). The linear range found applying the optimized conditions was 0.2 to 200 μM. The intra- and inter-day assay precision (R.S.D.) of CDA (10 μM) were 4.8 and 7.2% for rat plasma, and 4.0 and 4.9% for rat urine, respectively. The usefulness of the present method was proved by the application to plasma and urine samples. The study of the biokinetics of CDA in rats revealed that the elimination of CDA is due to urinary excretion.     

Post-translational protein modification by carotenoid cleavage products

Carotenoids are known to generate various aldehydes, known as carotenoid-derived aldehydes (CDAs), which could efficiently react with protein or DNA. In this in vitro model study, interaction between CDA and protein has been studied. Various proteins were incubated with CDA, and protein modification and adduct formation were confirmed by using matrix-assisted laser desorption and ionization time-of-flight, amino acid analysis, and measuring enzyme activity on modification with CDA. Using radiolabeled NaB((3) H)H(4) and Raney nickel as well as sulfhydryl assay (Ellman's reagent), we confirmed that CDA could conjugate with cysteine through a thioether linkage. The carbonyl assay using 2,4-dinitrophenylhydrazine revealed the possible involvement of Schiff's base reaction between CDA and lysine. The adducts formed between β-apo-8-carotenal (BA8C) and N-acetylcysteine and BA8C and N-acetyllysine were confirmed by HPLC and ESI-MS. Our results suggest that CDA could alter protein function by post-translational interaction with cysteine and lysine by thioether linkage and by schiff's based bonds, respectively. Thus, the formation of CDA adducts with proteins could alter functional properties of proteins responsible for maintaining cell homeostasis and thereby cause cellular toxicity. In view of these observations, further studies are required to understand the delicate balance between beneficial and/or harmful effects of carotenoids as a dietary supplement to slow age-related macular degeneration progression.     

Importance of hydrogen-bonding sites in the chiral recognition mechanism between racemic D3 terbium(III) complexes and amino acids

The perturbation of the racemic equilibrium of luminescent D3 terbium(III) complexes with chelidamic acid (CDA), a hydroxylated derivative of 2,6-pyridine-dicarboxylic acid (DPA), by added chiral biomolecules such as L-amino acids has been studied using circularly polarized luminescence and 13C NMR spectroscopy. It is shown in this work that the chiral-induced equilibrium shift of [Tb(CDA)3](6-) by L-amino acids (i.e. L-proline or L-arginine) was largely influenced by the hydrogen-bonding networks formed between the ligand interface of racemic [Tb(CDA)3](6-) and these added chiral agents. The capping of potential hydrogen-bonding sites by acetylation in L-proline led to a approximately 100-fold drop in the induced optical activity of the [Tb(CDA)3](6-):N-acetyl-L-proline system. This result suggested that the hydrogen-bonding networks serve as the basis for further noncovalent discriminatory interactions between racemic [Tb(CDA)3](6-) and added L-amino acids.     

The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1,1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography--electrospray tandem mass spectrometry

Varying concentrations of cyclopropane-1,1-dicarboxylic acid (CDA), an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase, added to the solid culture medium of tomato nodal shoot segments resulted in a reduction in the level of endogenous ethylene according to the concentration of inhibitor applied. Following treatment with inhibitor, plants were homogenised and the concentrations of CDA and of 1-aminocyclopropane-1-carboxylic acid (ACC) were measured simultaneously in the resulting juice using an HPLC-ESI/MS-MS method. The levels of CDA and ACC measured in the plant tissues were associated with the concentration of inhibitor added to the solid medium. The HPLC-ESI/MS-MS method described produced limits of detection of 0.8 pmol for ACC and of 4 pmol for CDA.     

Localization of the congenital dyserythropoietic anemia II locus to chromosome 20q11.2 by genomewide search.

Congenital dyserythropoietic anemias (CDA) are genetic disorders characterized by anemia and ineffective erythropoiesis. Three main types of CDA have been distinguished: CDA I and CDA III, whose loci have been already mapped, and CDA II (MIM 224100), the most frequent among CDAs, which is transmitted as an autosomal recessive trait and is known also as "HEMPAS" (hereditary erythroblast multinuclearity with positive acidified serum). We have recruited a panel of well-characterized CDA II families and have used them to search for the CDA II gene by linkage analysis. After the exclusion of three candidate genes, we ob-tained conclusive evidence for linkage of CDA II to microsatellite markers on the long arm of chromosome 20 (20q11.2). A maximum two-point LOD score of 5.4 at a recombination fraction of .00 was obtained with marker D20S863. Strong evidence of allelic association with the disease was detected with the same marker. Some recombinational events established a maximum candidate interval of approximately 5 cM.     

Chemo- and regioselective peptide cyclization triggered by the N-terminal fatty acid chain length: the recombinant cyclase of the calcium-dependent antibiotic from Streptomyces coelicolor

Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2). This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides. The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA. Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol. The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition. The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr(2) and Ser(1) residues onto the C-terminus of the acyl-enzyme intermediate. This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far. Substitution of either Ser(1) or Thr(2) by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only l-configured Ser(1) and Thr(2) for cyclization. Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization. Elongation of the fatty acyl group of our thioester substrate from C(2) to C(6) as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.     

Paraneoplastic syndrome in mice bearing high-angiogenic variant of Lewis lung carcinoma: relations with tumor derived VEGF

Background

It is well-known that tumor exerts nonmetastatic systemic effect on organism caused the development of paraneoplastic syndrome (PNS). Recent findings point to relationships between development of PNS and tumor-derived vascular endothelial growth factor (VEGF).

Aim

Comparative study of PNS manifestations in mice with transplanted two variants of Lewis lung carcinoma with different angiogenic potential.

Methods

Plasma VEGF level was determined by immunoenzyme method, hematological indices were estimated with the use of hematological analyzer, the weight and cellularity of spleen and thymus were registered and histological analysis of tissue section of these organs was performed.

Results

Manifestations of anemia, extramedullary hemopoiesis and tumor-associated inflammatory disease was observed in animals with high angiogenic LLC/R9 variant and was not registered in low angiogenic LLC. The emergence of PNS symptoms correlated with elevated level of circulating VEGF at the early stages of LLC/R9 growth.

Conclusion

Manifestation of the paraneoplastic hematological syndrome most likely is conditioned on the ability of cancer cell to secrete VEGF in a high rate.     

FFCD-1004 Clinical Trial: Impact of Cytidine Deaminase Activity on Clinical Outcome in Gemcitabine-Monotherapy Treated Patients

Purpose

Because cytidine deaminase (CDA) is the key enzyme in gemcitabine metabolism, numerous studies have attempted to investigate impact of CDA status (i.e. genotype or phenotype) on clinical outcome. To date, data are still controversial because none of these studies has fully investigated genotype-phenotype CDA status, pharmacokinetics and clinical outcome relationships in gemcitabine-treated patients. Besides, most patients were treated with gemcitabine associated with other drugs, thus adding a confounding factor. We performed a multicenter prospective clinical trial in gemcitabine-treated patients which aimed at investigating the link between CDA deficiency on the occurrence of severe toxicities and on pharmacokinetics, and studying CDA genotype-phenotype relationships.

Experimental design

One hundred twenty patients with resected pancreatic adenocarcinoma eligible for adjuvant gemcitabine monotherapy were enrolled in this study promoted and managed by the Fédération Francophone de Cancérologie Digestive. Toxicities were graded according to National Cancer Institute’s Common Terminology Criteria for Adverse Events Version 4. They were considered severe for grade ≥ 3, and early when occurring during the first eight weeks of treatment. CDA status was evaluated using a double approach: genotyping for 79A>C and functional testing. Therapeutic drug monitoring of gemcitabine and its metabolite were performed on the first course of gemcitabine.

Results

Five patients out of 120 (i.e., 4.6%) were found to be CDA deficient (i.e., CDA activity <1.3 U/mg), and only one among them experienced early severe hematological toxicity. There was no statistically significant difference in CDA activity between patients experiencing hematological severe toxicities (28.44%) and patients who tolerated the treatment (71.56%). CDA genetic analysis failed in evidencing an impact in terms of toxicities or in CDA activity. Regarding pharmacokinetics, a wide inter-individual variability has been observed in patients.

Conclusion

This study, which included only 4.6% of CDA-deficient patients, failed in identifying CDA status as a predictive marker of toxicities with gemcitabine. A lack of statistical power because of smoothing effect of CDA variability as compared with real life conditions could explain this absence of impact.

Trial Registration

ClinicalTrials.gov NCT01416662     

Reorganization of dynamic self-assemblies of cellulose diacetate in solution: dynamical critical-like fluctuations in the lower critical solution temperature system

Dynamics of cellulose diacetate (CDA, the total degree of substitution (TDS) = 2.44) in dimethylacetamide (DMAc) in dilute solution was investigated at 2, 10, 20, 30, 40, 49.7, and 61.5 degrees C through dynamic light scattering in the quiescent state. The following three facts were made clear. First, CDA existed in three types of structures in the polar solvent, DMAc; one is a single CDA chain, and the others are dynamic structures, or self-assemblies, which were formed temporarily and locally by the solvent-mediated hydrogen bonding between the intermolecular C-6 position hydroxyls of the anhydroglucose units in the CDA backbone. Second, CDA showed a nature of low-temperature solubility in DMAc, that is, CDA is expected to dissolve molecularly below -12 degrees C but to take a phase separation above 65 degrees C, where two structures such as collapses of a single CDA chain and an aggregate appear. Third, a reorganization in the dynamic structures was detected at the temperature T = 33.8 degrees C. At this temperature, two dynamic structures showed the discontinuity in their correlation lengths, whereas the single CDA realized an uncorrelated chain state in the dynamical sense. In view of the low-temperature solubility of CDA in DMAc, this abnormal behavior around T was explained by dynamical critical-like fluctuations if T were treated as a kind of lower critical solution temperature (LCST) in the CDA/DMAc system. Here, the self-assemblies arise as the dynamical fluctuations under the spinodal decomposition situation and the competition between the hydrogen bonding (HB) and the hydrophobic interaction (HPhI) makes the conformation of CDA chains change drastically. In this scheme, the solvent-mediated HB and HPhI play important roles in the structure reorganization of cellulose derivatives in strong electronegative solvents, though HB and HPhI cooperate with the inherent chain helicality.     

Unsaturated Fatty Acid,cis-2-Decenoic Acid,in Combination with Disinfectants or Antibiotics Removes Pre-Established Biofilms Formed by Food-Related Bacteria

Biofilm formation by food-related bacteria and food-related pathogenesis are significant problems in the food industry. Even though much disinfection and mechanical procedure exist for removal of biofilms, they may fail to eliminate pre-established biofilms. cis-2 decenoic acid (CDA), an unsaturated fatty acid messenger produced by Pseudomonas aeruginosa, is reportedly capable of inducing the dispersion of established biofilms by multiple types of microorganisms. However, whether CDA has potential to boost the actions of certain antimicrobials is unknown. Here, the activity of CDA as an inducer of pre-established biofilms dispersal, formed by four main food pathogens; Staphylococcus aureus, Bacillus cereus, Salmonella enterica and E. coli, was measured using both semi-batch and continuous cultures bioassays. To assess the ability of CDA combined biocides treatments to remove pre-established biofilms formed on stainless steel discs, CFU counts were performed for both treated and untreated cultures. Eradication of the biofilms by CDA combined antibiotics was evaluated using crystal violet staining. The effect of CDA combined treatments (antibiotics and disinfectants) on biofilm surface area and bacteria viability was evaluated using fluorescence microscopy, digital image analysis and LIVE/DEAD staining. MICs were also determined to assess the probable inhibitory effects of CDA combined treatments on the growth of tested microorganisms'' planktonic cells. Treatment of pre-established biofilms with only 310 nM CDA resulted in at least two-fold increase in the number of planktonic cells in all cultures. While antibiotics or disinfectants alone exerted a trivial effect on CFU counts and percentage of surface area covered by the biofilms, combinational treatments with both 310 nM CDA and antibiotics or disinfectants led to approximate 80% reduction in biofilm biomass. These data suggests that combined treatments with CDA would pave the way toward developing new strategies to control biofilms with widespread applications in industry as well as medicine.     

CDA基因沉默对人慢性髓系白血病细胞增殖和凋亡的影响

目的:探讨胞苷脱氨酶(CDA)基因沉默在治疗人慢性髓系白血病(CML)中的潜在价值。方法:通过RT-PCR和Western blot检测CML患者和造血干细胞移植供体的骨髓单个核细胞中的CDA表达。对CML KCL-22细胞系转染shRNA和过表达CDA的p BS/U6-Neo质粒来诱导CDA基因沉默或过表达。通过细胞计数试剂盒8(CCK-8)测定和细胞集落形成实验评价细胞增殖,通过流式细胞仪检测细胞凋亡。此外,将0.2 m L不同处理的细胞悬浮液(106个细胞/m L)注射到裸鼠中建立裸鼠肿瘤异种移植模型。结果:与造血干细胞移植供体相比,CML患者的骨髓单个核细胞中的CDA m RNA和蛋白表达显著升高(P 0.05)。转染shRNA-CDA显著降低了KCL-22细胞的细胞活力和细胞集落数(P0.05)。与对照组(4.32%)相比,shRNA-CDA组(13.45%)的细胞凋亡率显著升高(P0.05)。与对照组相比,shRNA-CDA组的BCL-2蛋白表达水平显著降低,而cleaved caspase-3显著升高(P0.05)。与对照组相比,shRNA-CDA组的PI3K蛋白表达水平和Akt磷酸化水平显著降低(P0.05)。接种30 d后,与对照组相比,shRNA-CDA组裸鼠的肿瘤重量和肿瘤体积均显著降低(P0.05)。结论:CDA在人慢性髓系白血病中高表达,CDA基因沉默可在体内和体外抑制肿瘤细胞的生长。其机制与抑制PI3K/Akt信号通路的激活有关。