Protection against peroxynitrite-induced DNA damage by mesalamine: implications for anti-inflammation and anti-cancer activity

Mesalamine (5-aminosalicylic acid, 5-ASA) is known to be the first-line medication for treatment of patients with ulcerative colitis. Studies have demonstrated that ulcerative colitis patients treated with 5-ASA have an overall decrease in the risk of developing colorectal carcinoma. However, the mechanisms underlying 5-ASA-mediated anti-inflammatory and anti-cancer effects are yet to be elucidated. Because peroxynitrite has been critically involved in inflammatory stress and carcinogenesis, this study was undertaken to investigate the effects of 5-ASA in peroxynitrite-induced DNA strand breaks, an important event leading to peroxynitrite-elicited cytotoxicity. Incubation of φX-174 plasmid DNA with the peroxynitrite generator 3-morpholinosydnonimine (SIN-1) led to the formation of both single- and double-stranded DNA breaks in a concentration-dependent manner. The presence of 5-ASA at 0.1 and 1.0 mM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by SIN-1 was found to not be affected by 5-ASA at 0.1–50 mM, indicating that 5-ASA at these concentrations is not involved in the auto-oxidation of SIN-1 to form peroxynitrite. It is observed that 5-ASA at 0.1–1 mM showed considerable inhibition of peroxynitrite-mediated luminol chemiluminescence in a dose-dependent fashion, suggesting that 5-ASA is able to directly scavenge the peroxynitrite. Electron paramagnetic resonance (EPR) spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap resulting in the formation of DMPO-hydroxyl radical adduct from peroxynitrite, and 5-ASA only at higher concentration (1 mM) inhibited the hydroxyl radical adduct while shifting EPR spectra, indicating that 5-ASA at higher concentrations may generate a more stable free radical species rather than acting purely as a hydroxyl radical scavenger. Taken together, these studies demonstrate for the first time that 5-ASA can potently inhibit peroxynitrite-mediated DNA strand breakage, scavenge peroxynitrite, and affect peroxynitrite-mediated radical formation, which may be responsible, at least partially, for its anti-inflammatory and anti-cancer effects.     

Palaeoecological and sedimentological characteristics of the Lower Tortonian scleractinian reef corals of Gavdos Island,southern Greece

Early Late Miocene coral assemblages from five outcrops of Gavdos Island, Southern Greece, are investigated with respect to their palaeoecological implications. Small patch reefs with Porites assemblage are a common feature of the low-diversity coral occurrences. The determined hermatypic colonies indicate a nearshore palaeoecosystem prevailing in a tropical to subtropical coastal sea at depths ranging from 5 to 50 m with an average temperature of 22–26 °C. Microfacially, the studied Scleractinian patch reefs are represented by Coral Framestones-Floatstones. The reefal facies has been affected by syndepositional processes (boring activities-micritization), as well as by post-depositional diagenesis in the meteoric realm (dissolution, cementation and intense pedogenesis). The palaeoecological and sedimentological analysis indicates a restricted to open-marine inner platform setting of moderate to high energy, possibly of ramp-type (inner-mid ramp). Coral reef growth took place mainly during stages of accommodation (i.e., transgressive episode, cycle 3.1 of Vail curve) and of relatively low siliciclastic input.     

Interactions of bacterial flagellar chaperone–substrate complexes with FlhA contribute to co‐ordinating assembly of the flagellar filament

Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C‐terminal cytoplasmic domain of FlhA (FlhA_C), a membrane component of the export apparatus, provides a binding‐site for these chaperone–substrate complexes but it remains unknown how it co‐ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhA_C is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhA_C for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14‐fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhA_C for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.     

Continuous ATP Regeneration Using Immobilized Yeast Cells

Arthrobacter simplex was screened as an α-keto-δ-guanidinovalerate (ketoarginine) assimilating organism. A characteristic feature was its growth on ketoarginine as a carbon source; it began to grow after an extremely long lag. Its growth was stimulated by addition of 0.02% yeast extract to the medium.

The results indicated the transamination of arginine-α-ketoglutarate (α-KGA) and the hydrolyzing reaction of ketoarginine into α-keto-δ-aminovalerate and urea. Two intermediates, ketoarginine and α-keto-δ-aminovalerate, were isolated and identified by various procedures. Coupling of the two reactions was demonstrated in cell-free extracts of arginine-grown cells; ketoarginine formed from arginine by transamination with α-KGA was hydrolyzed directly to α-keto-δ-aminovalerate and urea. The metabolic routes of arginine in microorganisms were discussed.     

Fungal Proteolytic Enzymes

Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.     

Epithelial Cell Proliferation Arrest Induced by Lactate and Acetate from Lactobacillus casei and Bifidobacterium breve

In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either Lactobacillus or Bifidobacterium as bona fide symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by L. casei and B. breve respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA) were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.     

cljam: a library for handling DNA sequence alignment/map (SAM) with parallel processing

Background

Next-generation sequencing can determine DNA bases and the results of sequence alignments are generally stored in files in the Sequence Alignment/Map (SAM) format and the compressed binary version (BAM) of it. SAMtools is a typical tool for dealing with files in the SAM/BAM format. SAMtools has various functions, including detection of variants, visualization of alignments, indexing, extraction of parts of the data and loci, and conversion of file formats. It is written in C and can execute fast. However, SAMtools requires an additional implementation to be used in parallel with, for example, OpenMP (Open Multi-Processing) libraries. For the accumulation of next-generation sequencing data, a simple parallelization program, which can support cloud and PC cluster environments, is required.

Results

We have developed cljam using the Clojure programming language, which simplifies parallel programming, to handle SAM/BAM data. Cljam can run in a Java runtime environment (e.g., Windows, Linux, Mac OS X) with Clojure.

Conclusions

Cljam can process and analyze SAM/BAM files in parallel and at high speed. The execution time with cljam is almost the same as with SAMtools. The cljam code is written in Clojure and has fewer lines than other similar tools.