Phenolic compounds isolated from Pilea microphylla prevent radiation-induced cellular DNA damage

AbstractSix phenolic compounds namely, quercetin-3-O-rutinoside (1), 3-O-caffeoylquinic acid (2), luteolin-7-O-glucoside (3), apigenin-7-O-rutinoside (4), apigenin-7-O-β-d-glucopyranoside (5) and quercetin (6) were isolated from the whole plant of Pilea microphylla using conventional open-silica gel column chromatography and preparative HPLC. Further, these compounds were characterized by 1D, 2D NMR techniques and high-resolution LC–MS. Compounds 1–3 and 6 exhibited significant antioxidant potential in scavenging free radicals such as DPPH, ABTS and SOD with IC50 of 3.3–20.4μmol/L. The same compounds also prevented lipid peroxidation with IC50 of 10.4–32.2μmol/L. The compounds also significantly prevented the Fenton reagent-induced calf thymus DNA damage. Pre-treatment with compounds 1–3 and 6 in V79 cells attenuated radiation-induced formation of reactive oxygen species, lipid peroxidation, cytotoxicity and DNA damage, correlating the antioxidant activity of polyphenols with their radioprotective effects. Compounds 1, 3 and 6 significantly inhibited lipid peroxidation, presumably due to 3′,4′-catechol ortho-dihydroxy moiety in the B-ring, which has a strong affinity for phospholipid membranes. Oxidation of flavonoids, with catechol structure on B-ring, yields a fairly stable ortho-semiquinone radical by facilitating electron delocalization, which is involved in antioxidant mechanism. Hence, the flavonoid structure, number and location of hydroxyl groups together determine the antioxidant and radioprotection mechanism

Formation of gutingimycin: analytical investigation of trioxacarcin A-mediated alkylation of dsDNA

Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin–DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3′-end and an oligonucleotide with a phosphorylated 5′-end

In vitro phosphorylation as tool for modification of silk and keratin fibrous materials

An overview is given of the recent work on in vitro enzymatic phosphorylation of silk fibroin and human hair keratin. Opposing to many chemical "conventional" approaches, enzymatic phosphorylation is in fact a mild reaction and the treatment falls within "green chemistry" approach. Silk and keratin are not phosphorylated in vivo, but in vitro. This enzyme-driven modification is a major technological breakthrough. Harsh chemical chemicals are avoided, and mild conditions make enzymatic phosphorylation a real "green chemistry" approach. The current communication presents a novel approach stating that enzyme phosphorylation may be used as a tool to modify the surface charge of biocompatible materials such as keratin and silk

Genome Fragmentation Is Not Confined to the Peridinin Plastid in Dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3′-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates

The replication of plastid minicircles involves rolling circle intermediates

Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as ‘minicircles’ of ∼2–3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250–500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6–8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2–3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6–8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles

The replication of plastid minicircles involves rolling circle intermediates

Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as ‘minicircles’ of ∼2–3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250–500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6–8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2–3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6–8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles

Induction of Larval Metamorphosis of the Coral Acropora millepora by Tetrabromopyrrole Isolated from a Pseudoalteromonas Bacterium

The induction of larval attachment and metamorphosis of benthic marine invertebrates is widely considered to rely on habitat specific cues. While microbial biofilms on marine hard substrates have received considerable attention as specific signals for a wide and phylogenetically diverse array of marine invertebrates, the presumed chemical settlement signals produced by the bacteria have to date not been characterized. Here we isolated and fully characterized the first chemical signal from bacteria that induced larval metamorphosis of acroporid coral larvae (Acropora millepora). The metamorphic cue was identified as tetrabromopyrrole (TBP) in four bacterial Pseudoalteromonas strains among a culture library of 225 isolates obtained from the crustose coralline algae Neogoniolithon fosliei and Hydrolithon onkodes. Coral planulae transformed into fully developed polyps within 6 h, but only a small proportion of these polyps attached to the substratum. The biofilm cell density of the four bacterial strains had no influence on the ratio of attached vs. non-attached polyps. Larval bioassays with ethanolic extracts of the bacterial isolates, as well as synthetic TBP resulted in consistent responses of coral planulae to various doses of TBP. The lowest bacterial density of one of the Pseudoalteromonas strains which induced metamorphosis was 7,000 cells mm−2 in laboratory assays, which is on the order of 0.1 –1% of the total numbers of bacteria typically found on such surfaces. These results, in which an actual cue from bacteria has been characterized for the first time, contribute significantly towards understanding the complex process of acroporid coral larval settlement mediated through epibiotic microbial biofilms on crustose coralline algae

A Chemical Genetic Screen for Modulators of Asymmetrical 2,2′-Dimeric Naphthoquinones Cytotoxicity in Yeast

BACKGROUND: Dimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones. METHODOLOGY/PRINCIPAL FINDINGS: Exposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC(50)s in the microM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC(50). Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS). Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(P)H dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ. CONCLUSIONS/SIGNIFICANCE: In yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(P)H:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells

The Kolumbo submarine volcano of Santorini island is a large pool of bacterial strains with antimicrobial activity

Microbes in hydrothermal vents with their unique secondary metabolism may represent an untapped potential source of new natural products. In this study, samples were collected from the hydrothermal field of Kolumbo submarine volcano in the Aegean Sea, in order to isolate bacteria with antimicrobial activity. Eight hundred and thirty-two aerobic heterotrophic bacteria were isolated and then differentiated through BOX-PCR analysis at the strain level into 230 genomic fingerprints, which were screened against 13 different type strains (pathogenic and nonpathogenic) of Gram-positive, Gram-negative bacteria and fungi. Forty-two out of 176 bioactive-producing genotypes (76 %) exhibited antimicrobial activity against at least four different type strains and were selected for 16S rDNA sequencing and screening for nonribosomal peptide (NRPS) and polyketide (PKS) synthases genes. The isolates were assigned to genus Bacillus and Proteobacteria, and 20 strains harbored either NRPS, PKS type I or both genes. This is the first report on the diversity of culturable mesophilic bacteria associated with antimicrobial activity from Kolumbo area; the extremely high proportion of antimicrobial-producing strains suggested that this unique environment may represent a potential reservoir of novel bioactive compounds