Assessment of Microbial Richness in Pelagic Sediment of Andaman Sea by Bacterial Tag Encoded FLX Titanium Amplicon Pyrosequencing (bTEFAP)

Microbial diversity of 1,000 m deep pelagic sediment from off Coast of Andaman Sea was analyzed by a culture independent technique, bacterial tag encoded FLX titanium amplicon pyrosequencing. The hypervariable region of small subunit ribosomal rRNA gene covering V6–V9, was amplified from the metagenomic DNA and sequenced. We obtained 19,271 reads, of which 18,206 high quality sequences were subjected to diversity analysis. A total of 305 operational taxonomic units (OTUs) were obtained corresponding to the members of firmicutes, proteobacteria, plantomycetes, actinobacteria, chloroflexi, bacteroidetes, and verucomicrobium. Firmicutes was the predominant phylum, which was largely represented with the family bacillaceae. More than 44 % of sequence reads could not be classified up to the species level and more than 14 % of the reads could not be assigned to any genus. Thus, the data indicates the possibility for the presence of uncultivable or unidentified novel bacterial species. In addition, the community structure identified in this study significantly differs with other reports from marine sediments.     

A DNA extraction procedure that allows mite specimens to be slide mounted: phytoseiid species evaluated as a model

Four protocols for extracting DNA from mites, using phytoseiid species as exemplars, were evaluated to determine whether the DNA obtained could be used to amplify nuclear, mitochondrial or Random Amplified Polymorphic DNA (RAPD) markers from males, females and eggs. Protocol 3 was identified as the best and this allowed High-fidelity PCR (Hf-PCR) and Hf-RAPD PCR to be used successfully; it left behind the intact body of adult mites so they could be slide mounted for morphological analyses, although the eggs had to be pricked in order to yield sufficient DNA for amplifications. Protocol 3 involved soaking intact specimens in a GuSCN buffer and using a silica matrix, which binds nucleic acids, to yield DNA for amplification. The DNA isolated could be stored up to a month, indicating that the quality was good. This DNA extraction protocol will allow researchers to collect mites, store them in 95% ethanol, and subsequently extract sufficient DNA from single adults or eggs to provide diagnostic PCR products from both nuclear and mitochondrial DNA, yet leave the bodies intact for morphological analyses.     

Genome Sequence of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida S11

Here we report the genome sequence of a plant growth-promoting rhizobacterium, Pseudomonas putida S11. The length of the draft genome sequence is approximately 5,970,799 bp, with a G+C content of 62.4%. The genome contains 6,076 protein-coding sequences.     

Superantigen profiles of emm and emm-like typeable and nontypeable pharyngeal streptococcal isolates of South India

Background

Toxins derived from jellyfishes have been exploited as a model for the development of new drug promising applications to treat neurodegenerative diseases. The present work is aimed to evaluate the acute toxicity of crude venom of Pelagia noctiluca and then to screen the analgesic and antibutyrylcholinestrasic (anti-BuChE) activities of the crude venom and its fractions.

Methods

Sephadex G75 gel was used to separate crude venom of Pelagia noctiluca, which led to some fractions. In addition, in vivo analgesic and in vitro plasma antibutyrylcholinestrasic activities were carried out with Pelagia crude venom and its fractions respectively.

Results

The crude venom and its fractions displayed analgesic and anti-BuChE activities at different doses without inducing acute toxicity. Fraction 2 possesses the highest analgesic and antibutyrylcholinestrasic properties. The crude venom and fraction 1 had shown to possess less significant inhibitory activity against analgesic and antibutyrylcholinestrasic models.

Conclusions

Based on this study, the crude venom of Pelagia noctiluca is found to be a useful tool for probing pharmacological activity. The purification and the determination of chemical structures of compounds of active fractions of the venom are under investigation.     

Aurora B controls kinetochore-microtubule attachments by inhibiting Ska complex-KMN network interaction

The KMN network (named according to the acronym for KNL1, Mis12, and Ndc80) and the more recently identified Ska complex (Ska1-3) have been shown to mediate kinetochore (KT)-microtubule (MT) attachments. How these two complexes cooperate to achieve stable end-on attachments remains unknown. In this paper, we show that Aurora B negatively regulates the localization of the Ska complex to KTs and that recruitment of the Ska complex to KTs depends on the KMN network. We identified interactions between members of the KMN and Ska complexes and demonstrated that these interactions are regulated by Aurora B. Aurora B directly phosphorylated Ska1 and Ska3 in vitro, and expression of phosphomimetic mutants of Ska1 and Ska3 impaired Ska KT recruitment and formation of stable KT-MT fibers (K-fibers), disrupting mitotic progression. We propose that Aurora B phosphorylation antagonizes the interaction between the Ska complex and the KMN network, thereby controlling Ska recruitment to KTs and stabilization of KT-MT attachments.     

Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.     

HP1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry

The chromosomal passenger complex (CPC) is directed to centromeres during mitosis via binding to H3T3ph and Sgo1. Whether and how heterochromatin protein 1α (HP1α) influences CPC localisation and function during mitotic entry is less clear. Here, we alter HP1α dynamics by fusing it to a CENP‐B DNA‐binding domain. Tethered HP1 strongly recruits the CPC, destabilising kinetochore–microtubule interactions and activating the spindle assembly checkpoint. During mitotic exit, the tethered HP1 traps active CPC at centromeres. These HP1‐CPC clusters remain catalytically active throughout the subsequent cell cycle. We also detect interactions between endogenous HP1 and the CPC during G₂. HP1α and HP1γ cooperate to recruit the CPC to active foci in a CDK1‐independent process. Live cell tracking with Fab fragments reveals that H3S10ph appears well before H3T3 is phosphorylated by Haspin kinase. Our results suggest that HP1 may concentrate and activate the CPC at centromeric heterochromatin in G₂ before Aurora B‐mediated phosphorylation of H3S10 releases HP1 from chromatin and allows pathways dependent on H3T3ph and Sgo1 to redirect the CPC to mitotic centromeres.     

Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes

Coordination of cell growth and proliferation in response to nutrient supply is mediated by mammalian target of rapamycin (mTOR) signaling. In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles. Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects. Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression.     

Oxidatively generated DNA damage after Cu(II) catalysis of dopamine and related catecholamine neurotransmitters and neurotoxins: Role of reactive oxygen species

There is increasing evidence supporting a causal role for oxidatively damaged DNA in neurodegeneration during the natural aging process and in neurodegenerative diseases such as Parkinson and Alzheimer. The presence of redox-active catecholamine neurotransmitters coupled with the localization of catalytic copper to DNA suggests a plausible role for these agents in the induction of oxidatively generated DNA damage. In this study we have investigated the role of Cu(II)-catalyzed oxidation of several catecholamine neurotransmitters and related neurotoxins in inducing oxidatively generated DNA damage. Autoxidation of all catechol neurotransmitters and related congeners tested resulted in the formation of nearly a dozen oxidation DNA products resulting in a decomposition pattern that was essentially identical for all agents tested. The presence of Cu(II), and to a lesser extent Fe(III), had no effect on the decomposition pattern but substantially enhanced the DNA product levels by up to 75-fold, with dopamine producing the highest levels of unidentified oxidation DNA products (383±46 adducts/10(6) nucleotides), nearly 3-fold greater than 8-oxo-7,8-dihydro-2'-deoxyguanosine (122±19 adducts/10(6) nucleotides) under the same conditions. The addition of sodium azide, 2,2,6,6-tetramethyl-4-piperidone, tiron, catalase, bathocuproine, or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease (>90%) in oxidation DNA product levels, indicating a role for singlet oxygen, superoxide, H(2)O(2), Cu(I), and Cu(I)OOH in their formation. Whereas the addition of N-tert-butyl-α-phenylnitrone significantly decreased (67%) dopamine-mediated oxidatively damaged DNA, three other hydroxyl radical scavengers, ascorbic acid, sodium benzoate, and mannitol, had little to no effect on these oxidation DNA product levels, suggesting that free hydroxyl radicals may have limited involvement in this dopamine/Cu(II)-mediated oxidatively generated DNA damage. These studies suggest a possible contributory role of oxidatively generated DNA damage by dopamine and related catechol neurotransmitters/neurotoxins in neurodegeneration and cell death. We also found that a naturally occurring broad-spectrum antioxidant, ellagic acid, was substantially effective (nearly 50% inhibition) at low doses (1μM) at preventing this dopamine/Cu(II)-mediated oxidatively generated DNA damage. Because dietary ellagic acid has been found to reduce oxidative stress in rat brains, a neuroprotective role of this polyphenol is plausible.