REACTIVE OXYGEN SPECIES AS POSSIBLE MEDIATOR OF ANTIBACTERIAL ACTIVITY OF PARKIA JAVANICA, AGAINST BACTERIAL SPECIES PREDOMINANTLY FOUND IN CHRONIC WOUND.

The crude methanol extract of Parkia javanica was screened for antibacterial activity. against bacterial species predominantly found in chronic wound, by serial dilution technique. Growth kinetics study was performed and percentage of ROS production was measured by NBT reduction assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were obtained with a range of IC100 5-40 mg/ml in case of standard bacterial strains. The lag phase of all extract treated bacteria is extended compared to untreated cells. The normalized % of ROS is increased in presence of crude extract. This study suggests that the crude methanol extract of Parkia javanica possesses promising antimicrobial substances which are having activity against Standard ATCC bacterial species and ROS induced DNA damage could be the possible mediator of its antimicrobial activity. Keywords: Parkia javanica, antibacterial activity, standard ATCC bacterial strains, growth curve, ROS, DNA damag

Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks

WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and −467 in WRN as major phosphorylation sites mediated by DNA-PK. In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and −467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair

Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro , and Its Catalytic Activities Are Regulated by Phosphorylation

Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 3'-->5' exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb

In Vitro Structural and Functional Evaluation of Gold Nanoparticles Conjugated Antibiotics

Bactericidal efficacy of gold nanoparticles conjugated with ampicillin, streptomycin and kanamycin were evaluated. Gold nanoparticles (Gnps) were conjugated with the antibiotics during the synthesis of nanoparticles utilizing the combined reducing property of antibiotics and sodium borohydride. The conjugation of nanoparticles was confirmed by dynamic light scattering (DLS) and electron microscopic (EM) studies. Such Gnps conjugated antibiotics showed greater bactericidal activity in standard agar well diffusion assay. The minimal inhibitory concentration (MIC) values of all the three antibiotics along with their Gnps conjugated forms were determined in three bacterial strains,Escherichia coli DH5α,Micrococcus luteusandStaphylococcus aureus. Among them, streptomycin and kanamycin showed significant reduction in MIC values in their Gnps conjugated form whereas; Gnps conjugated ampicillin showed slight decrement in the MIC value compared to its free form. On the other hand, all of them showed more heat stability in their Gnps conjugated forms. Thus, our findings indicated that Gnps conjugated antibiotics are more efficient and might have significant therapeutic implications

The processing of Holliday junctions by BLM and WRN helicases is regulated by p53.

BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373-383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser(376) or Ser(378) completely abolishes this inhibition. Following blockage of DNA replication, Ser(15) phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases

Nucleolin Inhibits G4 Oligonucleotide Unwinding by Werner Helicase

The Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA).These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Virulence gene profiles, biofilm formation, and antimicrobial resistance of Vibrio cholerae non-O1/non-O139 bacteria isolated from West Bengal, India

Vibrio cholerae is the causative agent of acute dehydrating diarrhoeal disease cholera. Among 71 V. cholerae non-O1/non-O139 isolates, all yielded negative results for ctxA, ctxB and tcpA genes in PCR assay. Few strains were positive for stn (28.38%), and ompU (31.08%) genes. While all isolates were negative for ace gene, only two were positive for zot gene. All strains expressed toxR and toxT genes. It was also found that all isolates were slime-producer and these were capable of forming moderate to high biofilm. Biofilm formation was controlled positively by the transcriptional regulators VpsR and VpsT and was regulated negatively by HapR, as well as CRP regulatory complex. These isolates were resistant to ampicillin, furazolidone, doxycycline, vancomycin, erythromycin, while these were susceptible to ciprofloxacin, gentamycin, kanamycin, polymixin B, norfloxacin, chloramphenicol, sulphamethoxazole-trimethoprim, tetracycline, nalidixic acid, and streptomycin. Indeed, 69.01% isolates were resistant to multiple antibiotics (MAR: resistance to 3 or more antibiotics). Treatment protocols for cholera patients should be based on local antibiogram data

Anti-leukemic activity of betulinic acid from bulk to self-assembled structure

The Ziziphus jujuba tree is one of the major sources of betulinic acid (BA). After isolation, the bulk structure of the compound was converted to a self-assembled nanofibers (SA-BA) configuration which showed better anti-leukemic efficacy than its bulk form. After internalization in leukemic cells, SA-BA elevated reactive oxygen species (ROS) and pro-inflammatory cytokine secretion which ultimately activated apoptosis pathway. The SA-BA showed potent ameliorative role against acute chemotherapeutic toxicity induced by doxorubicin in human peripheral blood lymphocytes through the mechanism totally opposite to said pathway. Thus, SA-BA showed cell specific distinct effects. It was also revealed that the SA-BA had potent immunomodulatory affected on T cells and macrophages by polarizing the cytokine balance toward Th1 at a slightly higher dose. SA-BA arrested the growth of in vivo cancer by increasing the CD4 + cells in associated with increased cytotoxic T-cell response. SA-BA was also selectively internalized in folate receptor overexpressing leukemic cells. For this purpose, folic acid (FA) and polyethylene glycol (PEG) were conjugated on the nanostructured of SA-BA. After internalization, the conjugate (FA-PEG-SA-BA) diminished the cellular redox system and generated an excess amount of ROS which induced tumor necrosis factor-alpha-mediated cell death through activation of caspase 8 and 3 cascade system. Throughout all these studies, no toxic effects of the conjugates toward normal cells were observed. Thus, the whole study enlightens the multifunctional role of SA-BA in different aspects of anti-leukemic therapy which may be useful in future treatment policies