Free radical scavenging activity,total phenolic content,total antioxidant status,and total oxidant status of endemic Thermopsis turcica

Thermopsis turcica, endemic to Turkey, is in danger of extinction. Studies on this species are very few due to the fact that it was only discovered in 1983 and grows in a small circumscribed area in Turkey. In this study, free radical scavenging activity, total phenolic content, total oxidant status (TOS), and total antioxidant status (TAS) of methanol (TTM) and acetone (TTA) extracts of T. turcica were measured spectroscopically. Free radical scavenging activity was determined according to the elimination of DPPH radicals and total phenol content was determined by the Folin–Ciocalteu reaction. Total oxidant status (TOS) and total antioxidant status (TAS) were measured with commercially available kits. Methanol and acetone extracts of T. turcica were found to have a specific radical scavenging effect. This effect was found to be related to the total phenolic content of the extracts. Since the TTA had a higher phenolic content than the methanol extract, it had a stronger radical scavenging effect. In addition, the total antioxidant capacity of the methanol extract was observed to be higher than that of its acetone counterpart. As a result, due to its antioxidative properties, T. turcica is thought to be a natural source of antioxidants.     

Three new species of the genus Probles F?rster (Hymenoptera,Ichneumonidae, Tersilochinae) from?South Korea

Three closely related species of the genus Probles Förster, P. fulgida sp. n., P. korusa sp. n. and P. rukora sp. n., belong to the subgenus Euporizon Horstmann and differ from other Palearctic species of the genus by a combination of long and apically weakly sinuate ovipositor and short temple. These three species are assigned to a newly designated fulgida species-group, and a portion of the key for identification of this species-group is provided. Based on the shape of the ovipositor apex, the fulgida species-group resemble members of the subgenus Microdiaparsis Horstmann but are distinct in having a much shorter temple.     

Ionizing radiation-inducible microRNA miR-193a-3p induces apoptosis by directly targeting Mcl-1

The functions of microRNAs (miRNAs) as either oncogenes or tumor suppressors in regulating cancer-related events have been established. We analyzed the alterations in the miRNA expression profile of the glioma cell line U-251 caused by ionizing radiation (IR) by using an miRNA array and identified several miRNAs whose expression was significantly affected by IR. Among the IR-responsive miRNAs, we further examined the function of miR-193a-3p, which exhibited the most significant growth-inhibiting effect. miR-193a-3p was observed to induce apoptosis in both U-251 and HeLa cells. We also demonstrated that miR-193a-3p induces the accumulation of intracellular reactive oxygen species (ROS) and DNA damage as determined by the level of γH2AX and by performing the comet assay. The induction of both apoptosis and DNA damage by miR-193a-3p was blocked by antioxidant treatment, indicating the crucial role of ROS in the action of miR-193a-3p. Among the putative target proteins, the expression of Mcl-1, an anti-apoptotic Bcl-2 family member, decreased because of miR-193a-3p transfection. A reporter assay using a luciferase construct containing the 3′-untranslated region of Mcl-1 confirmed that Mcl-1 is a direct target of miR-193a-3p. Down-regulation of Mcl-1 by siRNA transfection closely mimicked the outcome of miR-193a-3p transfection showing increased ROS, DNA damage, cytochrome c release, and apoptosis. Ectopic expression of Mcl-1 suppressed the pro-apoptotic action of miR-193a-3p, suggesting that Mcl-1 depletion is critical for miR-193a-3p induced apoptosis. Collectively, our results suggest a novel function for miR-193a-3p and its potential application in cancer therapy.     

p53 acetylation enhances Taxol-induced apoptosis in human cancer cells

Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.     

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.     

Grazing history influences biodiversity: a case study on ground-dwelling arachnids (Arachnida: Araneae, Opiliones) in the Natural Park of Alpi Marittime (NW Italy)

Alpine pastures are typical examples of “high nature value farmlands”, representing important habitats harbouring unique biological communities. Disturbance induced by overgrazing influences significantly ecosystem processes, in which invertebrates play a major role. To develop new models of sustainable management of pastures, more knowledge is needed of the animal communities, essential to the ecological functioning of pasture ecosystems. The apparent poor ecological state of several pastures in the Natural Regional Park of Alpi Marittime (NW-Italy) lead us to evaluate the influence of grazing history on biodiversity, using spider and harvestmen assemblages as key groups. Four different pastoral types characterized by four different grazing histories were identified using the Daget-Poissonet method. Spiders and harvestmen were collected by means of pitfall traps. Arachnid assemblages were characterized in terms of composition, abundance, species richness, richness of endemic species and taxonomic relatedness. Generalized linear mixed models (GLMM) were used to test differences among assemblages occurring in each pastoral type. Furthermore, we tested differences in terms of plant communities (species richness and percentage of zoogenic species). Specificity and fidelity of every spider and harvestmen species within pastoral types were explored by the IndVal (Indicator Value) procedure. Fifty-eight species of spiders and seven species of harvestmen were collected (2,304 individuals). Pastoral types related to intensive grazing were characterized by the dominance of diurnal wanderer spiders (namely Lycosidae) while, conversely, nocturnal wanderers (mainly Gnaphosidae) were more abundant in extensive pastoral types. Results show that both species richness and spider abundance were higher in abandoned areas of extensive grazing, while endemic assemblages were richer in extensive grazed areas, which also hosted the most diverse plant community. Furthermore, most of the indicator species of spiders of this type were endemic, characterized by more demanding ecological requirements.     

Mycobacterium kansasii-induced death of murine macrophages involves endoplasmic reticulum stress responses mediated by reactive oxygen species generation or calpain activation

Although pathogenic mechanisms of tuberculosis have been extensively studied, little is known about the pathogenic mechanisms of Mycobacterium kansasii. In this work the influence of virulence and ER-stress mediated apoptosis of macrophages during two different strains of M. kansasii infection was investigated. We show that M. kansasii infection is associated with ER stress-mediated apoptosis in the murine macrophage cell line RAW 264.7. Infection of RAW 264.7 cells in vitro with apoptosis-inducing a clinical isolate of M. kansasii SM-1 (SM-1) resulted in strong induction of ER stress responses compared with M. kansasii type strain (ATCC 12478)-infected RAW 264.7 cells. Interestingly, inhibition of calpain prevented the induction of CHOP and Bip in ATCC 12478-infected RAW 264.7 cells but not in RAW 264.7 cells infected with SM-1. In contrast, reactive oxygen species (ROS) were significantly increased only in RAW 264.7 cells infected with SM-1. We propose that ROS generation is important for triggering ER stress-mediated apoptosis during SM-1 infection, whereas ATCC 12478-induced, ER stress-mediated apoptosis is associated with calpain activation. Our results demonstrate that the ER stress pathway plays important roles in the pathogenesis of M. kansasii infections, and that different strains of M. kansasii induce different patterns of ER stress-mediated apoptosis.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Metabolic profiling and enhanced production of phytosterols by elicitation with methyl jasmonate and silver nitrate in whole plant cultures of Lemna paucicostata

In this study, the effects of methyl jasmonate (MJ) and silver nitrate (SN) treatment on metabolic profiles and yields of phytosterols such as campesterol, stigmasterol, and β-sitosterol in whole plant cultures of Lemna paucicostata were investigated using gas chromatography–mass spectrometry coupled with multivariate statistical analysis. The MJ and SN treatments retarded the growth of L. paucicostata plants, while they enhanced the yields of three phytosterols, compared to control. Higher yields of phytosterols were attained at day 28 compared to day 42. Moreover, stigmasterol yield was the highest at 0.85 mg/g from day 28 plants grown under MJ + SN co-treated culture. Among the various metabolites, the levels of palmitic and stearic acids, which might participate in a defense mechanism, were higher in the MJ + SN condition than in control. To determine the optimal timing of MJ + SN addition, MJ + SN was added on days 21, 28, and 35 after inoculation. The total yield and productivity of phytosterol reached maximum levels when the MJ + SN was added at day 35. The highest productivity of stigmasterol (6.08 mg/L) was also achieved when MJ + SN was added on day 35.