An Antioxidant Extract of Tropical Lichen,Parmotrema reticulatum,Induces Cell Cycle Arrest and Apoptosis in Breast Carcinoma Cell Line MCF-7

This report highlights the phytochemical analysis, antioxidant potential and anticancer activity against breast carcinoma of 70% methanolic extract of lichen, Parmotrema reticulatum (PRME). Phytochemical analysis of PRME confirms the presence of various phytoconstituents like alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, tannins, anthraquinones, and ascorbic acid; among which alkaloids, phenols and flavonoids are found in abundant amount. High performance liquid chromatography (HPLC) analysis of PRME revealed the presence of catechin, purpurin, tannic acid and reserpine. Antioxidant activity was evaluated by nine separate methods. PRME showed excellent hydroxyl and hypochlorous radical scavenging as well as moderate DPPH, superoxide, singlet oxygen, nitric oxide and peroxynitrite scavenging activity. Cytotoxicity of PRME was tested against breast carcinoma (MCF-7), lung carcinoma (A549) and normal lung fibroblast (WI-38) using WST-1 method. PRME was found cytotoxic against MCF-7 cells with an IC₅₀ value 130.03±3.11 µg/ml while negligible cytotoxicity was observed on A549 and WI-38 cells. Further flow cytometric study showed that PRME halted the MCF-7 cells in S and G2/M phases and induces apoptosis in dose as well as time dependent manner. Cell cycle arrest was associated with downregulation of cyclin B1, Cdk-2 and Cdc25C as well as slight decrease in the expression of Cdk-1 and cyclin A1 with subsequent upregulation of p53 and p21. Moreover PRME induced Bax and inhibited Bcl-2 expression, which results in increasing Bax/Bcl-2 ratio and activation of caspase cascade. This ultimately leads to PARP degradation and induces apoptosis in MCF-7 cells. It can be hypothesised from the current study that the antioxidant and anticancer potential of the PRME may reside in the phytoconstitutents present in it and therefore, PRME may be used as a possible source of natural antioxidant that may be developed to an anticancer agent.     

Tristetraprolin Mediates Radiation-Induced TNF-α Production in Lung Macrophages

The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP^Ser178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.     

Polysaccharide-rich fraction of Termitomyces eurhizus accelerate healing of indomethacin induced gastric ulcer in mice

The current study aims to determine the healing activity of water soluble polysaccharide-rich fraction of a wild mushroom, Termitomyces eurhizus (TEps) against the indomethacin induced gastric ulceration in mice model. Gastric tissue histology, myeloperoxidase (MPO) activity, cyclooxygenases (COX) 1 and 2 expression, prostaglandin E₂ (PGE₂) synthesis, and modulation of pro/anti inflammatory cytokines expression were studied for this purpose. Histological study shows that TEps (20 mg/kg) effectively healed the gastric ulceration. Based on biochemical results, the healing capacities of TEps could be attributed to reduction of MPO activity and protection of mucosal mucin content. Enhanced synthesis of PGE₂ by modulation of COX-1 and COX-2 expression and a prominent shift of cytokines expression from pro (TNF-α, IL-1ß) to anti inflammatory (IL-10) side are also held responsible for ulcer healing. The preliminary study highlights the anti-ulcerogenic property of polysaccharide-rich fraction of Termitomyces eurhizus and opens an alternative cure for NSAID induced gastroduodenal diseases.     

Variations in the Rhythms of Respiration and Nitrogen Fixation in Members of the Unicellular Diazotrophic Cyanobacterial Genus Cyanothece

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.Nitrogen fixation is an important global phenomenon by which molecular nitrogen, one of the most abundant components of the earth’s atmosphere, is converted into a more reduced form suitable for incorporation into living systems. The majority of this nitrogen fixation is achieved by biological means through the activity of microorganisms (Burris and Roberts, 1993; Raymond et al., 2004; Rubio and Ludden, 2008). This process is energy intensive, and nitrogenase, the enzyme complex involved in the biological nitrogen fixation reaction, is generally known to be extremely sensitive to oxygen (Robson and Postgate, 1980; Hill et al., 1981; Berman-Frank et al., 2005). Thus, most microbes participating in this process fix nitrogen only when suitable anaerobic or microaerobic conditions are established in an otherwise oxygen-rich environment. However, some nitrogen-fixing (diazotrophic) microbes have the advantage of being able to fix nitrogen in aerobic environments. Outstanding among these are the photosynthetic prokaryotes called cyanobacteria, an extremely successful group of microbes with plant-like traits. These microbes are considered to be the progenitors of plant chloroplasts. Cyanobacteria perform both oxygen-evolving photosynthesis and oxygen-sensitive nitrogen fixation, thereby providing a platform to power the most metabolically expensive biological process (Simpson and Burris, 1984) with solar energy.Among the nitrogen-fixing cyanobacteria, filamentous strains have been extensively studied for their contribution to the nitrogen cycle in marine and terrestrial ecosystems (Mulligan and Haselkorn, 1989; Kaneko et al., 2001; Meeks et al., 2001; Sañudo-Wilhelmy et al., 2001; Wong and Meeks, 2001; Gomez et al., 2005). Some of these filamentous strains develop specialized cells called heterocysts that allow the spatial segregation of photosynthesis and nitrogen fixation. These heterocysts also have higher rates of respiratory oxygen consumption, which results in a virtually anoxic environment conducive for the nitrogenase enzyme (Bergman et al., 1997). All heterocystous strains are known to fix nitrogen aerobically. In contrast, nonheterocystous cyanobacteria lack any specialized oxygen-free compartments and often require incubation under microoxic or anaerobic conditions for nitrogen fixation (Rippka and Waterbury, 1977; Rippka et al., 1979; Brass et al., 1992). However, some nonheterocystous cyanobacterial strains can fix nitrogen under aerobic conditions. These include some filamentous genera like Trichodesmium spp., Lyngbya spp., and Oscillatoria spp. (Jones, 1990; Janson et al., 1994; Finzi-Hart et al., 2009) as well as unicellular genera like Gloeothece spp. and Cyanothece spp. (Wyatt and Silvey, 1969; Rippka and Waterbury, 1977; Huang and Chow, 1988; Van Ni et al., 1988; Schütz et al., 2004).In comparison with filamentous cyanobacteria, which have long been recognized for their nitrogen-fixing ability, the importance of unicellular cyanobacteria as key components of the environmental nitrogen cycle has only been recently uncovered. Studies over the last decade have established unicellular strains like Crocosphaera spp., Cyanothece spp., and UCYN-A as important players in the marine nitrogen cycle (Zehr et al., 2001; Montoya et al., 2004; Zehr, 2011). Since unicellular diazotrophic cyanobacteria utilize the same cellular platform for photosynthesis and nitrogen fixation, they are required to adjust their cellular metabolism to accommodate these two antagonistic processes. Systems-level studies in the unicellular genus Cyanothece have revealed a temporal separation of the two processes, photosynthesis occurring during the day and nitrogen fixation occurring at night (Stöckel et al., 2008; Toepel et al., 2008; Welsh et al., 2008). Cellular respiration plays a critical role during the transition from one phase to the next, rapidly freeing the intracellular environment of the photosynthetically generated oxygen and rendering it conducive for the induction of nitrogenase activity. In addition, respiration also sustains the process of nitrogen fixation, not only by maintaining a low-oxygen environment required for the functioning of the nitrogenase enzyme but also by mobilizing the stored solar energy to fuel this energy-intensive process.Unicellular diazotrophs exhibit great diversity in the efficiency of nitrogen fixation as well as in the physiological regulation of the process. For instance, members of the genus Gloeothece fix nitrogen aerobically during the day, but at 0% dissolved oxygen concentration, nitrogen fixation is shifted entirely to the dark period (Ortega-Calvo and Stal, 1991; Taniuchi et al., 2008). In contrast, some Synechococcus spp. strains can fix nitrogen only when incubated under anoxic conditions (Steunou et al., 2006). Members of the genus Cyanothece have been reported to engage in both aerobic and anaerobic nitrogen fixation, with nitrogenase activity peaking during the night (Reddy et al., 1993; Bergman et al., 1997; Turner et al., 2001). This suggests that, in addition to the regulations imposed by the diurnal cycle, strain-specific intracellular cues govern the process of nitrogen fixation in unicellular cyanobacteria, which may vary according to the genotype or the ecotype of the strains.Members of the unicellular cyanobacterial genus Cyanothece are diazotrophs that thrive in marine as well as terrestrial environments. This genus was originally grouped together with Synechococcus spp. but was later separated on the basis of distinct morphological and biochemical differences between the two genera (Komárek, 1976; Rippka and Cohen-Bazire, 1983). Some of the features that define the largely heterogeneous genus Cyanothece are oval to cylindrical cells, larger than 3 µm in size (they can be as large as 24 µm in diameter), radially arranged thylakoids, and a mucilaginous layer surrounding the cells (Komárek and Cepák, 1998; Porta et al., 2000; Liberton et al., 2011).It was recently demonstrated that Cyanothece sp. ATCC 51142, a member of the genus Cyanothece, has the unique ability to produce molecular hydrogen at exceptionally high rates under aerobic conditions (Bandyopadhyay et al., 2010). This striking observation was attributed to the nitrogenase enzyme system of Cyanothece sp. ATCC 51142. Our study also indicated that high rates of respiration in this strain might contribute to its nitrogenase-mediated aerobic hydrogen production. Glycerol was found to be an efficient source of reductants and energy for this process. In an effort to investigate if this atypical cyanobacterial trait was a characteristic of the genus Cyanothece, five additional Cyanothece spp. strains from different ecological habitats were sequenced to completion. The six strains display more than 90% identity in their 16S ribosomal RNA sequence but exhibit striking variability with respect to their genome sizes (with the largest genome being 7.8 Mb and the smallest being 4.4 Mb), the number of plasmids, and the percentage of pseudogenes (Bandyopadhyay et al., 2011). In addition, two of the strains possess linear chromosomal elements, features not known to occur in any other photosynthetic bacteria sequenced to date, which may impart niche-specific advantages to these strains. Analysis of the genome sequence of the Cyanothece spp. strains showed the presence of a nitrogenase gene cluster in all five strains, and preliminary analysis showed that four of the five strains were capable of aerobic nitrogen fixation and hydrogen production (Bandyopadhyay et al., 2011). In this study, we have focused on the patterns of nitrogen fixation and respiration in six different Cyanothece spp. strains in an effort to elucidate the underlying differences and similarities in these processes in unicellular diazotrophic strains with similar genotypic but varied ecological backgrounds. Our study reveals inherent differences in the regulation of these processes, which are likely controlled by strain-specific cellular signals. However, despite the differences in the patterns of nitrogenase activity, aerobic nitrogen fixation and hydrogen production was found to be a characteristic of this genus, with most members exhibiting nitrogenase-mediated hydrogen production at rates higher than any other wild-type cyanobacterial strain.     

Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress

Peptidase N (PepN), the sole M1 family member in Escherichia coli, displays broad substrate specificity and modulates stress responses: it lowers resistance to sodium salicylate (NaSal)-induced stress but is required during nutritional downshift and high temperature (NDHT) stress. The expression of PepN does not significantly change during different growth phases in LB or NaSal-induced stress; however, PepN amounts are lower during NDHT stress. To gain mechanistic insights on the roles of catalytic activity of PepN in modulating these two stress responses, alanine mutants of PepN replacing E264 (GAMEN motif) and E298 (HEXXH motif) were generated. There are no major structural changes between purified wild type (WT) and mutant proteins, which are catalytically inactive. Importantly, growth profiles of ΔpepN upon expression of WT or mutant proteins demonstrated the importance of catalytic activity during NDHT but not NaSal-induced stress. Further fluorescamine reactivity studies demonstrated that the catalytic activity of PepN is required to generate higher intracellular amounts of free N-terminal amino acids; consequently, the lower growth of ΔpepN during NDHT stress increases with high amounts of casamino acids. Together, this study sheds insights on the expression and functional roles of the catalytic activity of PepN during adaptation to NDHT stress.     

The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.