The C-terminal domain of the Escherichia coli RNA polymerase α subunit plays a role in the CI-dependent activation of the bacteriophage λ pM promoter

The bacteriophage λ pM promoter is required for maintenance of the λ prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the λ repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of pM. CI activates pM through cooperative binding to the OR1 and OR2 sites within the OR operator, with the OR2-bound CI dimer making contact with domain 4 of the RNA polymerase σ subunit (σ4). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase α subunit (αCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of pM. We also show that the location of αCTD at the pM promoter changes in the presence of CI. Thus, in the absence of CI, one αCTD is located on the DNA at position −44 relative to the transcription start site, whereas in the presence of CI, αCTD is located at position −54, between the CI-binding sites at OR1 and OR2. These results suggest that contacts between CI and both αCTD and σ are required for efficient CI-dependent activation of pM

A role for accessory genes rI.-1 and rI.1 in the regulation of lysis inhibition by bacteriophage T4

Lysis inhibition (LIN) is a known feature of the T-even family of bacteriophages. Despite its historical role in the development of modern molecular genetics, many aspects of this phenomenon remain mostly unexplained. The key element of LIN is an interaction between two phage-encoded proteins, the T holin and the RI antiholin. This interaction is stabilized by RIII. In this report, we demonstrate the results of genetic experiments which suggest a synergistic action of two accessory proteins of bacteriophage T4, RI.-1, and RI.1 with RIII in the regulation of LIN

Regulation of the switch from early to late bacteriophage lambda DNA replication

There are two modes of bacteriophage k DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (teta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wildtype cells the replication starting from orik proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from orik is predominantly unidirectional. The regulation of directionality of replication from orik is mediated by positive control of lambda pR promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the ptet promoter instead of pR was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional teta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional teta mechanism early after infection due to efficient transcriptional activation of ori lambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of teta genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of orik resulting in unidirectional teta replication followed by sigma type replication

S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux

Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells

Effects of Diet and Exercise on Endocrine Function of Skeletal Muscle

Skeletal muscle has been recognized as an endocrine tissue that releases appreciable amounts of circulating proteins, called myokines. Currently, we know that the skeletal muscles synthesize several hundreds of peptides classified as myokines, and muscle contraction stimulates their release [1,2]. Myokines can act in autocrine, paracrine or endocrine mode and there is an increasing number of data showing that they can affect different organs and tissues, e.g., the brain, bones, adipocyte tissue, heart artery, and many others [3]. For instance, the interleukins IL-6 and IL-10, released by the muscles during exercise, exert powerful local and systemic anti-inflammatory effects. Furthermore, IL-10 has been shown to provide cardio-and neuroprotection, which is mediated by the activation of anti-apoptotic protein kinase B (PKB or Akt) [4,5]. In addition, myokines like SPARC and oncostatin M show inhibitory activity against colon and breast cancer cells, respectively. Skeletal muscles represent the largest organ of the human body (the muscles constitute approximately 40% of total body mass), thus their role in the regulation of metabolic processes via myokines appears to be very important. Unfortunately, there is a limited amount of data demonstrating the effects of nutraceuticals on exercise-induced release of myokines. It has been shown that release of IL-6 from skeletal muscle was inhibited in persons supplemented with vitamin C and E. We hypothesize that natural compounds may exert their protective activity against some human diseases by modulating myokine synthesis

Influence of Hypoxia on Radiosensitization of Cancer Cells by 5-Bromo-2′-deoxyuridine

Radiotherapy is a crucial cancer treatment, but its outcome is still far from satisfactory. One of the reasons that cancer cells show resistance to ionizing radiation is hypoxia, defined as a low level of oxygenation, which is typical for solid tumors. In the hypoxic environment, cancer cells are 2–3 times more resistant to ionizing radiation than normoxic cells. To overcome this important impediment, radiosensitizers should be introduced to cancer therapy. When modified with an electrophilic substituent, nucleosides may undergo efficient dissociative electron attachment (DEA) that leaves behind nucleoside radicals, which, in secondary reactions, are able to induce DNA damage, leading to cancer cell death. We report the radiosensitizing effect of one of the best-known DEA-type radiosensitizers—5-bromo-2′-deoxyuridine (BrdU)—on breast (MCF-7) and prostate (PC3) cancer cells under both normoxia and hypoxia. MCF-7 and PC3 cells were treated with BrdU to investigate the effect of hypoxia on cell proliferation, incorporation into DNA and radiosensitivity. While the oxygen concentration did not significantly affect the efficiency of BrdU incorporation into DNA or the proliferation of tumor cells, the radiosensitizing effect of BrdU on hypoxic cells was more evident than on normoxic cells. Further mechanistic studies performed with the use of flow cytometry showed that under hypoxia, BrdU increased the level of histone H2A.X phosphorylation after X-ray exposure to a greater extent than under normal oxygenation conditions. These results confirm that the formation of double-strand breaks in hypoxic BrdU-treated cancer cells is more efficient. In addition, by performing stationary radiolysis of BrdU solution in the presence of an ●OH radical scavenger, we compared the degree of its electron-induced degradation under aerobic and anaerobic conditions. It was determined that radiodegradation under anaerobic conditions was almost twice as high as that under aerobic conditions

Antibacterial and anticancer activities of acetone extracts from in vitro cultured lichen-forming fungi

Abstract Background Lichens that were used in traditional medicine for ages produce numerous secondary metabolites, however our knowledge about biological activities of substances secreted by separated bionts is scarce. The main objectives of this study were to isolate and find optimal conditions for the growth of mycelia from three common lichen-forming fungi, i.e. Caloplaca pusilla, Protoparmeliopsis muralis and Xanthoria parietina and to evaluate antibacterial and antiproliferative activities of their acetone extracts. Methods Agar disc diffusion and broth microdilution methods were used to test antimicrobial activity against six species of bacteria. MTT method, flow cytometry assay and DAPI staining were applied to test antiproliferative activity of selected extracts against MCF-7 (human breast adenocarcinoma), PC-3 (human prostate cancer) and HeLa (human cervix adenocarcinoma) cancer cells. Results P. muralis strongly inhibited the growth of Gram-positive bacteria, i.e. Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis (MICs from 6.67 to 100.00 μg mL−1). X. parietina grown on PDA and G-LBM media decreased HeLa or MCF-7 cancer cells viability with IC50 values of about 8 μg mL−1, while C. pusilla grown on G-LBM medium showed the highest potency in decreasing MCF-7 (7.29 μg mL−1), PC-3 (7.96 μg mL−1) and HeLa (6.57 μg mL−1) cancer cells viability. We also showed induction of apoptosis in HeLa, PC-3 and MCF-7 cell lines treated with increasing concentrations of C. pusilla extract. Conclusion We showed that selected acetone extracts demonstrated a strong antimicrobial and anticancer effects that suggests that aposymbiotically cultured lichen-forming fungi can be a source of antibacterial and antiproliferative compounds

PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity.

Bacillus subtilis is a Gram-positive bacterium with a relatively large number of protein phosphatases. Previous studies have shown that some Ser/Thr phosphatases play an important role in the life cycle of this bacterium [Losick and Stragier (1992) Nature (London) 355, 601-604; Yang, Kang, Brody and Price (1996) Genes Dev. 10, 2265-2275]. In this paper, we report the biochemical properties of a putative, previously uncharacterized phosphatase, PrpE, belonging to the PPP family. This enzyme shares homology with other PPP phosphatases as well as with symmetrical diadenosine tetraphosphatases related to ApaH (symmetrical Ap(4)A hydrolase) from Escherichia coli. A His-tagged recombinant PrpE was purified from E. coli and shown to have Ni(2+)-dependent and okadaic acid-resistant phosphatase activity against a synthetic phosphorylated peptide and hydrolase activity against diadenosine 5',5"'-tetraphosphate. Unexpectedly, PrpE was able to remove phosphate from phosphotyrosine, but not from phosphothreonine or phosphoserine