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Cellular senescence is an irreversible side effect of some pharmaceuticals which can contribute to tissue degeneration. Objective To determine whether pharmaceutical glucocorticoids induce senescence in tenocytes. Methods Features of senescence (β-galactosidase activity at pH 6 (SA-β-gal) and active mammalian/mechanistic target of rapamycin (mTOR) in cell cycle arrest) as well as the activity of the two main pathways leading to cell senescence were examined in glucocorticoid-treated primary human tenocytes. Evidence of senescence-inducing pathway induction in vivo was obtained using immunohistochemistry on tendon biopsy specimens taken before and 7 weeks after subacromial Depo-Medrone injection. Results Dexamethasone treatment of tenocytes resulted in an increased percentage of SA-βgal-positive cells. Levels of phosphorylated p70S6K did not decrease with glucocorticoid treatment indicating mTOR remained active. Increased levels of acetylated p53 as well as increased RNA levels of its pro-senescence effector p21 were evident in dexamethasone-treated tenocytes. Levels of the p53 deacetylase sirtuin 1 were lower in dexamethasone-treated cells compared with controls. Knockdown of p53 or inhibition of p53 activity prevented dexamethasone-induced senescence. Activation of sirtuin 1 either by exogenous overexpression or by treatment with resveratrol or low glucose prevented dexamethasone-induced senescence. Immunohistochemical analysis of tendon biopsies taken before and after glucocorticoid injection revealed a significant increase in the percentage of p53-positive cells (p=0.03). The percentage of p21-positive cells also tended to be higher post-injection (p=0.06) suggesting glucocorticoids activate the p53/p21 senescence-inducing pathway in vivo as well as in vitro. Conclusion As cell senescence is irreversible in vivo, glucocorticoid-induced senescence may result in long-term degenerative changes in tendon tissue.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert -butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

Employing the tetracycline repressor tetR and the wild-type hCMV major immediate-early promoter, we have developed a highly sensitive tetracycline-inducible transcription switch in mammalian cells (T-REx; Invitrogen, Carlsbad, CA, USA). In view of the previous difficulty in achieving regulatable gene expression in recombinant HSV vector systems, we constructed a T-REx-encoding replication-defective HSV-1 recombinant, QR9TO-lacZ, that encodes two copies of the tetR gene controlled by the HSV-1 immediate-early ICP0 promoter and a reporter, the LacZ gene, under the control of the tetO-bearing hCMV major immediate-early promoter. Infection of cells, such as Vero, PC12, and NGF-differentiated PC12 cells, with QR9TO-lacZ led to 300- to 1000-fold tetracycline-regulated gene expression. Moreover, the expression of the LacZ gene by QR9TO-lacZ can be finely controlled by tetracycline in a dose-dependent fashion. Efficiently regulated gene expression can also be achieved in vivo following intracerebral and footpad inoculations in mice. The demonstrated capability of T-REx for achieving high levels of sensitively regulated gene expression in the context of the HSV-1 genome will significantly expand the utility of HSV-based vector systems for studying gene function in the nervous system and delivering regulated gene expression in therapeutic applications, particularly in the treatment of CNS diseases.

To investigate the efficient transduction of tumor cells which remains a major limitation of cancer gene therapy. In this study, we tested whether treatment with antimetabolic drugs 5-FU and hydroxyurea (HU) could improve adenovirus-mediated gene expression in tumor cells. We found that 5-FU and HU treatment significantly increased beta-gal activity in adenovirus (Ad.CMVBG)-infected human colon carcinoma (LoVo) and hepatocellular carcinoma (SMMC7721) cells in a dose- and time-dependent manner. These increases were maximized at 5.01+/-0.42-fold and 3.32+/-0.32-fold for 5-FU (50 microM), and at 6.60+/-0.50-fold and 4.82+/-0.43-fold for HU (5 mM) treatment, respectively, after 48 h infection. Transient increases in viral uptake, determined by real-time PCR for viral DNA content and by confocal microscopy for viral particles, were observed in 5-FU or HU-treated cells that partially contribute to the overall increases of beta-gal expression. Moreover, mRNA levels for the beta-gal gene in infected cells were significantly increased in both LoVo and SMMC7721 cells by 5-FU and HU treatment in contrast to the inhibition of viral DNA replication and the unchanged mRNA levels for alpha-actin gene. The induction appeared to be the result of enhanced transcription since beta-gal mRNA half-life was not affected by drug treatment. However, similar induction was not detected in CMV-beta-gal-expressing stable cells, suggesting that an adenovirus-associated mechanism might be involved in this induction. Our findings suggest that it may be possible to improve tumor cell transduction by adenovirus using chemotherapy.

Abstract We have identified the cuiD gene in Salmonella enterica serova Typhimurium that codes for a putative multicopper oxidase. Expression of cuiD is induced by copper ion and its promoter/operator has sequence similarity to the promoters controlled by the transcriptional regulators of the MerR family. We also identified and isolated a gene from S. enterica serova Typhimurium that encodes a 138-amino acid residue protein, sctR, a new member of the MerR family of transcriptional regulators. Transposon-insertional disruption of sctR shows sensitivity to copper ion and no response of cuiD expression. Copper-responsive induction and copper tolerance were restored by providing sctR in trans, suggesting that SctR plays an important role in intracellular copper detoxification.

Abstract Escherichia coli growing under anaerobic conditions produces H2 and CO2 by the enzymatic cleavage of formate catalyzed by formate hydrogenlyase (FHL) consisting of a molybdoenzyme formate dehydrogenase H (fdhF), hydrogenase 3 (hyc), and intermediate electron carriers (hyc). Transcription of both the fdhF and hyc operons requires the activator, FhlA protein, as well as formate and molybdate. Several fhlA mutants with an altered response to the required effector molybdate were isolated and these FhlA mutated proteins activated hyc transcription in the absence of molybdate, but only in the presence of formate. Mutated protein FhlA126 carries a single mutation (R495C) in the conserved central domain of the modular, σ54-dependent, enhancer-binding protein. FhlA57 contains two mutations; one in the unique N-terminal domain (E205K) and a second in the central domain (P442S). Both mutations in FhlA132 are located in the N-terminal domain (A42T and E363K). Both FhlA126 and FhlA132 proteins activated the hyc operon even in the absence of ModE and MoeA, two components of Mo-metabolism which are required for hyc-lac expression in wild-type E. coli. Based on these results, a model is proposed in which the native FhlA protein interacts with a unique form of Mo (MoeA product?) as a second effector for optimum expression of the hyc operon in E. coli.

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus cultures were treated with ethanol and tested for viability and beta-galactosidase activity. Exposure of the biomass of test cultures to 30%-55% ethanol (vol/vol) caused a 100% loss of viability and up to 15-fold increase in measurable beta-galactosidase activity in both streptococci and lactobacilli. Ethanol-treated cell suspensions could be stored for up to 6 months without loss of enzyme activity. The nonviable permeabilized biomass of the more active S. thermophilus was used to achieve up to 80% hydrolysis of lactose in aqueous solutions and non-fat milk

The influence of carbonation on the evolution of lactose, galactose and glucose in fermented milks with added probiotic bacteria (Lactobacillus casei, Lactobacillus acidophilus and/or Bifidobacterium bifidum) was evaluated and related to β-galactosidase activity of starter strains. During incubation and first days of refrigeration, lactose hydrolysis resulting in the liberation of galactose and glucose occurred in CT (Streptococcus thermophilus/Lb. casei), AT (Str. thermophilus/Lb. acidophilus) and ABT fermented milks (Str. thermophilus/Lb. acidophilus/Bifid. bifidum). Levels of galactose were higher than those of glucose and could be related to the preferential consumption of glucose by actively growing bacteria. Through the incubation, lactose and monosaccharide levels were not affected by milk carbonation. However, during refrigerated storage the presence of this gas was associated with slightly lower content of lactose and higher levels of galactose and glucose in AT and ABT products but not in CT fermented milks. Through the refrigeration galactose was moderately utilised by Lb. acidophilus in AT products whereas the presence of Bifid. bifidum seems to prevent the consumption of this sugar in ABT fermented milks. Glucose remained constant, with minor variations in CT products but a continuous increase of this sugar occurred in carbonated AT and ABT fermented milks during storage. β-Galactosidase activity displayed by Str. thermophilus strains was similar at pH 6·5 (initial pH of non-carbonated samples) and pH 6·3 (initial pH of carbonated samples) whereas Lb. acidophilus LaA3 showed greater β-galactosidase activity at pH 6·3 than at higher pH values. Thus, the enhanced metabolic activity of Lb. acidophilus caused by the low initial pH of carbonated milk also promoted higher cellular β-galactosidase activity that could have released greater amounts of galactose and glucose from lactose in AT and ABT fermented milks through the refrigerated period. In CT fermented milks, similar β-galactosidase activity levels of Str. thermophilus at pH 6·5 and 6·3 together with the absence of β-galactosidase activity in Lb. casei could explain the lack of differences on glucose and galactose content between carbonated and non-carbonated samples.

Abstract The Pas protein plays a key role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC), being required for the secretion of the Esp proteins. Here, the transcriptional regulation of the pas gene was analyzed through the construction of a pas::lacZ translational fusion. When bacteria were grown in Luria Bertani medium or tissue culture medium supplemented with HEPES, a bimodal activation curve was observed. The early phase of induction was not significantly modified by the incubation temperature (either 25 or 37°C), whereas the second phase, which overlaps with the late exponential growth phase, was enhanced at 37°C. The early phase was also stimulated by growth on tissue culture medium and by the addition of Ca2+, Mn2+or Mg2+ to the M9-glucose minimal medium. Primer extension analysis showed the presence of two major starts of transcription, which were located 58 and 60 bp upstream of the ATG-start codon of the Pas protein, respectively. Although these sites are very close to each other, the transcripts produced during the early induction phase mainly start on the −60 position, whereas the −58 start was activated during the second induction phase.