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  • Open access
  • 90 Reads
Vacuolar Sorting Mechanisms are Differently Influenced by Detoxification Processes

Glyphosate is a non-selective herbicide that inhibits the shikimate pathway’s enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) preventing the production of aromatic amino acids. This herbicide is largely used and appreciated because it controls a wide range of annual and perennial weeds but it has a minimal environmental impact when compared with other herbicides. Initially it was thought that resistance to glyphosate was not easy to evolve but the continuous applications, as it happened for other herbicides, have induced the development of several glyphosate-resistant weeds.

Glyphosate resistance can be developed as target-site and non-target-site mechanisms. In the target-site mechanism of resistance, either a mutation on the EPSPS enzyme (enzyme modification) or the overexpression of the EPSPS enzyme have been found to confer resistance. In the non-target-site mechanism of glyphosate resistance, the herbicide translocation and neutralization is observed. Pumping glyphosate into vacuoles via membrane transporters has been suggested as a possible process involved in the restricted glyphosate translocation. As a consequence, a different vacuolar organization or plasticity could be an interesting character or marker to correlate to glyphosate resistance.

Vacuolar markers AleuGFP (Sar1 dependent sorting) or GFPChi (Sar1 independent sorting) respectively can be used to monitor independent vacuolar sorting mechanisms during glyphosate induced stress. We observed that the adaptive reaction of tobacco protoplasts vacuolar system to the treatment with glyphosate, can be mimicked by the overexpression of a Triticum durum TdGST gene. Previous analysis evidenced that the herbicide glyphosate increased TdGST expression, confirming the role of GST in the protection against xenobiotics. Non-target-site glyphosate resistance mechanisms may correlate with an independent regulation of cell compartmentalization and herbicide induced genes may have a direct effect on it.

  • Open access
  • 101 Reads
EGF Receptor Transactivation is Crucial for Cholinergic MAP Kinase Signaling in Human Keratinocytes

Acetylcholine and its receptors are not only essential for the nervous system but also act as mediators of cell communication between non-neuronal cells. Signal transduction after cholinergic stimulation is mediated by two types of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the group of G protein coupled receptors (GPCR). Their activation can lead to initiation of the mitogen-activated protein (MAP) kinase cascade which contributes to cell survival, differentiation and other important cellular responses. The non-neuronal acetylcholine plays a substantial role in the human skin and regulates cell adhesion and migration, as well as proliferation and differentiation of keratinocytes. We here show that in the human keratinocyte cell line HaCaT, the muscarinic acetylcholine receptors are mediators of mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to an extensive activation of the MAP kinase ERK, together with an activation of the protein kinase Akt. These signaling pathways are dependent on the transactivation of the epidermal growth factor receptor (EGFR), and EGFR transactivation even appears to be the only pathway through which muscarinic receptors facilitate ERK activation in these cells. The transactivation pathway involves a triple-membrane-passing process, proceeding through the activation of matrix metalloproteases and an extracellular EGF-like ligand release.

  • Open access
  • 95 Reads
The effect of irradiation time on the viability of Bacillus subtilis by cationic porphyrin compounds

The photodynamic therapy (PDT) is a combination of using a photosensitizer agent, light and oxygen that can cause oxidative cellular damage. This technique is applied in several cases, including microbial control. Various porphyrin and metalloporphyrin compounds are used as photosensitizer agents. In this study, the effect of a tetra-cationic porphyrin and its zinc compound was investigated on Bacillus subtilis under irradiation with a tungsten lamp as a visible light and the illumination time varied from 0 min to 30 min. Increasing the illumination time reduced the colony number, through the disorganisation of the bacterial cell wall. 

  • Open access
  • 81 Reads
Co-presence of Mesenchymal Stem Cells in an in vivo engineered liver pocket-scaffold increases neo-angiogenesis and albumin production

Introduction: Fabrication of three-dimensional (3D) liver tissue is limited by many factors, one of them is the lack of vascularization in the tissue-engineered constructs. To overcome these limitations, an in vivo engineered liver pocket-scaffold's ability to increase neo-angiogenesis was considered in the present study. Methods: Hyaluronan-besed scaffolds enriched with human Mesenchymal Stem Cells (hMSCs) and hepatocytes were implanted into rats. Angiogenesis and hepatocyte function were monitored. Results: The formation of de novo vascular networks within scaffolding matrices was noted as well as a better albumin production by the implanted hepatocytes. Conclusions: Our results emphasized that the presence in culture of hMSC increased tissue concentrations of growth factors and may promote angiogenesis resulting in a higher density of blood vessels coupled with a better metabolic support of hepatocytes.

  • Open access
  • 62 Reads
Anti-Inflammatory Effect of Quercetin on RAW 264.7 Mouse Macrophages Induced with Polyinosinic-Polycytidylic Acid

Quercetin (3,3,4,5,6-Pentahydroxyflavone) is one of well-known antioxidants and a flavonol found in many fruits, leaves, and vegetables. Quercetin is also well-known to have anti-inflammatory effects on lipopolysaccharide-induced macrophages. However, the effects of quercetin on virus-induced macrophages are not fully reported. In this study, the anti-inflammatory effect of quercetin on double-stranded RNA (dsRNA)-induced macrophages was examined. Quercetin at concentrations of up to 50 μM significantly inhibited the production of nitric oxide (NO), IL-6, MCP-1, IP-10, RANTES, GM-CSF, G-CSF, TNF-α, LIX,VEGF, and MIP-1α as well as calcium release in dsRNA [50 µg/mL of polyinosinic-polycytidylic acid]-induced RAW 264.7 mouse macrophages (P < 0.05). Quercetin at concentrations of up to 50 μM also significantly inhibited mRNA expression of signal transducer and activated transcription 1 (STAT1) and STAT3 in dsRNA-induced RAW 264.7 (P < 0.05). In conclusion, quercetin has alleviating effects on viral inflammation concerned with its inhibition of NO, cytokines, chemokines, and growth factors in dsRNA-induced macrophages via the calcium-STAT pathway.

  • Open access
  • 102 Reads
Effects of photon hormesis on cells irradiated by alpha particles

Hormetic responses generally refer to biphasic dose-response relationships showing low-dose stimulation and high-dose inhibition (Calabrese and Baldwin, Hum. Exp. Toxicol., 2002, 21, 91-97; Calabrese and Linda, Nature 2003, 421, 691-692; Calabrese, Environ. Toxicol. Chem. 2008, 27, 1451-1474). In relation, “photon hormesis” refers to the phenomenon in which a low-dose photon irradiation mitigates the cellular damages induced by other ionizing radiations (Rithidech and Scott, Dose Response 2008, 6, 252-271). “Photon hormesis” can mean gamma-ray hormesis or X-ray hormesis depending on the origin of the photons, and gamma-ray hormesis has been proposed as an explanation for the suppression of lung cancers induced by alpha particles (Scott, Dose Response 2008, 6, 299-318; Scott et al. J. Am. Physicians Surg. 2008, 13, 8-11). In the present study, X-ray hormesis was demonstrated in CHO and HeLa cell lines by comparing the number of p53 binding-protein 1 (53BP1) foci per cell occurred in the cells which were (1) irradiated by a toxic dose of alpha particles from an 241Am source, and (2) irradiated by the same dose of alpha particles with an additional small X-ray dose (10 mGy). With the additional X-ray dose, the numbers of 53BP1 foci per cell were significantly reduced at 24 h post-irradiation, which confirmed the presence of X-ray hormesis. Moreover, photon hormesis was also studied using HCT116 and HCT 116 p53-/- cells using the methodology described above, which showed that p53 played an important role in the photon hormesis.

  • Open access
  • 73 Reads
Light Exposure in Microscopy – How can Cell Survival be Increased?
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Challenges in modern microscopy are (1) increasing resolution down to the nanometre range and (2) 3D imaging of cells and tissues with multiple light exposures. However, in both cases light doses may exceed the limits of non-phototoxic doses by several orders of magnitude. Therefore, we determined limits for maintaining cell viability upon application of different wavelengths of light as well as various fluorescent dyes or fluorescent proteins. In a further step we adapted wide field and laser scanning microscopy for light doses compatible with live cell imaging.

If in 3D microscopy various layers of a sample are recorded, light sheet fluorescence microscopy (LSFM) is preferred to laser scanning or structured illumination microscopy, since only those planes are exposed to light which are recorded simultaneously, thus limiting the light dose even upon multiple exposure or long-term experiments. Appropriate LSFM modules have been developed which may be combined with any wide field microscope. If in 2D microscopy studies are focused on cell surfaces, e.g. plasma membranes, total internal reflection fluorescence microscopy (TIRFM) appears to be an appropriate method, since the limited penetration depth of the evanescent wave may also reduce phototoxic cell damage. Here, a variable-angle TIRFM method is suggested for measuring cell-substrate topology.

  • Open access
  • 61 Reads
Interferon alpha induces the apoptosis of cervical cancer HeLa cells by activating both the Intrinsic Mitochondrial pathway and ER Stress-induced pathway

The interferon alpha (IFN-α) has been used for treatment of various malignancies such as hepatocellular carcinoma (HCC), malignant melanoma and renal cell cancer (RCC), and has been shown to promote the apoptosis and inhibit the growth of several tumor cell types. However, the effect of IFN-α on cervical cancer is completely unknown. In this study, HeLa cells were used as a testing model for the treatment of IFN-α on cervical cancer. The results indicate that IFN-α could markedly inhibit the proliferation and induce the apoptosis of HeLa cells. The activation of caspase 3, the up-regulation of Bim and the cleaved PARP1, the down-regulation of Bcl-xL as well as the release of cytochrome C from mitochondria to cytosol were significantly induced upon IFN-α treatment. In addition, caspase 4 which is implicated in ER stress-induced apoptosis was cleaved with IFN-α treatment. Knockdown of caspase 4 by siRNA markedly reduced the cell apoptosis rate, but not affected the mitochondrial apoptotic pathway. Moreover, no significant changes on he expression of caspase 8, caspase 10, Fas/Fas-L were observed upon IFN-α treatments, indicating the apoptosis caused by IFN-α may be independent of extrinsic apoptotic pathway. These findings suggest that IFN-α may possess the capacity of an anti cervical cancer compound by mainly activating both the intrinsic mitochondrial pathway and ER stress-induced pathway.

  • Open access
  • 97 Reads
AhR/IL-24 signaling is associated with susceptibility to dioxins
Published: 15 October 2015 by MDPI in 1st Electronic Conference on Molecular Science session Posters

Dioxins are a class of highly toxic and persistent environmental pollutants that cause multiple adverse health effects in humans, mainly through binding to the ligand-activated transcription factor, aryl hydrocarbon receptor (AhR). Genetic variation in AhR may modulate the susceptibility to dioxins, and little is known about the downstream signaling pathways that lead to multiple adverse health effects. In the present study, we evaluated the effect of a single nucleotide polymorphism (SNP) –130C/T in the AhR promoter on dioxin-inducible gene transcription and downstream signaling pathways using primary human fibroblast. We found that AhR SNP –130C/T modulates AhR mRNA and protein expression in normal human chorionic stromal cells. The gene expression profiles in the cells exposed to a representative dioxin compound TCDD were further determined by using microarray technology. Several genes associated with human disorders including interleukin 24 (IL-24) were highly up-regulated in cells with the TT genotype. Higher up-regulation of IL-24 protein in cells with the TT genotype was also observed. We further evaluated the genotype, dioxins concentrations and serum IL-24 levels in the blood sample from 64 Yusho patients who were accidentally exposed to high concentrations of dioxins via the ingestion of contaminated rice oil in a famous incident of contamination happened in 1968 in Japan.We found that AhR SNP –130C/T affects serum IL-24 levels, independently of serum dioxins concentrations in Yusho patients. Our observations demonstrated the association of AhR SNP –130C/T with dioxin susceptibility in individuals. In particular, we found that IL-24, which is associated with the inflammatory response, acts as an AhR downstream effector. Our investigation provides new insights into the understanding of the mechanisms of health impairments in Yusho patients and genetic susceptibility to dioxins.

  • Open access
  • 81 Reads
Expression of human H ferritin prompts the identification of a hitherto elusive yeast orthologue and enables parsing of distinct iron-induced cell death pathways in Saccharomyces cerevisiae
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The ferritin sub-family of iron binding proteins form multi-subunit nanotype iron-storage cages that serve to store iron and prevent iron toxicity. Of importance ferritin can prevent cell death in mammalian cells in response to many apoptotic stresses such as hydrogen peroxide. Here we describe the identification of human H-ferritin as a suppressor of the pro-apoptotic murine Bax sequence in yeast. Using a combination of spot and vital dye-based assays, we demonstrate that ferritin is a general pro-survival sequence since it also prevents the effects other stresses including copper and rapamycin in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Eukarya, Bacteria and Archaea, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. Our observation that heterologous human H-ferritin retains pro-survival functions when expressed in yeast indicates either ferritin is self-functioning or that yeast has a yet to be uncovered ferritin. To address the later possibility, we carried out a detailed in silico analysis of the yeast proteome leading to the identification of a protein of unknown function to be a strong candidate for the elusive yeast ferritin (yFer1). In addition to sharing 20% sequence identity with the 183 residue human H-ferritin, yFer1 also has similar functional properties as ferritin when overexpressed in yeast including preventing the effects of Bax and copper. The study of pro-survival sequences offers information not only on the function of pro-apoptotic sequences but also on the nature of the different forms and sub-forms of PCD. For example, copper and rapamycin likely induce similar forms of apoptotic-like PCD given that their lethal effects can be prevented by the overexpression of the same anti-apoptotic sequences including human ferritin and the putative yeast ferritin. In contrast we find that cell death induced by iron is distinctly different in part because it can only be prevented by a small subset of pro-survival proteins including the newly identified putative yeast ferritin. A detailed comparison of the copper and iron mediated PCD subroutines, including potential areas of cross-talk are under investigation and will be discussed in more detail.

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