DNAzyme Based Amplified Biosensor on Ultrasensitive Fluorescence Detection of Pb (II) Ions from Aqueous System.

A label -free DNAzyme amplified biosensor is found to be highly selective and sensitive towards fluorescent detection of Pb2+ ions in aqueous media. The DNAzyme complex has designed by the hybridization of the enzyme and substrate strand. In the presence of Pb2+, the DNAzyme activated and cleaved the substrate strand of RNA site (rA) into two oligonucleotide fragments. Further, the free fragment was hybridized with a complementary strand on the surface of MBs. After magnetic separation, SYBER Green I was added and readily intercalate with the dsDNA to gives a bright fluorescence signal with intensity directly proportional to the concentration of Pb2+ions. A detection limit of 5 nM in Pb2+ the detection range 0 to 500 nM was obtained. This label- free fluorescent biosensor has been successfully applied to the determination of environmental water samples. Then results open up the possibility for real-time quantitative detection of Pb2+ with convenient potential applications in the biological and environmental field. Graphical Abstract.

Localization of zinc after in vitro mineralization in osteoblastic cells.

The present study was designed to investigate the incorporation of zinc (Zn) into cultured UMR-106 osteoblasts in response to mineralization caused by the addition of beta-glycerophosphate. As a result of the induced mineralization, the contents of calcium (Ca), phosphorus (P), and Zn in the monolayer increased, whereas the magnesium (Mg) content did not change. The activity of alkaline phosphatase (ALP) also increased during the process. The zinc distribution in the cell monolayer was studied using Zinquin, a fluorescent zinc ion chelator. The double fluorescent labeling with Zinquin and calcein revealed that zinc was localized both as intracellular vesicles and extracellular clusters, whereas calcium was colocalized with extracellular zinc. These results suggest that zinc is involved in the mineralization process of UMR-106 cells.

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride.

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.

Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells.

It has been suggested that oxidative stress plays a major role in various forms of cell death, including necrosis and apoptosis. We have previously reported that fluoride (NaF) induces apoptosis in HL-60 cells by caspase-3 activation. The main focus of this investigation was to arrive at a possible pathway of the apoptosis induced by NaF upstream of caspase-3, because the mechanism is still unknown. The present study showed that after exposure to NaF, there was an increase in MDA and 4-HNE and a loss of mitochondrial membrane potential (deltaPsi(m)) was also observed in NaF-treated cells. There was a significant increase in cytosolic cytochrome c, which is released from the mitochondria. We have reported a downregulation of Bcl-2 protein in NaF-treated cells. The antioxidants N-acetyl cysteine (NAC), glutathione (GSH) protected the cells from loss of deltaPsi(m), and there was no cytochrome c exit or Bcl-2 downregulation, and we suggest that these antioxidants prevent apoptosis induced by NaF. These results suggested that perhaps NaF induced apoptosis by oxidative stress-induced lipid peroxidation, causing loss of deltaPsi(m), and thereby releasing cytochrome c into the cytosol and further triggering the caspase cascade leading to apoptotic cell death in HL-60 cells.

Subcytocidal attack by staphylococcal alpha-toxin activates NF-kappaB and induces interleukin-8 production.

Formation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-kappaB. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-kappaB. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.

Transcription of krox-20/egr-2 is upregulated after exposure to fibrous particles and adhesion in rat alveolar macrophages.

Alveolar macrophages meet various types of particulate substances deposited deep in the lung. We report differences in biologic responses of alveolar macrophages between phagocytosis of fine spherical and fibrous particles. Although titanium dioxide (TiO(2)) is thought to be biologically inert, the cytotoxicity of fibrous TiO(2) (F-TiO(2)) was much higher than spherical TiO(2) (S-TiO(2)). Differential display and the subsequent Northern blot analysis indicated that transcription of krox-20/egr-2 gene was slightly and greatly upregulated in S- and F-TiO(2)-exposed alveolar macrophages, respectively. The messenger RNA (mRNA) level of krox-20/egr-2 increased up to 8 h in F-TiO(2)-exposed alveolar macrophages, whereas krox-20/egr-2 mRNA level was transiently increased in response to adhesion to the culture dish. Stimulation with lipopolysaccharide also increased krox-20/egr-2 mRNA level transiently, although the mRNA level rebounded after 8 h. The analysis with 5' rapid amplification of complementary DNA ends suggested that there is a heterogeneity in the upstream region of this gene (krox-20/egr-2 and krox-20H1; accession numbers AB032420 and AB032419, respectively). The polymerase chain reaction analysis with specific primers for krox-20/egr-2 and krox-20H1 indicated that both genes were almost equally upregulated after either adhesion to the plastic dish or phagocytosis of F-TiO(2). These results suggest that both krox-20/egr-2 and krox-20H1 are implicated in adhesion and phagocytosis, and that the expression of krox-20 may reflect interaction with foreign substances and adhesion in alveolar macrophages.

Fluoride induces apoptosis by caspase-3 activation in human leukemia HL-60 cells.

Even though fluoride toxicity is increasingly being considered to be important, very little information is available on the mechanism of action of fluoride. In the present study, the toxicity of fluoride on human leukemia (HL-60) cells was investigated and the involvement of caspase-3 was also studied. Fluoride induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Annexin staining and DNA ladder formation on agarose gel electrophoresis further revealed that HL-60 cells underwent apoptosis on exposure to 2-5 mM fluoride. Western blotting using polyclonal anti-caspase-3 antibody and mouse anti-human poly(ADP-ribose) polymerase (PARP) monoclonal antibody was performed to investigate caspase-3 and PARP activity. Fluoride led to the activation of caspase-3 which was evident by the loss of the 32 kDa precursor and appearance of the 17 kDa subunit. Furthermore, intact 116 kDa PARP was cleaved by fluoride treatment as shown by the appearance of a cleaved 89 kDa fragment. The results clearly suggest that fluoride causes cell death in HL-60 cells by causing the activation of caspase-3 which in turn cleaves PARP leading to DNA damage and ultimately cell death.

RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation.

RGD motif-containing peptides have been used in various studies of cell adhesion and growth. We report that RGD triggered apoptosis at a concentration of 1 mmol/L, whereas RAD-containing peptides failed to induce apoptosis in HL-60 cells. RGD-treated cells revealed internucleosomal DNA fragmentation. Western blot reveals caspase-3 activation in RGD peptide-treated cells. A caspase-3 inhibitor z-VAD-FMK completely blocked the apoptosis, but a caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-2 inhibitor (z-VDVAD-FMK) did not block the apoptosis, suggesting that caspase-3 might have a critical role in the execution process of apoptosis induced by RGD. RGD peptides have been used extensively to inhibit tumor metastasis. Our results should help in further understanding the RGD peptide-induced apoptosis, which is important since RGD peptides have a potential role in therapies of the future.

Serum protein, ascorbic acid & iron & tissue collagen in oral submucous fibrosis--a preliminary study.

A study of 36 patients with oral submucous fibrosis, revealed that all patients had the habit of chewing betel nut, pan masala or the traditional mixture (betel nut, betel leaf and lime) suggesting a link between fibrosis and arecanut. There was an increase in the globulin fraction of protein and hence a decreased A/G ratio in these patients. There was a significant increase in total protein levels possibly due to the increase in globulin fractions and other serum proteins. Ascorbate and iron levels decreased perhaps because of their utilisation in collagen synthesis. The total tissue collagen content increased significantly in patients with advanced disease and, it increased with the progression of the disease leading to hypomobility of the tongue, lips, cheeks, soft palate and faucial pillars.