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Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins. [ABSTRACT FROM AUTHOR]

Abstract: We report here the use of albumin-based biosensor chips for the determination of metal content and characterization of metal–protein interaction by surface plasmon resonance. Bovine serum albumin was immobilized onto a carboxymethylated dextran matrix and used for metal detection. The temperature for the analysis was defined and the highest interaction was observed at 25°C. The albumin sensor chip binds cadmium, zinc or nickel in a concentration-dependent manner, but not magnesium, manganese and calcium. The optimal buffer condition used for the analysis contains 0.01M HEPES, pH 7.4, 1mM NaCl and 0.005% Tween-20. Using this condition, a linear calibration curve within the range of 10−8 to 10−4 M can be established for the metals. However, a dramatic increase in binding capacity was observed when metal concentration was higher than 10−4 M and reached a plateau at 10−2 M. The detection limit for Cd can reach as low as 1ppb. When measuring a solution containing two species of metal ions with the albumin chip, an additive effect was observed for Ni and Zn. However, 20–30% reduction in resonance response was found upon mixing Cd with Zn or Ni. These observations are consistent with the binding characteristics of albumin. The feasibility of measuring serum metal content by the albumin chip was examined. A linear calibration curve can be established if the samples are boiled and passed through a gel filtration column. The binding affinity of metal with albumin can also be achieved by using the sensor chip. The binding affinity follows the order of Ni>Zn>Cd. These results indicate that the albumin-based sensor chip is useful not only in the quantification of metal content, but also in the characterization of the biochemical properties of albumin. [Copyright &y& Elsevier]

A biosensor based on mammalian metallothionein (MT) for the detection of metal ions was developed and characterized. MT was immobilized onto a carboxymethylated dextran matrix as a biosensor for the detection of metal ions by surface plasmon resonance (SPR). The optimal pH for the immobilization step was determined to be 4. The temperature for the analysis was also defined, and the highest interaction was observed at 30 °C. The MT sensor chip binds cadmium (Cd), zinc (Zn) or nickel (Ni), but not magnesium (Mg), manganese (Mn) and calcium (Ca). Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection is at the micromolar level. The interaction between the metal ions and the sensor chip is influenced significantly by the presence of NaCl, Tween 20 and the pH of the reaction buffer. By decreasing the NaCl in the reaction buffer to 1 mM, the MT chip effectively differentiates cadmium from zinc and nickel. Kinetic parameters of the metal–MT interactions were also determined by using this chip. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel, which is the same as that determined for MT in solution. Thus, the MT chip can be an effective biosensor for the detection and measurement of several metal ions. [Copyright &y& Elsevier]

Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins. [ABSTRACT FROM AUTHOR]

Leptin is an adipokine predominantly secreted by adipocytes and has many physiological roles, including in energy homeostasis. We identified that AM630, a cannabinoid receptor 2 (CB2) antagonist, down-regulated leptin expression in mature adipocytes differentiated from either stromal vascular fractions isolated from inguinal fat pads of C57BL/6J mice or 3T3-L1 preadipocytes. However, the leptin-suppressive effects of AM630 preserved in CB2-deficient adipocytes indicated the off-target activity of AM630 in leptin expression. Pharmacological and genetic studies, cheminformatics, and docking simulation were applied to identify the potential protein target of AM630 that modulates leptin expression in differentiated primary preadipocytes. Screening of the reported off-targets of AM630 identified a synthetic cannabinoid WIN55212-2 exerting the same function. Target deconvolution and docking simulation suggested that AM630 and WIN55212-2 were both inhibitors of lipocalin-type prostaglandin D2 synthase (L-PGDS). Further studies showed that L-PGDS positively regulates leptin expression. Although glucocorticoid and aldosterone were previously reported to induce expression of both L-PGDS and leptin, our data demonstrated that L-PGDS mediates only glucocorticoid-induced leptin expression in differentiated primary preadipocytes. No effect was observed after aldosterone treatment. This newly discovered glucocorticoid – L-PGDS – leptin pathway may provide insights into current clinical use of glucocorticoid and management of their undesired effects such as obesity. [ABSTRACT FROM AUTHOR]

Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1 kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5′ to 3′) shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators. [ABSTRACT FROM AUTHOR]

Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE. [ABSTRACT FROM PUBLISHER]

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases. [ABSTRACT FROM AUTHOR]

Abstract: Ischemic shock and acute kidney injury (AKI) are the primary causes of high mortality in the intensive care unit (ICU) patients. In a rat model, ischemia is able to disrupt the actin cytoskeleton of proximal tubule cells. This effect is associated with the expression and excretion of actin depolymerizing factor (ADF)/cofilin. However, the human evidence of ADF/cofilin excreted in ICU patients with ischemic shock and/or AKI remains to be addressed. Here we developed a 96-well high-throughput, localized surface plasmon-coupled fluorescence biosensor (HT-LSPCFB) combining a sandwich immunoassay to measure the urine cofilin-1 in 57 ICU patients and 8 healthy controls. A linear relationship between 1 and 105 pg/mL of human cofilin-1 recombinant protein was determined (R 2 =0.9845) in this study. The highest normalized cofilin-1 levels obtained in the ICU patients and healthy adults were 1.985 and 0.726, respectively. The mean normalized cofilin-1 level of total ICU patients (0.7403) was also significantly higher than that of healthy adults (0.4759) (p =0.0052). An examination of the receiver operating characteristic (ROC) curve and area under curve (AUC) showed that cofilin-1 is acceptable for assessing the ICU patients (AUC=0.775) and shock (AUC=0.713), but not for AKI (AUC=0.681). Therefore, the HT-LSPCFB is suitable for detecting the urine cofilin-1 which potentially becomes a prognostic factor for ICU patients with shock. [Copyright &y& Elsevier]

Abstract: Water-soluble germanium nanoparticles (wsGeNPs) with allyamine-conjugated surfaces were fabricated and emit blue fluorescence under ultraviolet light. The wsGeNP was physically and chemically stable at various experimental conditions. Cytotoxicity of the fabricated wsGeNP was examined. MTT assay demonstrated that wsGeNP possessed high toxicity to cells and clonogenic survival assay further indicated that this effect was not resulted from retarding cell growth. Flow cytometric analysis indicated that wsGeNP did not alter the cell cycle profile but the sub-G1 fraction was absent from treated cells. Results from DNA fragmentation and propidium iodide exclusion assays also suggested that apoptotic cell death did not occur in cells treated with wsGeNP. Addition of a necrosis inhibitor, necrostatin-1, attenuated cell damage and indicated that wsGeNP caused necrotic cell death. Cell signaling leads to necrotic death was investigated. Intracellular calcium and reactive oxygen species (ROS) levels were increased upon wsGeNP treatment. These effects can be abrogated by BAPTA-AM and N-acetyl cysteine respectively, resulting in a reduction in cell damage. In addition, wsGeNP caused a decrease in mitochondrial membrane potential (MMP) which could be recovered by cyclosporine A. The cellular signaling events revealed that wsGeNP increase the cellular calcium level which enhances the production of ROS and leads to a reduction of MMP, consequentially results in necrotic cell death. [Copyright &y& Elsevier]

Purpose: The aim of this work is to compare the radiosensitizing effect between organic and inorganic germanium compounds and to investigate whether nanometer-sized germanium particles can act as radiosensitizers. Materials and methods: Bis (2-carboxyethylgermanium) sesquioxide (Ge-132), germanium oxide (GeO2) and germanium nanoparticles were used in this study. Cell viability was determined by clonogenic survival assay. Cellular DNA damage was evaluated by alkaline comet assay, confocal microscopy and the cellular level of phospho-histone H2AX (γ-H2AX). Results: Nanometer-sized germanium particles were fabricated. They have a similar radiosensitizing effect as that of GeO2. Conversely, Ge-132 did not enhance the radiosensitivity of cells. Comet assay was employed to evaluate the level of DNA damage and confirmed that inorganic germanium compounds enhanced cellular radiosensitivity. Notably, the comet assay indicated that the nanoparticle itself caused a higher level of DNA damage. The possibility that germanium nanoparticles per se caused DNA damage was ruled out when the cellular level of γ-H2AX was examined. Conclusions: We demonstrated that inorganic but not organic germanium compounds exerted radiosensitizing effect in cells. Nanometer-sized germanium particles were fabricated and were able to enhance the radiosensitivity of cells. Confounding effect may occur when comet assay is used to estimate the level of DNA damage in the presence of germanium nanoparticles. [ABSTRACT FROM AUTHOR]

Abstract: Cadmium (Cd) induces necrotic death in Chinese hamster ovary (CHO) K1 cells and we have established the responsible signaling pathway. Reportedly, necrostatin-1 (Nec-1) rescues cells from necrotic death by mediating through the death domain receptor (DR) signaling pathway. We show here that Nec-1 also effectively attenuates necrotic death triggered by Cd. Two other treatments that cause necrotic cell death, one can (z-VAD-fmk/TNF-α on U937 cells) and the other cannot (etherynic acid (EA) on DLD-1 cells) be rescued by Nec-1, were also studied in parallel for comparison. Results show that Nec-1 is ineffectual in modulating intracellular calcium contents, calpain activity (a downstream protease), or reactive oxygen species production. It can counteract the reduction in mitochondrial membrane potential (MMP) caused by treating CHO K1 or U937 cells with necrosis-inducing agent. However, this effect was not found in EA-treated DLD-1 cells. Notably, Nec-1 elevates NF-κB activity in the presence or absence of necrosis-inducing agents. Our study shows that, in addition to DR-mediated necrosis, Nec-1 is effective in attenuating Cd-induced necrosis. It rescues cells with reduced MMP implying that mitochondrion is its major acting site. [Copyright &y& Elsevier]

Chinese hamster ovary K1 (CHO K1) cells are very sensitive to cadmium (Cd) toxicity. They were used to investigate the effect of Cd on cell cycle progression. Cells were cultured with 0.1, 0.4, 1 or 4μM Cd for various time intervals. There was no difference in growth rate when less than 0.4μM Cd was given within 24h. A dose-dependent reduction of cell proliferation was observed when more than 0.4μM of Cd was given. The cells were pulse-labeled with 5-bromodeoxyuridine (BrdU), and the labeled cells were cultured in the presence of increasing concentrations of Cd. Cell cycle progression was retarded as a function of Cd concentration. G2/M arrest was observed when the BrdU-labeled cells were treated with 1μM Cd for 8h, whereas cells receiving 4μM Cd stopped at the S phase within 4h. Cell cycle analysis of cells treated with Cd for 24h showed that G2/M arrest occurred only when cells received 0.8 to 2μM Cd. Despite the occurrence of G2/M arrest in the Cd treatment, only a limited proportion of the cells were blocked in the M phase. However, the increase in M phase cells coincided with an elevation in the cyclin-dependent kinase 1 activity. To examine whether Cd acts on cells at a specific cell stage, they were synchronized at the G1 or G2/M phase then treated with 1μM Cd for 12h. The cells were blocked at the G2/M and G1/S phase, respectively. This finding indicates that Cd toxicity is global and not cell phase specific. We also investigated the involvement of Cd-induced reactive oxygen species (ROS) with the occurrence of G2/M block and found a lack of correlation between cell cycle arrest and ROS production. We measured the Cd content that caused G2/M arrest from a series of Cd treatments and determined the ranges of cumulative Cd concentrations that could result in cell cycle arrest. [Copyright &y& Elsevier]

Summary background Previous studies have suggested that serum ferritin is one of the components of the insulin resistance syndrome in Caucasians. Because serum ferritin levels differ significantly between men and women, variation in the role of ferritin in insulin resistance between the sexes, particularly in Asian populations, is still unknown. objective To examine whether the association between serum ferritin and insulin resistance differs between men and women in randomly selected non-diabetic Chinese subjects. design A retrospective study. patients Four hundred and seventeen non-diabetic Chinese subjects (140 men and 277 women) were studied. measurements Fasting plasma glucose, insulin, lipoproteins and serum ferritin concentrations, as well as plasma glucose and insulin responses to a 75-g oral glucose test (n = 219), were determined. results Fasting serum ferritin concentrations (mean ± SEM) were significantly higher in men than in women (504 ± 33 vs. 242 ± 18 pmol/l, P < 0·001). In women, fasting serum ferritin concentrations correlated significantly with age, body mass index (BMI), amount of body fat, fasting plasma glucose, insulin, cholesterol, triglyceride concentrations, glucose response to an oral glucose load, and homeostasis model assessment (HOMA) of insulin resistance but not with blood pressure, high density lipoprotein (HDL) cholesterol levels and insulin response to oral glucose. On the contrary, none of the above anthropometric and metabolic variables was related to fasting serum ferritin levels in men. HOMA insulin resistance increased progressively across three different tertiles for measured serum ferritin concentrations in women (P < 0·003). In men, HOMA insulin resistance levels were not different among three differing measured serum ferritin levels (P = 0·424). Adjustment for age, BMI and menopause status did not change the significant relationship between HOMA insulin resistance and serum ferritin in... [ABSTRACT FROM AUTHOR]

We report here for the first time that germanium oxide (GeO2) blocks cell progression. GeO2 is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO2 arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO2 when treated for 12 h, but decreased at GeO2 concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO2 delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO2 treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO2-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO2, cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO2 treatment in CHO cells. However, GeO2 decreased p34cdc2 kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO2 removal and correlated with the transition of G2/M–G1 phase of the cells. This result suggests that GeO2 treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells. [Copyright &y& Elsevier]

Abstract: Contamination of the environment with heavy metal ions has been an important worldwide concern for decades. Among them, mercury ion (Hg2+) is one of the heavy metal ion maintained in the detection because of its toxicity. In this study, we have designed the gold nanoparticle (AuNP) probes of carboxylic modified protein and employed the lateral flow assay to detect the Hg2+ without complicated instrumentations and technical expertise. The captured AuNP probes on the test zone and control zone of the sensor produced the characteristic red bands, enabling visual discrimination of Hg2+ concentrations. The demand of Hg2+ analysis in environmental applications requires parallel analytical strategies; such a method with simplicity and rapidity has enormous potential for the application of Hg2+ monitoring in environment, water, and food samples. [Copyright &y& Elsevier]