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Fluorescence anisotropy is a homogeneous, sensitive, ratiometric, and real-time analytical technology. However, it is a great challenge to produce a large fluorescence anisotropy change upon the presence of target small molecules without nanoparticles-dependent amplification. This work reports a nanoparticle-free and multiple G-enhanced fluorescence anisotropy assay for detection of DNAzyme activity. A Pb(2+)-dependent GR-5 DNAzyme was used as a model. We hybridized the rA-cleavable substrate strand containing a TMR label at the 5'-end with the DNAzyme strand containing an extended three G bases at the 3'-end. By this design, we demonstrate that both fluorescence quenching and the enhanced DNAzyme activity contribute to a Pb(2+)-induced large fluorescence anisotropy change (|Δr| = 0.168). The limit of detection for Pb(2+) is estimated to be about 100 pM with a dynamic range from 200 pM to 100 nM. The interference from the other nine divalent metal ions of 1000-times excess amount is negligible. Moreover, we show an extended assay for evaluation of the interactions of Pb(2+) with cysteine and glutathione by the detection of GR5 DNAzyme activity. Collectively, we developed a novel fluorescence anisotropy amplification assay, enabling us to detect DNAzyme activity and associated cofactors and inhibitors and to characterize the Pb(2+)-chelation capability of free thiols.

Intermittent and low-dose parathyroid hormone (PTH) injection to stimulate bone formation has been used in the treatment of osteoporosis. The N-terminal fragment 1-34 of PTH is quite similar in structure and function to N-terminal PTH-related protein (PTHrP). PTH(1-34) and PTHrP also share a coreceptor, the PTH/PTHrP receptor. Therefore, some studies have suggested that PTHrP could effectively stimulate bone formation, similar to PTH. We used an ovariectomized (OVX) rat model of osteoporosis to study the effects of PTHrP(1-34) on bone metabolism by measuring bone mineral density (BMD), bone histomorphometrics, and biomechanical parameters. We found that subcutaneous injection of PTHrP(1-34) (40 or 80 μg/kg body weight every day) in OVX rats increased lumbar and femoral BMD, improved bone biomechanical properties, enhanced bone strength, and promoted bone formation. We selected 40 μg/kg as the preferred therapeutic dose of PTHrP(1-34) and investigated the effects of frequency of treatment (per 1, 2, 3, or 7 days) on bone metabolism in OVX rats. We found that injection of PTHrP(1-34) once per day or every other day significantly improved the BMD and strength of OVX rats. Serum calcium and phosphate levels in all treated rats did not vary significantly from control rats. Based on our results, intermittent low-dose PTHrP(1-34) injection promoted bone formation in OVX rats, suggesting a high potential for therapeutic use in osteoporosis patients.

Glibenclamide, a blocker of ATP-sensitive potassium channels, has been found to induce apoptosis in some cell types, including pancreatic beta-cells. Since autophagy plays doubleedged roles in pancreatic beta-cell survival, frequently through cross-talking with apoptosis, we investigated if glibenclamide induced autophagy in INS-1 rat insulinoma cells and the influence of autophagy on apoptosis. Mammalian target of rapamycin (mTOR) is a negative regulator of autophagy. As one of the substrates of mTOR, p70 S6 kinase (p70 S6K) is phosphorylated upon mTOR activation. Our results showed that glibenclamide induced an elevated protein level of the autophagy marker LC3-II, while decreasing phosphorylated p70 S6K, indicative of inhibition on mTOR signaling in INS-1 cells. Furthermore, inhibiting glibenclamide-induced autophagy via knocking down the autophagy essential gene Atg7 decreased cell viability and increased apoptosis in INS-1 cells. Our results indicate that glibenclamide induces autophagy in INS-1 cells, and that autophagy activation is exerting a protective activity against apoptosis.

Iron overload has been associated with bone mass loss. To elucidate the effects of excess iron on bone metabolism, an iron-overloading mouse model was established by administering iron-dextran at 250 mg/kg to female BALB/c mice. After 4 weeks, the mice were sacrificed and the biomechanical properties of the femurs were examined. The results suggested a notable decrease of the maximal bending stress and the modulus of bending elasticity in the femurs obtained from the excess iron-treated mice compared to the control mice. The levels of the serum osteocalcin, C-telopeptide of type I collagen (CTX-1) and tumor necrosis factor-α (TNF-α) were measured in order to investigate the underlying mechanism responsible for the excess iron-induced bone strength reduction. Overall, the results suggested that iron overload resulted in a marked reduction of bone load-bearing capacity through a TNF-triggered osteoclast differentiation and resorption mechanism.

Reproducible, low loss and high-yield purification process for single-walled carbon nanotubes (SWNTs) was developed by coupling of oxidation in wet air and treatment with gas phase nitric acid. Oxidation in wet air can burn out the carbonaceous particles and gas phase nitric acid treatment can etch away the catalytic metals. The as-synthesized SWNTs and the purified SWNTs were characterized by field emission scanning electron microscopy, high resolution transmission electron microscopy, Raman spectrophotometer and thermal gravimetric analysis. It was shown that such a facile and controllable gas phase process for purifying SWNTs can lead to the minimal losses and minimal destruction of SWNTs, thus highly purified SWNTs without amorphous carbon and with very low metal content were obtained.

The comparative analysis of the most widely used methods of mesoporosity characterization of two activated carbon fibers is presented. Not only the older methods are used, i.e. Barrett, Joyner and Halenda (BJH), Dubinin (the so-called first variant-D-1ST and the so-called second variant-D-2ND), Dollimore and Heal (DH), and Pierce (P) but the recently developed ones, i.e. the method of Nguyen and Do (ND) and that developed by Do (Do) are also applied. Additionally, the method of the characterization of fractality is put to use (fractal analog of FHH isotherm). The results are compared and discussed. (C) 2002 Elsevier Science B.V. All fights reserved.

A comparative analysis of the results for the estimation of the pore-size distribution based on the methods of Do and co-workers (ND) and density functional theory (DFT) is given. A new algorithm (ASA, Adsorption Stochastic Algorithm) is adopted and it is shown that this algorithm can be successfully applied for the determination of the PSD curve from the ND method. The obtained results show that, generally, the ND and DFT methods lead to almost the same PSD curves and this similarity is observed for carbons of different origin and possessing different pore structures. However, if the contribution of micropores to the PSD increases, the differences in the fit of ND and DFT to the experimental data are more pronounced.

The tailoring of pore surface chemistry of activated carbon fibers is shown to be an effective method for improving the adsorption efficiency of various volatile chemical compounds (VOCs). An oxidation treatment with air resulted in a significant increase in the adsorption capacities and breakthrough time for Rayon-based activated carbon fibers (ACFs) in removal of formaldehyde. The porous structure parameters of Rayon-based ACFs were determined with standard nitrogen adsorption analysis. The pore surface chemistry of samples under study was analyzed by Fourier Transform Infrared spectra (FTIR). Thus to some extent, the relationship between the adsorption properties, porous structure and pore surface chemistry was revealed.

Chlorpyrifos (CPF) is commonly used for agricultural and domestic applications, and its contamination is widely detected in environmental media, and in a broad spectrum of field crops, fruits and vegetables. CPF exposure causes many adverse effects on human health including hepatoxicity, neurotoxicity and endocrine disruption. However, few studies have been conducted thus far to investigate the potential influence of CPF exposure on iron metabolism at concentrations that do not trigger significant cell death. Iron metabolism is concertedly governed by the hepcidin-ferroportin axis, where hepcidin is the central hormone involved in the regulation of iron absorption and release, while ferroportin is the only known iron exporter that functions by iron egress from cells. In the present study, we demonstrated that CPF treatment at a non-toxic concentration greatly enhanced ferroportin gene transcription in human macrophage THP-1 cells. CPF significantly inhibited hepcidin expression in human hepatocyte HepG2 cells. As a result, the intracellular labile iron pool (LIP) was largely reduced in THP-1 and HepG2 cells. The combined data of this study identified a novel finding of CPF that disrupts iron homeostasis by altering ferroportin and hepcidin expression. These findings would be useful in understanding the biological effects of CPF exposure, especially under relatively low and non-toxic doses.

Cadmium (Cd) is a heavy metal that has detrimental effects on various organs. The widespread contamination of Cd in the environment, crops and food sources poses a severe threat to human health. Acute toxicities of Cd have been extensively investigated; however, the health impact of chronic low‑dose exposure to Cd, particularly exposure under non‑toxic concentrations, has yet to be elucidated. Furthermore, the toxic threshold of Cd is currently unknown. Ferroportin is the only known iron exporter in vertebrate cells, and it has an essential role in controlling iron egress from cells. To the best of our knowledge, the present study is the first to verify the regulation of ferroportin by Cd. Treatment with low‑dose Cd (i.e. at sublethal concentrations, without undermining cell viability) increased the protein expression of ferroportin in macrophages, and this was associated with depleted cellular iron levels. Mechanistic investigations revealed that Cd modulated the ferroportin concentration at the translational level, via the iron responsive element located at the 5'‑untranslated region of ferroportin. In conclusion, these data provide evidence for the molecular basis by which Cd alters cellular iron availability through elevating concentrations of ferroportin.

Osteoporosis is one of the leading disorders among aged people. Bone loss results from a number of physiological alterations, such as estrogen decline and aging. Meanwhile, iron overload has been recognized as a risk factor for bone loss. Systemic iron homeostasis is fundamentally governed by the hepcidin-ferroportin regulatory axis, where hepcidin is the key regulator. Hepcidin deficiency could induce a few disorders, of which iron overload is the most representative phenotype. However, there was little investigation of the effects of hepcidin deficiency on bone metabolism. To this end, hepcidin-deficient (Hamp1(-/-)) mice were employed to address this issue. Our results revealed that significant iron overload was induced in Hamp1(-/-) mice. Importantly, significant decreases of maximal loading and maximal bending stress were found in Hamp1(-/-) mice relative to wildtype (WT) mice. Moreover, the levels of the C-telopeptide of type I collagen (CTX-1) increased in Hamp1(-/-) mice. Therefore, hepcidin deficiency resulted in a marked reduction of bone load-bearing capacity likely through enhancing bone resorption, suggesting a direct correlation between hepcidin deficiency and bone loss Targeting hepcidin or the pathway it modulates may thus represent a therapeutic for osteopenia or osteoporosis. (C) 2014 Elsevier B.V. All rights reserved.

We recently developed a two-site immunofluorometric assay (IFMA) of salmon calcitonin (SCT) by DELFIA (dissociation enhancement lanthanide fluoroimmunoassay) technique using the same polyclonal antibodies both for “catching” the antigen and for signaling. In the present study we used a monoclonal antibody to SCT 1–11 as the capture antibody. This antibody was biotinylated before use in streptavidin-coated microtitration plates. The polyclonal antibody labeled with Eu chelate was used as a signaling marker. This combination of antibodies resulted in an assay that was three to four times more sensitive than the previous IFMA, with a detection limit of 0.3 pmol/L serum. Intact SCT 1–32 was detected by the assay (recoveries 94–96%), but not the fragments SCT 1–11 and SCT 10–32 or human calcitonin. Dilutions of plasma samples containing SCT were parallel to the calibration curve of SCT 1–32. Pharmacokinetic studies of SCT, 100 IU administered intramuscularly to 10 men, indicated peak serum concentrations of 32–128 pmol/L within 10–20 min with apparent half-life of 56 ± 18 min (mean ± SD). This new assay will allow study of the pharmacokinetics of new calcitonin preparations that do not require injection.

Iron is essential for the human being, involving in oxygen transport, energy metabolism and DNA synthesis. Iron homeostasis is tightly governed by the hepcidin-ferroportin axis, of which hepcidin is the master regulator. Excess iron is associated with various diseases including osteopenia and osteoporosis, which are closely related to the alternation of the endogenous estrogen level. To verify the biological effect of estrogen on iron metabolism, we established a mouse model of estrogen deficiency by ovariectomy. We demonstrated that the hemoglobin content and serum iron level decreased, whereas the tissue iron level in liver and spleen increased in the ovariectomized mice. Moreover, the transcription of hepatic hepcidin was elevated in ovariectomized mice compared to the control mice. We further demonstrated that there was an estrogen response element (ERE) in the promoter region of the hepcidin gene. The assay using the luciferase reporter system confirmed the existence of a functional ERE in the hepcidin promoter, as the estradiol treatment reduced hepcidin expression in cells transfected with ERE-intact construct, with no response to estradiol in cells transfected with ERE-devoid construct. In conclusion, estrogen greatly contributes to iron homeostasis by regulating hepatic hepcidin expression directly through a functional ERE in the promoter region of hepcidin gene. These findings might help build a better understanding towards the etiology of postmenopausal osteoporosis accompanied by excess tissue iron (such as iron retention of osteoclasts in bone) under estrogen deficiency.

A two-site assay was developed by use of the "dissociation and enhancement lanthanide fluoroimmunoassay" (DELFIA) technique for determination of salmon calcitonin (SCT) in serum after administration to osteoporotic patients. Polyclonal antibodies were produced in rabbits immunized with SCT coupled to ovalbumin. After affinity purification, the antibodies were used both as immobilized capture antibodies and as Eu-chelate-labeled signal antibodies. A sensitive assay with a detection limit of 1.1 pmol/L was achieved, and no cross-reaction with human calcitonin was observed. The intra- and interassay CVs were < 12% (n = 10) and < 15% (n = 4), respectively. Analytical recovery of SCT added to serum was 91% +/- 3% (mean +/- SD, n = 4). SCT was measurable in all the samples from eight osteoporotic patients after subcutaneous SCT administration. We conclude that this new sensitive and specific two-site DELFIA can reliably measure SCT in serum.

This study was performed to characterize the bone metabolism in ten 6-month-old male Goto-Kakizaki (GK) rats, a spontaneous type 2 diabetic model, with ten age- and sex-matched non-diabetic Wistar rats as controls. The femora and the fifth lumbar vertebrae were analyzed by a dual energy X-ray absorptiometry for bone mineral density. Histomorphometrical analyses were performed on the sections from the tibia embedded in methylmethacrylate. Biomechanical characterizations were made by a three-point bending test and a compressive test on the femur and the fifth vertebral body respectively. Compared to the control rats, the bone mineral density was significantly deceased and the histomorphometrical studies showed significantly decreased trabecular bone volume, trabecular thickness and number, osteoid surface and thickness, mineralizing surface, mineral apposition rate and bone formation rate, and also a significant increase in mineralization lag time in the diabetic rats. Strength in both bones and elastic modulus of vertebral body significantly decreased in the diabetic rats as well. In addition, the serum osteocalcin levels were significantly decreased and the serum tartrate-resistant acid phosphatase activity was significantly increased. In conclusion, the 6-month-old GK diabetic rats developed osteopenia with an increased risk of fracture owing to the decreased bone formation, and might be a useful model for unraveling the effects of diabetes on bones independent of obesity frequently seen in the type 2 diabetic patients.

The influence of heat treatment of rayon-based activated carbon fibers on the adsorption behavior of formaldehyde was studied. Heat treatment in an inert atmosphere of nitrogen for rayon-based activated carbon fibers (ACFs) resulted in a significant increase in the adsorption capacities and prolongation of breakthrough time on removing of formaldehyde. The effect of different heat-treatment conditions on the adsorption characteristics was investigated. The porous structure parameters of the samples under study were investigated using nitrogen adsorption at the low temperature 77.4 K. The pore size distributions of the samples under study were calculated by density functional theory. With the aid of these analyses, the relationship between structure and adsorption properties of rayon-based ACFs for removing formaldehyde was revealed. Improvement of their performance in terms of adsorption selectivity and adsorption rate for formaldehyde were achieved by heat post-treatment in an inert atmosphere of nitrogen.

Parathyroid hormone-related protein (PTHrP) is a key factor behind humoral hypercalcemia of malignancy (HHM). It is produced in most breast tumors and may be an important local mediator of skeletal metastases due to breast cancer. PTHrP may mediate local bone destruction in the absence of increased circulating PTHrP. Calcitonin (CT) is used for treatment of HHM, but there are data showing that CT can increase PTHrP expression and secretion in vitro. We have therefore studied the effect of CT on PTHrP gene expression and secretion in MCF-7 breast cancer cells. PTHrP mRNA decreased significantly after 4, 8, and 16 h incubation with 10 nM salmon calcitonin (sCT) when compared with the respective controls. PTHrP mRNA also decreased significantly and dose-dependently after incubation with sCT at 0.1 to 10 nM for 16 h. The PTHrP levels in the conditioned medium also decreased in a similar dose-dependent manner. The adenylate cyclase agonist forskolin lowered the PTHrP mRNA dose-dependently. In cells exposed to varying concentrations of sCT for 15 min, the cAMP levels increased dose-dependently. In conclusion, sCT can suppress PTHrP gene expression in MCF-7 breast cancer cells. The suppressive effect is probably exerted mainly via the cAMP-protein kinase A pathways.

Using dissociation and enhancement time-resolved lanthanide fluorometry, we have developed a quantitative competitive (QC)-PCR for measuring parathyroid hormone-related protein (PTHrP) mRNA after reverse transcription. A cloned PTHrP cDNA target was also modified by deletion of 10 bp and insertion of 21 bp in the midregion of the fragment and cloned for use as a competitor (i.e., internal standard). Two primers spanning 362 bp of target and 373 bp of competitor were designed and one of the primers was biotinylated. Two oligonucleotide probes, one recognizing the target and the other hybridizing to the competitor, were labeled with Eu chelate. Two equal aliquots of PCR products were assayed with each probe separately in streptavidin-coated wells. After 35 PCR cycles, the competitor signal decreased exponentially (y = e(3.74 −0.624x); r2 = 0.965) and the target signal increased exponentially (y = e(1.14 + 0.497x); r2 = 0.984) when 1000 copies/tube of the competitor and 0–100 000 copies/tube of the target DNA were added. Log-transformed data for the ratio of target to competitor signals (y) and the copies of the target DNA added (x) were used for plotting the linear calibration curve (y = 2.79+2.76x; r2 = 0.976). This QC-PCR enables analysis of multiple samples simultaneously and can be used to study PTHrP gene expression in malignancy and physiology.

Objective: High concentrations of parathyroid hormone-related protein (PTHrP) have been found in goat milk but it is not known whether it can enter the circulation of the neonate. In this study we have developed a sensitive two-site lanthanide immunofluorometric assay (IFMA) using dissociation and enhancement time-resolved fluorometry to address this question. Method: Affinity-purified anti-PTHrP 38–67 raised in rabbit was biotinylated and immobilized in streptavidin-coated microtitration wells as a 'capture' antibody. As a signal, affinity-purified anti-PTHrP 1–34, raised in sheep, was labeled with an europium chelate. A sensitivity of 0·3 pmol/l was achieved. PTHrP levels were determined in the plasma of eleven neonatal, seven parturient and six non-pregnant, non-lactating goats as well as in goat milk. Results: The circulating PTHrP levels (mean±s.d.) were significantly increased at day 1 (6·1± 1·7 pmol/l; P< 0·01) and day 3 (3·5±0·6 pmol/l; P< 0·05) after birth in the male kids (n = 8) bottle-fed with milk from the dams, compared with before (2·2±0·7 pmol/l)and 30 min after (2·0±0·6 pmol/l) the first feeding and 14 days (2·4±0·8 pmol/l) later. In the female kids (n = 3) fed with formula there was no such increase and the concentrations remained between 1·6–1·9 pmol/l. In the parturient goats the mean±s.d. PTHrP levels before, during and after parturition were 2·9±1·7. 4·2±2·4 and 3·7±2·2 pmol/l respectively (n = 7) which demonstrated that plasma PTHrP was higher during and after parturition in comparison with before (P < 0·05). The levels in non-pregnant, non-lactating goats were 3·3±1·5 pmol/l (n = 6). PTHrP levels in goat milk were in the nanomolar range and were highest in the colostrum. Conclusions: A significant increase of plasma PTHrP was observed in goat kids fed with milk from their dams and this increase was not found in kids fed with formula. Plasma PTHrP was also increased during parturition. European Journal of Endocrinology 136 546–551

Bucht E, Rong H, Bremme K, Granberg B, Rian E, Tørring 0. Midmolecular parathyroid hormonerelated peptide in serum during pregnancy, lactation and in umbilical cord blood. Eur J Endocrinol 1993;132:438–43. ISSN 0804–4643 Indications of an important physiological role of parathyroid hormone-related peptide (PTHrP) for fetal calcium homeostasis, maternal–fetal calcium transport and reproduction have accumulated over recent years. The PTHrP concentrations were measured by an earlier developed midregion radioimmunoassay in serum from lactating healthy females and umbilical cord blood and compared with levels in age-matched non-pregnant or lactating females. The PTHrP concentrations could be measured in all samples after silica cartridge C18 extraction of 10–12 ml of serum. The concentrations were significantly higher during lactation (mean ± sd: 0.72 ± 0.14 pmol/l, N = 22) and in umbilical cord blood (0.85 ± 0.18 pmol/l, N =12) compared with healthy age-matched women (0.48 ± 0.09 pmol/l, N = 10, p< 0.001). The molecular forms of PTHrP were also studied in an agematched control group, in pregnant women and in umbilical cord blood by gel chromatography in a fast protein liquid chromatography system of Sep-Pak-extracted pooled serum. In all three groups we found heterogeneity of the molecular forms with two predominant peaks. The smallest fragment had a molecular weight of 4–6 kD while the largest form appeared as a high-molecular-weight molecule. In conclusion, the concentrations of midmolecule PTHrP fragments in serum are elevated during lactation and in umbilical cord blood. Because the midregion of PTHrP has unique actions, our results indicate that PTHrP may play an important physiological role for the mother and for the maternal–fetal calcium transport. Elisabet Bucht, Department of Molecular Medicine, Unit of Endocrinology and Diabetology, Karolinska Hospital, S-171 76 Stockholm, Sweden

Objective To study the changes on bone metabolic characteristics, bone mineral density (BMD) and bone biomechanics in type 2 diabetic model of female rats, and investigate the relationships between these indexes. Methods Type 2 diabetic model of rats was set up by feeding high-glucose-high-fat diet and by injecting intraperitoneally with a low-dose streptozotocin (STZ).All rats were sacrificed after 20 weeks. The BMD of femora and lumbar vertebra, the ratio of ash weight to dry weight, the biomechanical parameters and the bone metabolic related indexes, such as serum Ca, BGP, TRAP, urinary calcium/creatinine(Ca/Cr),urinary hydroxyproline/creatinine(HYP/Cr), and fasting blood glucose, GHbA1c, TG, TC, HDL-C,LDL-C,fasting serum insulin, IGF-Ⅰwere measured. Results Compared with normal control group, the BMD of femora and lumbar vertebra, the ratio of ash weight to dry weight and the bone biomechanical indexes of type 2 diabetic rats decreased rapidly. Insulin resistance was induced in rats of type 2 diabetes mellitus as the level of FPG, FINS, HOMA-IR markedly increased. Serum concentrations of TRAP, TG, TC, LDL-C, GHbA1c markedly increased, and BGP,IGF-Ⅰ,HDL-C decreased. There were even significant differences. BMD of femora showed a positive correlation with BGP strongly.Conclusions The rat model of type 2 diabetes mellitus which is similar to human type 2 diabetes mellitus is induced by feeding high-glucose-high-fat diet and by intraperitoneal injection with a low-dose STZ. This rat model is characterized by moderate hyperglycemia, lipid disorder and insulin resistance. The Bone resorption was accelerated and the formation decreased in this type 2 diabetic model rats, which consequently resulted in the descending bone mass and the changes of the bone biomechanical properties.