Showing papers on "Alkaline phosphatase published in 2012"
TL;DR: An inhibition type strategy has been developed to sensitively detect ALP and indicates that the deposition of the electropolymerized phenol products may compete with the electrophoretic-driven adsorption of CdSe NPs on glassy carbon electrode and induce the ECL inhibition, which can be demonstrated by scanning electron microscopy, energy dispersive spectrometry, and anodic stripping voltammetry.
Abstract: Alkaline phosphatase (ALP) catalyzes the hydrolysis and transphosphorylation of a wide variety of phosphoric acid monoesters and plays an important role in clinical diagnosis. In this work, an ALP-responsive anodic electrochemiluminescence (ECL) system based on coreaction of CdSe nanoparticles (NPs) and triethylamine has been designed for facile detection of ALP. The substrate of ALP, i.e., phenyl phosphate salt, shows no effect on the ECL emission whereas its catalytic product of phenol may induce ECL inhibition. For the buffer containing phenyl phosphate, the ECL emission is found to decline in the presence of ALP with different incubation time. The mechanism investigations indicate that the deposition of the electropolymerized phenol products may compete with the electrophoretic-driven adsorption of CdSe NPs on glassy carbon electrode and induce the ECL inhibition, which can be demonstrated by scanning electron microscopy, energy dispersive spectrometry, and anodic stripping voltammetry. Therefore, an ...
149 citations
TL;DR: The collagenase/trypsin method of cell isolation yields a higher cell density than the others and expresses a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method ofcell isolation resulted in a highercell proliferation rate and activity compared to the other methods.
Abstract: Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.
147 citations
TL;DR: Morphological analysis showed that typically advanced plaques with extensive calcification lesions, abundant cholesterol crystals, and proliferative collagen were formed 4 months after the triple administrations of STZ-CML-HFD, and that CML deposition signals and the expression of receptor for advanced glycation end-products (RAGE) in the aortic wall were mainly restricted in the atherosclerotic plaques.
147 citations
TL;DR: Higher magnesium levels prevented BVSMC calcification, inhibited expression of osteogenic proteins, apoptosis and further progression of already established calcification and may have the potential to play a role for VC also in clinical situations.
Abstract: Background. Vascular calcification (VC), mainly due to elevated phosphate levels, is one major problem in patients suffering from chronic kidney disease. In clinical studies, an inverse relationship between serum magnesium and VC has been reported. However, there is only few information about the influence of magnesium on calcification on a cellular level available. Therefore, we investigated the effect of magnesium on calcification induced by b-glycerophosphate (BGP) in bovine vascular smooth muscle cells (BVSMCs). Methods. BVSMCs were incubated with calcification media for 14 days while simultaneously increasing the magnesium concentration. Calcium deposition, transdifferentiation of cells and apoptosis were measured applying quantification of calcium, von Kossa and Alizarin red staining, real-time reverse transcription–polymerase chain reaction and annexin V staining, respectively. Results. Calcium deposition in the cells dramatically increased with addition of BGP and could be mostly prevented by co-incubation with magnesium. Higher magnesium levels led to inhibition of BGP-induced alkaline phosphatase activity as well as to a decreased expression of genes associated with the process of transdifferentiation of BVSMCs into osteoblast-like cells. Furthermore, estimated calcium entry into the cells decreased with increasing magnesium concentrations in the media. In addition, higher magnesium concentrations prevented cell damage (apoptosis) induced by BGP as well as progression of already established calcification. Conclusions. Higher magnesium levels prevented BVSMC calcification, inhibited expression of osteogenic proteins, apoptosis and further progression of already established calcification. Thus, magnesium is influencing molecular processes associated with VC and may have the potential to play a role for VC also in clinical situations.
143 citations
TL;DR: IL-1β effectively and rapidly induced human MSC differentiation into osteoblasts and mineralization, mainly through the noncanonical Wnt-5a/Ror2 pathway, which suggests potential benefits of IL-1 β-treated human M SCs in the treatment of damaged bone as well as in the induction of self-renewal and self-repair of damaged tissue.
Abstract: Objective
Mesenchymal stem cells (MSCs) are considered to be a novel tool for the treatment of rheumatoid arthritis (RA) because of their multipotency to differentiate into osteoblasts and chondrocytes, their immunosuppressive effects, and availability. The aim of this study was to assess the mechanisms of human MSC differentiation into osteoblasts under inflammatory conditions.
Methods
Human MSCs were cultured in commercialized osteogenic induction medium with inflammatory cytokines for up to 10 days. Osteoblast differentiation was detected by alkaline phosphatase staining and messenger RNA (mRNA) expression of multiple osteoblast markers. Mineralization was assessed by alizarin red S staining.
Results
Among the various cytokines tested, interleukin-1β (IL-1β) induced differentiation of human MSCs into osteoblasts, which was confirmed by alkaline phosphatase activity, expression of RUNX2 mRNA, and strong alizarin red S staining. Among various molecules of the Wnt family, Wnt-5a and receptor tyrosine kinase–like orphan receptor 2 (Ror2), a major receptor of Wnt-5a, were significantly induced in human MSCs by IL-1β. Silencing of either WNT5A or ROR2 by small interfering RNA with 2 different sequences reduced alkaline phosphatase activity, RUNX2 expression, and alizarin red S staining of human MSCs induced by IL-1β.
Conclusion
IL-1β effectively and rapidly induced human MSC differentiation into osteoblasts and mineralization, mainly through the noncanonical Wnt-5a/Ror2 pathway. These results suggest potential benefits of IL-1β–treated human MSCs in the treatment of damaged bone as well as in the induction of self-renewal and self-repair of damaged tissue, including osseous tissue.
138 citations
TL;DR: Leptin predicted the serum levels of PTH, 1,25D, and FGF23, the mineral-regulating hormones, and BAP, a bone formation marker, in women with body mass index ranging from 13.9-65.8 kg/m(2).
Abstract: Background: Obesity is associated with hyperparathyroidism and increased bone mass and turnover, but their pathogeneses are unclear. Aims: Our aim was to determine in obesity interrelationships among serum levels of leptin, the mineral-regulating hormones, bone turnover markers, and sclerostin. Methods: This case-control study was performed in 20 women having bariatric surgery and 20 control women matched for race and age. Anthropometrics and fasting serum biochemistries were measured in controls and in bariatric patients the morning of surgery. Results: Body mass index (48.9 vs. 25.4 kg/m2), weight (128.6 vs. 71.9 kg), serum leptin (74.6 vs. 25.2 ng/ml), PTH (44.5 vs. 28.8 pg/ml), fibroblast growth factor 23 (FGF23) (42.4 vs. 25.9 pg/ml), and bone alkaline phosphatase (BAP) (25.8 vs. 17.5 U/liter) were higher, but height (162.3 vs. 167.7 cm) and 1,25-dihydroxyvitamin D (1,25D) (39.2 vs. 48.7 pg/ml) were lower in bariatric surgery patients than controls. There was no difference in serum sclerostin, amino-...
129 citations
TL;DR: It is reported that ECM derived from human umbilical vein endothelial cells (HUVEC) promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSC) and Cell-derived ECM-containing calcium phosphate scaffolds is a promising osteogenic-promoting bone void filler in bone tissue regeneration.
110 citations
TL;DR: Comp comparative DNA microarray analysis between mouse myoblastic C2C12 cells transfected with either stable empty vector or ALK2, the mutation that constitutively activates the bone morphogenetic protein (BMP) receptor, suggests that OGN may be a crucial humoral bone anabolic factor that is produced by muscle tissues.
98 citations
TL;DR: AGE2 and AGE3, but not high glucose, may inhibit the osteoblastic differentiation of stromal cells by decreasing osterix expression and partly by increasing RAGE expression, as well as inhibiting cell growth and increasing cell apoptosis.
Abstract: Diabetes mellitus is known to be associated with osteoporotic fractures through a decrease in osteoblastic bone formation rather than an increase in osteoclastic bone resorption. However, its precise mechanism is unknown, and we examined whether or not high glucose or advanced glycation end products (AGEs), which play key roles in the pathogenesis and complications of diabetes, would affect the osteoblastic differentiation, growth, and apoptosis of mouse stromal ST2 cells. Ten to 200 μg/mL AGE2 or AGE3 alone dose-dependently inhibited the mineralization. AGE2 or AGE3 alone (200 μg/mL) significantly inhibited alkaline phosphatase (ALP) activities as well as the mineralization of the cells (p < 0.01). In contrast, 22 mM glucose alone or in combination with 200 μg/mL AGE2 or AGE3 did not affect these cellular phenotypes. Real-time PCR showed that AGE2 or AGE3 alone (200 μg/mL) significantly decreased mRNA expressions of osteocalcin as well as osterix on day 14 (p < 0.01). Western blot analysis showed that AGE2 or AGE3 alone (200 μg/mL) also decreased the levels of Runx2 and osterix protein expressions on days 7 and 14. AGE2 or AGE3 significantly suppressed cell growth and increased apoptotic cell death in time- and dose-dependent manners (p < 0.01). Moreover, AGE3 alone (200 μg/mL) significantly increased mRNA expression of the receptor for AGEs (RAGE) on days 2 and 3 (p < 0.01). These results suggest that AGE2 and AGE3, but not high glucose, may inhibit the osteoblastic differentiation of stromal cells by decreasing osterix expression and partly by increasing RAGE expression, as well as inhibiting cell growth and increasing cell apoptosis.
87 citations
TL;DR: Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP) and increases Young's modulus, most profoundly for cPEG.
Abstract: Alkaline Phosphatase (ALP), an enzyme involved in mineralization of bone, was incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These were collagen type I, a mussel protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG-fumaric acid copolymer OPF. After incubation in calcium glycerophosphate (Ca-GP) solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES and von Kossa staining confirmed CaP formation. The amount of mineral formed decreased in the order cPEG > collagen > OPF. Mineral:polymer ratio decreased in the order collagen > cPEG > OPF. Mineralization increased Young’s modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel-CaP composites could find application as bone regeneration materials.
85 citations
TL;DR: Induced pluripotent stem cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins have potential applications in transgenic breeding and gene-modified animals.
Abstract: Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals.
TL;DR: In this paper, the authors investigated the potential mechanism of Arg-Gly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl4-induced hepatic fibrosis in rats.
Abstract: AIM: To investigate the potential mechanism of Arg-Gly-Asp (RGD) peptide-labeled liposome loading oxymatrine (OM) therapy in CCl4-induced hepatic fibrosis in rats.
METHODS: We constructed a rat model of CCl4-induced hepatic fibrosis and treated the rats with different formulations of OM. To evaluate the antifibrotic effect of OM, we detected levels of alkaline phosphatase, hepatic histopathology (hematoxylin and eosin stain and Masson staining) and fibrosis-related gene expression of matrix metallopeptidase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-1 as well as type I procollagen via quantitative real-time polymerase chain reaction. To detect cell viability and apoptosis of hepatic stellate cells (HSCs), we performed 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and flow cytometry. To reinforce the combination of oxymatrine with HSCs, we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM, and its targeting of HSCs was examined by fluorescent microscopy.
RESULTS: OM attenuated CCl4-induced hepatic fibrosis, as defined by reducing serum alkaline phosphatase (344.47 ± 27.52 U/L vs 550.69 ± 43.78 U/L, P < 0.05), attenuating liver injury and improving collagen deposits (2.36% ± 0.09% vs 7.70% ± 0.60%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). OM inhibited cell viability and induced apoptosis of HSCs in vitro. RGD promoted OM targeting of HSCs and enhanced the therapeutic effect of OM in terms of serum alkaline phosphatase (272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L, P < 0.05), liver injury, collagen deposits (0.26% ± 0.09% vs 2.36% ± 0.09%, P < 0.05) and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen (P < 0.05). Moreover, in vitro assay demonstrated that RGD enhanced the effect of OM on HSC viability and apoptosis.
CONCLUSION: OM attenuated hepatic fibrosis by inhibiting viability and inducing apoptosis of HSCs. The RGD-labeled formulation enhanced the targeting efficiency for HSCs and the therapeutic effect.
TL;DR: It is demonstrated that AS1 strain can modulate the development of DMH-induced rat colon carcinogenesis through an antioxidant-dependent mechanism.
Abstract: The relationship between antioxidant and anticancer properties of probiotic bacterium strain Lactobacillus plantarum AS1 (AS1) in colon cancer induced by 1,2-dimethylhydrazine (DMH) has been studied. In this study, an increased level of lipid peroxide (LPO) products and increased activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione-S transferase) and marker enzymes (alkaline phosphatase and acid phosphatase) in colon and plasma of cancer-bearing animals have been observed. AS1 was supplemented either before initiation or during initiation and selection/promotion phases of colon carcinogenesis and was found to be effective in altering lipid peroxidation and antioxidant enzyme activities and marker enzymes to a statistically significant level measured either in the colon and in the plasma. These alterations inclined towards normal in a time-dependent manner on AS1 supplementation. The mean tumor volume diameter and total number of tumors were found to be statistically decreased in AS1 pre- and post-treated rats. Furthermore, histopathological examination shows remarkable difference between control and treated groups. The in vitro antioxidant assay shows that AS1 has promising antioxidant property. These results demonstrate that AS1 strain can modulate the development of DMH-induced rat colon carcinogenesis through an antioxidant-dependent mechanism.
TL;DR: The results indicated that TSG may be effective in providing protection against osteoporosis associated with oxidative stress and inhibition of the release of bone-resorbing mediators and oxidative damage of the cells.
TL;DR: Results suggest that nicotine has a bimodal effect on the proliferation and osteoblast differentiation in hABMMSCs.
TL;DR: It is speculated that V10, which has a potential to be a potent effector for disease treatment, could have a role in treating Leishmania diseases.
TL;DR: It is demonstrated that NO effectively augments MSC ability to repair liver fibrosis induced by CCl4 in mice and therefore is a better treatment regimen to reduce Liver fibrosis.
Abstract: Liver fibrosis is a major health problem worldwide and poses a serious obstacle for cell based therapies. Mesenchymal stem cells (MSCs) are multipotent and important candidate cells for future clinical applications however success of MSC therapy depends upon their homing and survival in recipient organs. This study was designed to improve the repair potential of MSCs by transplanting them in sodium nitroprusside (SNP) pretreated mice with CCl4 induced liver fibrosis. SNP 100 mM, a nitric oxide (NO) donor, was administered twice a week for 4 weeks to CCl4-injured mice. MSCs were isolated from C57BL/6 wild type mice and transplanted in the left lateral lobe of the liver in experimental animals. After 4 weeks, animals were sacrificed and liver improvement was analyzed. Analysis of fibrosis by qRT-PCR and sirius red staining, homing, bilirubin and alkaline phosphatase (ALP) serum levels between different treatment groups were compared to control. Liver histology demonstrated enhanced MSCs homing in SNP-MSCs group compared to MSCs group. The gene expression of fibrotic markers; αSMA, collagen 1α1, TIMP, NFκB and iNOS was down regulated while cytokeratin 18, albumin and eNOS was up-regulated in SNP-MSCs group. Combine treatment sequentially reduced fibrosis in SNP-MSCs treated liver compared to the other treatment groups. These results were also comparable with reduced serum levels of bilirubin and ALP observed in SNP-MSCs treated group. This study demonstrated that NO effectively augments MSC ability to repair liver fibrosis induced by CCl4 in mice and therefore is a better treatment regimen to reduce liver fibrosis.
TL;DR: The data indicate that TNAP is increased in both sporadic and familial AD but not in the aged brain, indicating that the increase is likely a consequence of AD-associated changes in the brain.
Abstract: Background: Tissue non-specific alkaline phosphatase (TNAP) has been shown to promote the neurotoxicity of extracellular tau which contributes to the spread of pathology in Alzheime
TL;DR: It is suggested that TGFβ subtypes do affect terminal differentiation of in vitro cultured hBMSCs differently, with significantly less cartilage formation when 2.5 ng/ml of either TGF β subtype was used and no differences between 10 and 25 ng/ML.
Abstract: Human bone marrow stromal-derived mesenchymal stem cells (hBMSCs) will differentiate into chondrocytes in response to defined chondrogenic medium containing transforming growth factor-β (TGFβ). Results in the literature suggest that the three mammalian subtypes of TGFβ (TGFβ1, TGFβ2 and TGFβ3) provoke certain subtype-specific activities. Therefore, the aim of our study was to investigate whether the TGFβ subtypes affect chondrogenic differentiation of in vitro cultured hBMSCs differently. HBMSC pellets were cultured for 5 weeks in chondrogenic media containing either 2.5, 10 or 25 ng/ml of TGFβ1, TGFβ2 or TGFβ3. All TGFβ subtypes showed a comparable dose-response curve, with significantly less cartilage when 2.5 ng/ml was used and no differences between 10 and 25 ng/ml. Four donors with variable chondrogenic capacity were used to evaluate the effect of 10 ng/ml of either TGFβ subtype on cartilage formation. No significant TGFβ subtype-dependent differences were observed in the total amount of collagen or glycosaminoglycans. Cells from a donor with low chondrogenic capacity performed equally badly with all TGFβ subtypes, while a good donor overall performed well. After addition of β-glycerophosphate during the last 2 weeks of culture, the expression of hypertrophy markers was analysed and mineralization was demonstrated by alkaline phosphatase activity and alizarin red staining. No significant TGFβ subtype-dependent differences were observed in expression collagen type X or VEGF secretion. Nevertheless, pellets cultured with TGFβ1 had significantly less mineralization than pellets cultured with TGFβ3. In conclusion, this study suggests that TGFβ subtypes do affect terminal differentiation of in vitro cultured hBMSCs differently.
TL;DR: AA and GP provided an inductive effect for differentiation of osteoprecusor cells, while short-term application of DEX seemed to lead to a dose-dependent increase of cellular differentiation and long-term use of DEx seemed to reduce mineralization.
TL;DR: Protective effect of TSTA against CCl4-induced hepatic injury may rely on its effect on reducing oxidative stress, suppressing inflammatory responses and improving drug-metabolizing enzyme activity in liver.
TL;DR: The development and application of a novel fluorogenic live cell permeant substrate for AP (Live AP Stain), which shows specific and robust staining of pluripotent cells such as human EC, murine and human ESC and iPSC with minimal or noStaining of feeder cells and human fibroblasts.
Abstract: Alkaline phosphatases (AP) are a class of enzymes that hydrolyze phosphate containing molecules under alkaline conditions. In humans, there are primarily four different types of this enzyme; intestinal, placental, placental-like and non-tissue specific forms. The non-tissue specific isozyme of AP is expressed in liver, bone and kidney. A similar isozyme was identified in pluripotent stem cells when monoclonal antibodies, TRA-2-49/6E, recognizing determinants of human embryonal carcinoma (EC) cells showed specific reactivity to this isoform.1 AP is also known to be expressed at high levels in other pluripotent stem cell types such as embryonic germ cells (EG), embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC).2–5 Although definitive measures of pluripotency involves in-vitro tri-lineage differentiation and in-vivo teratoma formation, the most widely tested and validated panel for initial evaluation of ESC and iPSC consists of Stage Specific Embryonic Antigen SSEA4; Tumor Rejection Antigens TRA-1-60, TRA-1-81; AP; Oct4 and Nanog.6, 7
In the case of murine ESC, AP positive colony forming in-vitro assay is used as a measure of pluripotency to demonstrate the ability of cells to single cell clone, attach and proliferate.8 A similar assay has been adapted for hESC where the sensitivity of the AP positive Colony forming assay to detect loss of pluripotent hESC has been found to be more sensitive than marker expression.9 More recently, the onset of AP positive colonies during early stages of reprogramming is used as an initial indicator of successful reprogramming of cells. Furthermore, in some instances the number of AP positive colonies is used as a mark of reprogramming efficiency.10 Nevertheless, this marker alone is not a definitive marker for the established iPSC clones. Additional marker evaluation is necessary to identify and qualify bona fide iPSCs.11 AP expression levels is a less sensitive measure to differentiate between undifferentiated and early differentiating cells since its expression level is reported to be varied depending on the lineage of differentiation.12 AP staining has been used as a fast and easy method that results in a specific chromogenic or fluorescent staining of the pluripotent stem cells. However, the current methods using AP staining require cell fixation and/or result in end products that accumulate within the cells. As a result, these AP stained colonies often lose their morphology and cannot be propagated any further. Inability to further culture selected pluripotent colonies identified using AP staining is a serious disadvantage of this methodology. An ideal solution would be an AP substrate that stains cells without altering the integrity or characteristics of stem cells thereby allowing further expansion of the stained colonies.
Here in, we report the development and application of a novel fluorogenic live cell permeant substrate for AP (Live AP Stain). When incubated with cells for 20–30 min in basal culture media, this stain shows specific and robust staining of pluripotent cells such as human EC, murine and human ESC and iPSC with minimal or no staining of feeder cells and human fibroblasts. Stained colonies retain their morphology and preserve their cell health. The green fluorescence of the stained colonies is eliminated from cells 60–90 min after removal of the stain from the media. We have further utilized this stain in iPSC work flow to identify emerging iPSC clones during reprogramming of BJ human fibroblasts using CytoTune™; a Sendai-virus based non-integrating reprogramming method.13 Clones with robust AP staining were manually picked and propagated further. Expanded clones expressed other pluripotent markers, differentiated into cell types representative of the three germ layers and maintained a normal karyotype. These results indicate that AP Live Stain reported in this study does not alter the integrity or characteristics of the stained cells and is therefore an ideal tool to label early intermediates during iPSC generation or clonal populations of ESC for further selection and expansion.
TL;DR: Tissue nonspecific alkaline phosphatase (TNAP) activity can be described as more primitive bone marrow-derived cells and TNAP levels in MSCs can be used to predict chondrocyte hypertrophy or osteogenic capacity.
Abstract: Multiple surface markers have been utilized for the enrichment of bone marrow mesenchymal stromal cells (MSCs) and to define primitive stem cells. We classified human bone marrow-derived MSC populations according to tissue nonspecific alkaline phosphatase (TNAP) activity. TNAP expression varied among unexpanded primary MSCs, and its level was not related to colony-forming activity or putative surface markers, such as CD105 and CD29, donor age, or gender. TNAP levels were increased in larger cells, and a colony-forming unit-fibroblast assay revealed that the colony size was decreased during in vitro expansion. TNAP-positive (TNAP+) MSCs showed limited multipotential capacity, whereas TNAP-negative (TNAP−) MSCs retained the differentiation potential into 3 lineages (osteogenic-, adipogenic-, and chondrogenic differentiation). High degree of calcium mineralization and high level of osteogenic-related gene expression (osteopontin, dlx5, and cbfa1) were found in TNAP+ cells. In contrast, during chondrogenic di...
TL;DR: A rapid in situ method on live cells to measure activities of alkaline phosphatase, alanyl amino peptidase and sucrase is developed and can easily be adapted to robotised high throughput platforms.
TL;DR: Results suggested the oxidative stress induced liver damage which may be due to the regulation of multiple genes by Apigenin at higher doses in Swiss mice.
Abstract: Apigenin, a dietary flavonoid, is reported to have several therapeutic effects in different diseases including cancer. Toxicity of Apigenin is however, least explored, and reports are scanty in literature. This warrants dose-specific evaluation of toxicity in vivo. In the present study, Apigenin was administered intraperitoneally to Swiss mice at doses of 25, 50, 100 and 200 mg/kg. Serum levels of alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) were measured along with the examination of liver histology, reactive oxygen species (ROS) in blood, lipid peroxidation (LPO), glutathione level, superoxide dismutase activity, catalase activity, glutathione S-transferase activity and gene expression in liver tissue. Increase in ALT, AST, ALP, ROS, ratio of oxidized to reduced glutathione (GSSG/GSH) and LPO, altered enzyme activities along with damaged histoarchitecture in the liver of 100 or 200 mg/kg Apigenin treated animals were found. Microarray analysis revealed the differential expression of genes that correspond to different biologically relevant pathways including oxidative stress and apoptosis. In conclusion, these results suggested the oxidative stress induced liver damage which may be due to the regulation of multiple genes by Apigenin at higher doses in Swiss mice.
TL;DR: A new window towards cancer therapy with the combined control of elevated levels of alkaline phosphatase and granulomatous amoebic encephalitis in cancer patients is revealed.
Abstract: The biological significance of polyoxometalates is well renowned owing to their anticancer, antiviral and antibiotic properties. Here another therapeutic aspect of polyoxometalates has been explored as alkaline phosphatase inhibitors along with the remarked anticancer and amoebicidal properties. Synthesis and inhibitory studies of a set of seven polyoxotungstates against two major isozymes of alkaline phosphatase i.e. tissue specific and tissue non-specific alkaline phosphatase revealed their promising activity as alkaline phosphatase inhibitors. All compounds exhibited alkaline phosphatase inhibitory potency in nanomolar ranges. For tissue specific alkaline phosphatase, Na10[H2W12O42]·27H2O (A6) was found to be the most potent inhibitor (Ki value 313 ± 7 nM), while for tissue non-specific alkaline phosphatase Na33[H7P8W48O184]·92H2O (A3) showed the highest inhibition potency (Ki values 135 ± 10 nM). Moreover cytotoxicity evaluation of these compounds against lung carcinoma cells and immortalized human corneal epithelial cells demonstrated their anticancer potential with no cytotoxic effects on normal human cell lines. All anticancer drugs result in an impaired immune system and such immunocompromised persons become vulnerable to opportunistic infections specially Acanthamoeba which causes granulomatous amoebic encephalitis (GAE) which almost always results in death. The exclusive property of our tested polyoxotungstates is their strong amoebicidal activity against Acanthamoeba. Hence the study reveals a new window towards cancer therapy with the combined control of elevated levels of alkaline phosphatase and granulomatous amoebic encephalitis in cancer patients.
TL;DR: The results showed that bone turnover was significantly increased and bone mass was significantly decreased 8 weeks after ovariectomy in rats, proposing that the ovariectomized rat model be considered a reproducible and reliable model of osteoporosis.
Abstract: Objective The purpose of this study was to verify the appropriateness of ovariectomized rats as the osteoporosis animal model. Methods Twelve female Sprague-Dawley rats underwent a sham operation (the sham group) or bilateral ovariectomy [the ovariectomy (OVX) group]. Eight weeks after operations, serum biochemical markers of bone turnover were analyzed; osteocalcin and alkaline phosphatase, which are sensitive biochemical markers of bone formation, and C-terminal telopeptide fragment of type I collagen C-terminus (CTX), which is a sensitive biochemical marker of bone resorption. Bone histomorphometric parameters and microarchitectural properties of 4th lumbar vertebrae were determined by micro-computed tomographic (CT) scan. Results The OVX group showed on average 75.4% higher osteocalcin and 72.5% higher CTX levels than the sham group, indicating increased bone turnover. Micro-CT analysis showed significantly lower bone mineral density (BMD) (p=0.005) and cortical BMD (p=0.021) in the OVX group. Furthermore, the OVX group was found to have a significantly lower trabecular bone volume fraction (p=0.002). Conclusion Our results showed that bone turnover was significantly increased and bone mass was significantly decreased 8 weeks after ovariectomy in rats. Thus, we propose that the ovariectomized rat model be considered a reproducible and reliable model of osteoporosis.
TL;DR: The current evidence does not support ALP being proposed as a stand-alone means of monitoring treatment and it only achieves usefulness in a diagnostic and monitoring capacity when combined with other serum and imaging techniques.
Abstract: Alkaline phosphatase (ALP) is regularly measured in clinical practice. Changes in serum levels are observed in a number of clinical conditions. In neonatology, it has been proposed as a useful marker for both a diagnosis and an indication of the severity of metabolic bone disease (MBD) in infants born preterm. Nutritional practices, aimed at reducing the occurrence or severity of MBD, have led to ALP being proposed as a stand-alone means of monitoring treatment. The current evidence does not support this use: ALP only achieves usefulness in a diagnostic and monitoring capacity when combined with other serum and imaging techniques.
TL;DR: The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner, and osteogenesis was optimal in certain iron concentrations, which may be attributed to increased intracellular ROS levels.
Abstract: Bone metabolism has a close relationship with iron homeostasis. To examine the effects of iron excess and iron deficiency on the biological activities of osteoblast in vitro, human osteoblast cells (hFOB1.19) were incubated in a medium supplemented with 0–200 μmol/L ferric ammonium citrate and 0–20 μmol/L deferoxamine. The intracellular iron was measured by a confocal laser scanning microscope. Proliferation of osteoblasts was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptotic cells were detected using annexin intervention V/PI staining with a flow cytometry. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. The number of calcified nodules and mineral area was evaluated by von Kossa staining assay. The expressions of type I collagen and osteocalcin of cultured osteoblasts were detected by reverse transcriptase polymerase chain reaction and Western blot. Intracellular reactive oxygen species (ROS) was measured using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate by flow cytometry. The results indicated that excessive iron inhibited osteoblast activity in a concentration-dependent manner. Low iron concentrations, in contrast, produced a biphasic manner on osteoblasts: mild low iron promoted osteoblast activity, but serious low iron inhibited osteoblast activity. Osteogenesis was optimal in certain iron concentrations. The mechanism underlying biological activity invoked by excessive iron may be attributed to increased intracellular ROS levels.
TL;DR: Treatment with camel's milk alleviates alcohol-associated hazards and protects hepatic tissue from alcohol-induced toxicity, and results showed amelioration of all tested parameters following administration of CM.