Alkaline Phosphatase Reaction Research Articles

Detection of alkaline phosphatase in canine cells previously stained with Wright–Giemsa and its utility in differentiating osteosarcoma from other mesenchymal tumors

Osteosarcoma (OSA) is a common primary bone tumor in dogs. Demonstration of alkaline phosphatase (ALP) reactivity by tumor cells on unstained slides is useful in differentiating osteosarcoma from other types of sarcoma. However, unstained slides are not always available. The objectives of this study were to evaluate the diagnostic utility of detecting ALP expression in differentiating osteosarcoma from other sarcomas in dogs using cytologic material previously stained with Wright-Giemsa stain and to assess the sensitivity and specificity of ALP expression for diagnosing osteosarcoma using a specific protocol. Archived aspirates of histologically confirmed sarcomas in dogs that had been previously stained with Wright-Giemsa stain were treated with 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as a substrate for ALP. Cells were evaluated for expression of ALP after incubation with BCIP/NBT for 1 hour. Sensitivity and specificity of ALP expression for diagnosis of OSA were calculated. In samples from 83 dogs, cells from 15/17 OSAs and from 4/66 tumors other than OSA (amelanotic melanoma, gastrointestinal stromal tumor, collision tumor, and anaplastic sarcoma) expressed ALP. Sensitivity and specificity of ALP expression detected using BCIP/NBT substrate applied to cells previously stained with Wright-Giemsa stain for OSA were 88 and 94%, respectively. ALP expression detected using BCIP/NBT substrate applied to previously stained cells is useful in differentiating canine OSA from other mesenchymal neoplasms.

Mineralization of peptide amphiphile nanofibers and its effect on the differentiation of human mesenchymal stem cells

One of the important targets in regenerative medicine is to design resorbable materials that can promote formation of new bone in large skeletal defects. One approach to this challenge is to use a bioactive and biodegradable organic matrix that can promote cellular adhesion and direct differentiation. We have here studied matrices composed of peptide amphiphiles (PAs) that self-assemble into nanofibers and create self-supporting gels under cell culture conditions. The bioactivity of PAs was designed by incorporating in their peptide sequences phosphoserine residues, to promote hydroxyapatite formation in the culture medium, and the cell adhesion epitope RGDS. In osteogenic medium supplemented with calcium the PA nanofibers were found to nucleate spheroidal nanoparticles of crystalline carbonated hydroxyapatite approximately 100nm in diameter. This mineralization mode is not epitaxial relative to the long axis of the nanofibers and occurs in the presence of serine or phosphoserine residues in the peptide sequence of the amphiphiles. Mixing of the phosphoserine-containing PAs with 5wt.% RGDS-containing PA molecules does not inhibit formation of the mineral nanoparticles. Quantitative real time reverse transcription polymerase chain reaction and immunohistochemistry analysis for alkaline phosphatase (ALP) and osteopontin expression suggest that these mineralized matrices promote osteogenic differentiation of human mesenchymal stem cells. Based on ALP expression, the presence of phosphoserine residues in PA nanofibers seems to favor osteogenic differentiation.

Morphology, cytochemistry and immunocytochemistry of the circulating granulocytes of the loggerhead sea turtle Caretta caretta

The eosinophils and heterophils of loggerheads display a similar rounded shape, an eccentric and elongated or slightly lobulated nucleus and a cytoplasm containing acidophilic pleomorphic granules. The characterization of the different types of granulocytes from nine Mediterranean loggerheads (Caretta caretta), based on cytochemical and immunocytochemical reactions and ultrastructural studies, is reported. On the basis of their positivity after myeloperoxidase, alkaline phosphatase and chloroacetate esterase reactions (typical of mammalian neutrophils), the granulocytes were classified as heterophils, while those myeloperoxidase-negative, major basic protein-positive granulocytes (like mammalian eosinophils), displaying a weak expression of interleukin 5 (a growth and differentiation factor, activator and chemoattractant for eosinophils), were classified as eosinophils. The immunocytochemical and TEM studies allowed the identification of these two granulocyte types, both of which show an eosinophilic reaction with May–Grunwald–Giemsa, periodic acid–Schiff and neutral red staining positivity.

Automated detection of particle concentration and chemical reactions in EWOD devices

Real-time measurement of electrical properties are used to perform the first real-time detection of particle concentration and chemical reactions in electrowetting on dielectric devices without the need for optical access. For particle laden droplets, the change in both resistance and capacitance was found to be linear from zero to five-hundred particles with a resolution of approximately six particles for both measurements. Electrical properties were also measured for mixtures of alkaline phosphatase and p-Nitrophenyl Phosphate; reagents commonly used in immunoassays as the resultant chemical reaction produces a yellow precipitate. Experiments were performed with mixtures created off-chip and in droplet that were statically mixed on chip. The difference between the measured and expected capacitance was found to increase with the concentration of alkaline phosphatase and chemical reactions could be positively identified in mixtures made both on and off the chip. Real-time measurements of reagents mixed in a four electrode electrowetting on dielectric mixer were also taken. In these experiments, chemical equilibrium was reached after approximately 20 cycles and the difference between the measured and expected capacitance was over 8.5 times greater than the experimental uncertainty.

Mineralized Tissue Formation by Bone Morphogenetic Protein-7–transfected Pulp Stem Cells

Introduction Recombinant human bone morphogenetic protein-7 (BMP-7) is a well-established agent to induce bone and dentin formation. Little is understood until now whether BMP-7 could be used to genetically modify human dental pulp stem cells for tissue engineering applications. Methods This study was to determine the feasibility of mineralized tissue formation from human dental pulp-derived stem cells (DPSCs) transfected with adenoviral-mediated human BMP-7 gene through in vitro and in vivo evaluations. Results In vitro results of alkaline phosphatase, calcium content, and real-time polymerase chain reaction showed that BMP-7–transfected cells had the ability to differentiate towards the odontoblast phenotype and produce a calcified extracellular matrix. Transfected cell were seeded onto a porous ceramic scaffold and implanted subcutaneously in mice. Samples were retrieved after 4 and 8 weeks for histology evaluation. The results indicated that only the cultures with BMP-7 gene transfection showed obvious hard-tissue generation. Conclusions Our data suggest that adenovirus-mediated BMP-7 expression can induce odontogenic differentiation of human DPSCs and show effectively mineralized tissue formation in vivo, which may provide support for gene therapy candidate of BMP-7 in dental tissue engineering.

Affecting osteoblastic responses with in vivo engineered potato pectin fragments

Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides.

Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems

BackgroundWhole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round.ResultsWe show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated.ConclusionSince POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

Acute Skeletal Injury Is Necessary for Human Adipose-Derived Stromal Cell–Mediated Calvarial Regeneration

Studies have demonstrated that human adipose-derived stromal cells (ASCs) are able to repair acute calvarial injuries. The more clinically relevant repair of an established skeletal defect, however, has not been addressed. The authors sought to determine whether human ASCs could heal chronic (established) calvarial defects. Critical-sized (4 mm) mouse parietal defects were created. Human ASCs were engrafted either immediately postoperatively (acute defect) or 8 weeks following defect creation (established defect). Methods of analysis included microcomputer tomography scans, histology, and in situ hybridization. Finally, human ASCs were treated in vitro with platelet-rich plasma to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (alkaline phosphatase, alizarin red, and quantitative reverse transcriptase polymerase chain reaction). Nearly complete osseous healing was observed when calvarial defects were immediately engrafted with human ASCs. In contrast, when human ASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microcomputer tomography, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in an established human ASC-engrafted defect. In situ hybridization and quantitative reverse transcriptase polymerase chain reaction showed an increase in bone morphogenetic protein (BMP) expression (BMP-2, BMP-4, and BMP-7) acutely following calvarial defect creation. Finally, in vitro treatment of human ASCs with platelet-rich plasma enhanced osteogenic differentiation and increased BMP-2 expression. Although human ASCs can be utilized to heal an acute mouse calvarial defect, they do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated after injury may explain these differences in healing. Platelet-rich plasma enhances osteogenic differentiation of human ASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.

Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-D-ribose 5-phosphate (ribose 5-phosphate) and α-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator α-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn(2+)-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[(18)O(3)]phosphate and [U-(13)C(5)]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.

A novel double staining strategy for improved detection of testicular carcinoma in situ cells in human semen samples

Prompted by the recently reported expression of POU5F1 (OCT3/4) in epididymis, a panel of markers for carcinoma in situ (CIS) testis and testicular germ cell tumours (TGCT), including AP-2γ(TFAP2C), NANOG, OCT3/4, KIT, placental-like alkaline phosphatase (PLAP), M2A/PDPN and MAGE-A4 were examined by immunohistochemistry or in situ hybridisation in urogenital epithelia, which may interfere with detection of CIS cells in semen. In addition to OCT3/4, the expression of AP-2γ and NANOG or their variants was detected in urogenital epithelia, while other CIS markers, including PLAP/alkaline phosphatase were absent. A combination of immunocytological staining for AP-2γ or OCT3/4 and rapid cytochemical alkaline phosphatase reaction was subsequently developed. This approach was tested in 22 patients with TGCT. In 14 patients (63.6%), double stained cells were found and thus the method was proven suitable for the detection of CIS cells in semen. In conclusion, transcription factors related to pluripotency and undifferentiated state of cells, which most likely have several variants or modifications, are unexpectedly detected using currently available antibodies in urogenital epithelial cells which may be shed into semen. Combining the immunohistochemical nuclear markers with a rapid cytochemical alkaline phosphatase reaction for detection of CIS cells in ejaculates may provide a more reliable diagnostic method.

Diagnosis of testicular carcinoma in situ‘(intratubular and microinvasive)’ seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections

Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for the presence of invasive germ cell tumour components, and the precursor carcinoma in situ (CIS). Currently, diagnosis is based on paraffin-embedded tissue, therefore a delay in further surgery is mainly unavoidable. The aim was to develop an intraoperative assessment technique using alkaline phosphatase as a marker. A total of 4093 snap-frozen samples and matched paraffin-embedded tissue of 1500 patients were included. Besides standard haematoxylin and eosin (H&E) staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15 min) was applied on frozen sections, while H&E and immunohistochemistry for detection of OCT3/4, α-fetoprotein, human chorionic gonadotrophin (hCG) and cytokeratin was performed on the paraffin-embedded slides. Endothelial cells served as control for the dAP test. Positive staining was found in all CIS (n=965), seminoma (n=1035) and embryonal carcinoma (n=584), either intratubular, microinvasive or invasive. Differentiated non-seminomas (n=1238) showed variable staining. No staining was identified in spermatocytic seminomas (n=5), testicular lymphomas (n=42), testicular rhabdomyosarcomas (n=7), Leydig cell tumours (n=31), Sertoli-cell-only nodules (n=4), (epi) dermoid cyst (n=16), normal testicular parenchyma (n=116), testicular torsion (n=32) and inflammation of the epididymis (n=19). The dAP test results matched H&E-stained parallel sections, as well paraffin-embedded tissue analysis, including immunohistochemistry. The dAP test is an informative, reproducible and easy tool to diagnose CIS, (intratubular and microinvasive) seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery.

Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing

Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed for rapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern.

Osteogenic differentiation of human mesenchymal stem cells synergistically enhanced by biomimetic peptide amphiphiles combined with conditioned medium

An attractive strategy for bone tissue engineering is the use of extracellular matrix (ECM) analogous biomaterials capable of governing biological response based on synthetic cell–ECM interactions. In this study, peptide amphiphiles (PAs) were investigated as an ECM-mimicking biomaterial to provide an instructive microenvironment for human mesenchymal stem cells (hMSCs) in an effort to guide osteogenic differentiation. PAs were biologically functionalized with ECM isolated ligand sequences (i.e. RGDS, DGEA), and the osteoinductive potential was studied with or without conditioned medium, containing the supplemental factors of dexamethasone, β-glycerol phosphate and ascorbic acid. It was hypothesized that the ligand-functionalized PAs would synergistically enhance osteogenic differentiation in combination with conditioned medium. Concurrently, comparative evaluations independent of osteogenic supplements investigated the differentiating potential of the functionalized PA scaffolds as promoted exclusively by the inscribed ligand signals, thus offering the potential for therapeutic effectiveness under physiological conditions. Osteoinductivity was assessed by histochemical staining for alkaline phosphatase (ALP) and quantitative real-time polymerase chain reaction analysis of key osteogenic markers. Both of the ligand-functionalized PAs were found to synergistically enhance the level of visualized ALP activity and osteogenic gene expression compared to the control surfaces lacking biofunctionality. Guided osteoinduction was also observed without supplemental aid on the PA scaffolds, but at a delayed response and not to the same phenotypic levels. Thus, the biomimetic PAs foster a symbiotic enhancement of osteogenic differentiation, demonstrating the potential of ligand-functionalized biomaterials for future bone tissue repair.

Osteoinduction in Umbilical Cord- and Palate Periosteum-Derived Mesenchymal Stem Cells

Adult abdominoplasty (AA) fat is an ideal source for mesenchymal stem cells (MSCs) because it is discarded after surgery, abundant, and easy to harvest. Children however, do not have the same abundant quantities of fat as adults, nor are they likely to undergo a procedure during which fat is routinely discarded. Hence, finding an alternate source for MSCs in children is a reasonable strategy. Two such sources are the palate periosteum (PP) and the umbilical cord (UC). Advantages for PP as a source of MSCs are accessibility during palate repair, ease of harvest, and minimal risk to the patient. The UC, like AA, is a discarded tissue, with a theoretically unlimited supply, which can be harvested in children with craniofacial bone abnormalities in advance of reconstructive procedures. Our objective in this study is to characterize MSCs from 3 sources (AA, PP, and UC) by surface marker prevalence, and to assess osteoinductive capability. Institutional review board approval was obtained for harvest of AA, PP, and UC. The presence of MSCs was determined using immunostaining and flow cytometry for cell surface markers CD73, CD90, CD105, and SSEA-4. Osteogenesis was induced using osteogenic medium. Osteoinduction was evaluated using Alizarin red staining, and real-time polymerase chain reaction for bone morphogenetic protein-2, alkaline phosphatase, and osteocalcin at 7, 14, and 21 days. MSCs from AA, PP, and UC all stained positive for CD73, CD90, CD105, and SSEA-4. Flow cytometry demonstrated significant differences in expression of CD90 and SSEA-4 but similar values for CD73 and CD105. Following osteoinduction, MSCs from all sources stained positive for calcium deposition. In UC MSCs, reverse transcriptase-polymerase chain reaction demonstrated greater elevation in bone morphogenetic protein-2 and alkaline phosphatase mRNA beginning at day 7 and extending to day 21. Osteocalcin mRNA levels were comparable for all 3 sources of MSCs. For children with craniofacial bone defects, UC-derived MSCs may be ideal for tissue engineered bone: temporally, the UC can be harvested in advance of surgical timing for the need for bone, is readily available, easy to harvest, and leads to osteoinduction that is more robust than either AA or PP.

Gain of Function Mutations in Membrane Region M2C2 of KtrB Open a Gate Controlling K+ Transport by the KtrAB System from Vibrio alginolyticus

KtrB, the K(+)-translocating subunit of the Na(+)-dependent bacterial K(+) uptake system KtrAB, consists of four M(1)PM(2) domains, in which M(1) and M(2) are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M(2C) is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M(2C1) and M(2C3), which are connected by a nonhelical M(2C2) region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M(2C2) led to a huge gain of function of K(+) uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in V(max) for K(+) transport. Na(+) translocation by KtrB was not affected. Partial to complete deletions of M(2C2) also led to enhanced V(max) values for K(+) uptake via KtrB. However, several deletion variants also exhibited higher K(m) values for K(+) uptake and at least one deletion variant, KtrB(Delta326-328), also transported Na(+) faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M(2C2) forms a flexible structure within the membrane allowing M(2C3) to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M(2C2) forms a flexible gate controlling K(+) translocation at the cytoplasmic side of KtrB, and (ii) that M(2C2) is required for the interaction between KtrA and KtrB.

12-O-Tetradecanoylphorbol-13-acetate Inhibits Melanoma Growth by Inactivation of STAT3 through Protein Kinase C-activated Tyrosine Phosphatase(s)

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

Alkaline phosphatase reaction buffer (APRB)

Alkaline phosphatase reaction buffer (APRB)

Derivation and Characterization of Two Genetically Unique Human Embryonic Stem Cell Lines on In-House–Derived Human Feeders

This study describes the successful derivation of two human embryonic stem (hES) cell lines using 53 frozen and 18 fresh "slow-growing" surplus embryos, obtained from collaborating in vitro fertilization clinics, on in-house-derived human feeder layers. The cell lines have been derived by whole embryo culture followed by further expansion of manually dissected inner cell mass from the surrounding trophoectodermal cells. Immunocytochemical localization of cell surface markers like SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, staining for alkaline phosphatase and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of pluripotency state markers viz. Oct-4, TERT, Nanog, Rex1, and Sox2 indicate that both cell lines possess typical features of embryonic stem cells. Both cell lines exhibit normal female karyotype after 40 passages in culture. Pluripotent nature of the cell lines was confirmed both in vitro and in vivo. Embryoid bodies, formed in suspension culture, express markers for all three lineages as indicated by RT-PCR analysis for SOX 1 (ectoderm), HAND 1 (mesoderm), AFP (endoderm), and CDX2 (trophoectoderm). Teratoma formed in vivo in severe combined immunodeficient mice revealed cells of all the three embryonic germ layers. Comparison of the STR and human leukocyte antigen profiles of these cell lines with the existing human ES cell lines indicate that they are genetically distinct. The addition of our hES cell lines contributes usefully to the globally restricted repertoire of human ES cell lines.

Immunohistochemical analysis of ZAP-70 expression in chronic lymphocytic leukemia

This paper shows a protocol for the detection of ZAP-70 expression in B-CLL (B cell chronic lymphocytic leukemia) tumor cells by common immunohistochemical methods. The study was conducted on bone marrow trephine biopsies from 62 B-CLL patients at the time of diagnosis. Immunohistochemical reactions based on peroxidase and alkaline phosphatase reactions were used, as well as double immunofluorescent labeling for ZAP-70 detection as an indirect marker of mutated and unmutated CLL. Clinical relevance of the ZAP-70 expression detection method was assessed using chi2 test between ZAP-70 positivity data and other known prognostic factors, i.e., clinical and cytogenetics data. ZAP-70 was detected in 13 out of 62 patients. Statistically significant results were obtained for ZAP-70 positive cases and known indicators of worse prognosis. Immunohistochemical analysis supported by double immunofluorescent labeling, as shown here, is an easy and reliable technique for the detection of ZAP-70 expression in B-CLL tumor cells applicable in every hematopathology laboratory.

Entrapment and measurement of a biologically functionalized microbead with a microwell electrode

A chip with integrated electrophoretic and electrochemical systems was developed to manipulate either an individual microbead or a cell inside a microwell electrode (MWE) for electrochemical measurement. The optimal MWE geometry (30 microm diameter and 25 microm depth) was designed to accommodate the micro particles according to the simulated results. A chip device was sequentially built from a slide patterned with Pt electrodes, an adhesive tape defined with a flow channel (200 microm in width and 25 microm in height), and an indium tin oxide (ITO) cover. The MWE not only generated an active electrophoretic force to trap the particle but also provided a low flow velocity area (LFVA) to stabilize the trapped bead or cell in a continuous flow. Scanning electrochemical microscopy (SECM) theory was employed to explain the electrochemical behaviors of the MWE. An enhanced current was confirmed as the redox recycling effect on the conductive ITO cover. The catalytic reaction of an individual alkaline phosphatase coated microbead (ALP-bead) was electrochemically detected with the MWE after being trapped. The ALP on the trapped ALP-bead catalyzed the hydrolysis of p-aminophenylphosphate (PAPP) to p-aminophenol (PAP), and then a decaying amperogram (+0.3 V vs. Ag/AgCl) due to a tiny PAP quantity around the MWE was observed.