5′-Nucleotidase

Summary 5′-Nucleotidase and alkaline phosphatase activity was investigated in the developing kidney of the mouse by histochemical and electrophoretic methods. The growth of the kidneys was studied by determining the incorporation of radioactive thymidine by autoradiography. During development the isoenzyme patterns of 5′-nucleotidase and alkaline phosphatase behaved in a different way. In correlating the histochemical and electrophoretic changes, it has been found that the 5′-nucleotidase isoenzymes as well as the alkaline phosphatase isoenzymes are located in different parts of the kidney. In the convoluted part of the proximal tubule 5′-nucleotidase isoenzyme 3 and alkaline phosphatase isoenzyme 5 are present, while in the straight part of this tubule 5′-nucleotidase isoenzyme 5 and — upto three weeks — alkaline phosphatase isoenzyme 3 are located. So in tissue structures having different functional capacities, different isoenzymes of 5′-nucleotidase and alkaline phosphatase are found.     

A rapid micro method for counting cells “in situ” using a fluorogenic alkaline phosphatase enzyme assay

Summary A new method has been developed to count cells “in situ”, based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10⁴ to 5×10⁵ for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays. Editor's Statement This paper describes a quick method for quantitation of cell number in microcultures. Such procedures are valuable for the many situations in which minimizing cells and medium volume is desirable, although somewhat specialized equipment is required for the procedure. An alternative procedure for quantitation of cells in microtiter culture appeared previously in this journal (McCaffrey, et al., 24∶247–252).     

Bispecific-Ab-based immunoassay of thyroid-stimulating hormone

 A bispecific F(ab′)₂ fragment recognizing both human thyroid-stimulating hormone (TSH) and alkaline phosphatase (AP) was prepared by disulfide bond exchange between F(ab′)₂ fragments of IgG1 mAb against TSH and AP. AP was polymerized by glutaraldehyde, and a sandwich enzyme-linked immunosorbent assay for TSH was developed by using the AP polymers and the bispecific F(ab′)₂ fragment. In this assay, the preparation of covalently linked AP-mAb conjugates was not needed, and the interaction of mAb with non-specific proteins was greatly reduced. The sensitivity for TSH increased in proportion to the degree of AP polymerization, and the lower detection limit obtained with the AP trimer was 0.5 μU/ml. The use of the bispecific F(ab′)₂ fragment allows us to use monomers and polymers of AP and thereby regulates the sensitivity of the assay. Accepted: 14 October 1997     

Accurate non-invasive image-based cytotoxicity assays for cultured cells

Background  

The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay.     

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-β1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation. Received: 15 September 1997 / Accepted: 6 April 1998     

Determination of the number of endothelial cells in culture using an acid phosphatase assay

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

DNase,RNase, and phosphatase activities of antibodies formed upon immunization by DNA,DNase I,and DNase II

Relative DNase, RNase (efficiency of hydrolysis of ribo- and deoxyribooligonucleotides (ON)), and phosphatase (removal of the ON 5′ terminal phosphate) catalytic activities of antibodies (AB) obtained after rabbit immunization by DNA, DNase I, and DNase II were compared. It is shown that electrophoretically homogeneous preparations of polyclonal AB from non-immunized rabbits did not exhibit such activities. Immunization of rabbits by DNA, DNase I, and DNase II results in generation of IgG abzymes that exhibit high activity in the ON hydrolysis reaction and even higher activity in cleavage of 5′ terminal phosphate of ON. In this case K _m values for supercoiled plasmid DNA and ON found in reactions of their AB-dependent nuclease hydrolysis and phosphatase cleavage of 5′ terminal phosphate differ by 2–4 orders of magnitude. This shows that nuclease and phosphatase activities belong to different abzyme fractions within polyclonal AB. Thus, in this work data indicative of the possibility of a formation of antibodies exhibiting phosphatase activity after immunization of animals with DNA, DNase I, and DNase II, were obtained for the first time. Possible reasons for production of AB with phosphatase activity after immunization of rabbits with these immunogens are discussed.     

A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells

Summary A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or “weeding.” The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin≈laminin>gelatin>poly-d-lysine≈Matrigel>hyaluronic acid≈plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-d-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7±2.6% (n=6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of γ-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation. This work was funded by the National Institutes of Health grant RO1-NS-21076, and AHA-GIA 881134. Support for Ellen Gordon provided by the National Institutes of Health, NSO7144 and the Seattle Affiliate of the AHA (88-WA-111, 89-WA-112).     

Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Ternary borate–nucleoside complex stabilization by ribonuclease A demonstrates phosphate mimicry

Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate ester recognition sites on proteins might exhibit significant affinity for nonenzymatically formed borate esters. ¹¹B NMR studies and activity measurements on ribonuclease A (RNase A) in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A·3′-deoxycytidine–2′-borate ternary complex that mimics the complex formed between RNase A and a 2′-cytidine monophosphate (2′-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2′-deoxycytidine mixture, demonstrating the critical importance of the 2′-hydroxyl group for complex formation. Titration of the ternary complex with 2′-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2′-CMP. RNase A binding of a cyclic cytidine-2′,3′-borate ester, which is a structural homolog of the cytidine-2′,3′-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2′,3′-borate·RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cytidine-2′,3′-cyclic phosphate at pH 7, indicating considerably stronger binding. However, the value is 1,000-fold larger than the reported dissociation constant of the RNase A complex with uridine–vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters supports enzyme catalysis.     

Apolipoprotein A-I mimetic peptide D-4F promotes human endothelial progenitor cell proliferation,migration, adhesion though eNOS/NO pathway

Circulating endothelial progenitor cells (EPCs) have a critical role in endothelial maintenance and repair. Apolipoprotein A-I mimetic peptide D-4F has been shown to posses anti-atherogenic properties via sequestration of oxidized phospholipids, induction of remodeling of high density lipoprotein and promotion of cholesterol efflux from macrophage-derived foam cells. In this study, we test the effects of D-4F on EPC biology. EPCs were isolated from the peripheral venous blood of healthy male volunteers and characterized by 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL uptake and ulex europaeus agglutinin binding and flow cytometry. Cell proliferation, migration, adhesion, nitric oxide production and endothelial nitric oxide synthase (eNOS) expression in the absence and presence of D-4F or simvastatin (as a positive control), were assayed. We demonstrated that D-4F significantly enhanced EPC proliferation, migration and adhesion in a dose-dependent manner compared with vehicle. However, all of the favorable effects of D-4F on EPCs were dramatically attenuated by preincubation with NOS inhibitor L-NAME. Further, D-4F also increased nitric oxide production in culture supernatant and the levels of eNOS expression and phosphorylation. The stimulatory effects of D-4F (10 μg/ml) on EPC biology were comparable to 0.5 μM simvastatin. These results suggest that eNOS/NO pathway mediates the functional modulation of EPC biology in response to D-4F treatment and support the notion that the beneficial role of D-4F on EPCs may be one of the important components of its anti-atherogenic potential.     

Stabilities of complexes formed between lead(II) and simple phosphonate or phosphate monoester ligands including some pyrimidine-nucleoside 5′-monophosphates (CMP2–, UMP2–, dTMP2–)

 The stability constants of the 1 : 1 complexes formed between Pb²⁺ and several simple phosphate monoesters (4-nitrophenyl phosphate, phenyl phosphate, d-ribose 5-monophosphate, n-butyl phosphate) or phosphonate ligands (methylphosphonate, ethylphosphonate) (R-PO^2– ₃) were determined by potentiometric pH titrations in aqueous solution (25  °C;I=0.1 M, NaNO₃). The construction of a log K ^P _P ^b _b(R-PO3) versus pK ^H _H(R-PO3) plot for the mentioned ligand systems results in a straight line on which the data pairs (the corresponding equilibrium constants were also measured) for uridine 5′-monophosphate (UMP^2–) and thymidine 5′-monophosphate (dTMP^2–) also fall; this result shows that in the Pb²⁺ complexes of UMP^2– and dTMP^2– the nucleobase residues do not interfere, in neither a positive nor a negative way, with the binding of Pb²⁺ and that the stability of all these complexes is determined by the basicity of the phosph(on)ate group. The mentioned straight-line correlation (as defined by the least-squares procedure) allowed us to demonstrate (via constants determined now) that the stability of the Pb²⁺ complex of cytidine 5′-monophosphate (CMP^2–) is also solely determined by the basicity of its phosphate group. A similar evaluation, based on literature data, for the Pb(HPO₄) complex reveals that its stability corresponds closely to the expectations based on the Pb(R-PO₃) data, though there is a slight hint that Pb(HPO₄) may be somewhat more stable [which would be in agreement with previous observations of other M(HPO₄) complexes]; clearly, more such comparisons are possible with the reference line given now. Based on the stability constants of the monoprotonated Pb(H;CMP)⁺ complex and the Pb(cytidine)²⁺ species (which was also measured now), it is concluded that in Pb(H;CMP)⁺ the proton is located at the phosphate group and Pb²⁺ mainly at the N3/(C2)O site of the cytosine residue. Regarding nucleic acids in solution, it is further concluded that the affinity of Pb²⁺ towards the negatively mono-charged phosphate unit, —O—P(O)₂ ^–—O—, of a nucleic acid backbone is comparable to that of the cytosine moiety, the affinity towards other nucleobase residues being smaller. This information may prove helpful regarding the properties of lead ribozymes. Received: 16 April 1999 / Accepted: 2 June 1999     

A novel fluorometric assay for ADP-ribose pyrophosphatase activity

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.     

Effect of βTCP filled polyetheretherketone on osteoblast cell proliferation in vitro

Summary Non-resorbable thermoplastic polymers have become more important for reconstructive surgery due to their excellent chemical and physical properties. Polyetheretherketone-β-tricalcium phosphate (βTCP-PEEK) composites were developed as alternative materials for load-bearing applications. This study presents the effect of polyetheretherketone (PEEK) specimens incorporated with 5, 10, 20 and 40 wt% β-tricalcium phosphate (βTCP) and processed by injection molding on cultivated osteoblast cells. Normal human osteoblast (NHOst) cells were seeded onto polymer discs to evaluate cell viability and proliferation after 24, 72 and 120 h of cultivation by employing the WST-1 assay. Standard tissue culture plastic was used as a control. The osteoblast cells were found to be viable in all PEEK groups, while the cell proliferation was progressively inhibited due to the incorporated β-tricalcium phosphate. βTCP-PEEK showed concentration independent decrease of cell proliferation compared to the unfilled PEEK and the control group. In summary, this study confirms the non-toxic nature of pure PEEK, whereas this could not definitely be verified for βTCP-PEEK as a composite material in chosen concentrations of β-tricalcium phosphate in vitro.     

A simple colorimetric determination of the manganese content in photosynthetic membranes

The functional Mn content of intact photosystem II membrane fragments was measured as 4.06 ± 0.13 Mn/reaction center when determined using a simple, sensitive colorimetric assay that will also work with thylakoids and core complexes. This procedure requires minimal sample material, does not need expensive assay equipment, requires four simple steps, and only takes 20–30 min to perform. These include (a) removal of the adventitious Mn ions by CaCl₂ treatment of the membranes, (b) extraction of the Mn from the O₂-evolving complex with hydrochloric acid, (c) purification of the extract by centrifugation followed by filtration of the supernatant through an Acrodisc syringe filter (0.2 μm nylon membrane), and (d) colorimetric determination of Mn in the extract using the reaction of the chromogenic agent, 3,3′,5,5′-tetramethylbenzidine, with previously oxidized Mn(II) cations carried out at high pH. The colorimetric assay itself has been used previously by Serrat (Mikrochim Acta 129:77–80, 1998) for assaying Mn concentrations in sea water and drinking water.     

The reduction of water-soluble tetrazolium salt reagent on the plasma membrane of epidermal keratinocytes is oxygen dependent

The water-soluble tetrazolium salt (WST-1) assay is frequently used to assess cell proliferation. However, our study showed that in normal and cancerous keratinocytes, this assay is more responsive to changes in oxygenation than to rates of cell growth. Stimulation of keratinocyte proliferation by low Ca²⁺ and suppression of proliferation by nocodazole resulted in modest changes in WST-1 readings, whereas gradually reducing the level of oxygen in the cellular environment from ambient (21%) to near anoxic (0.1%) revealed a very strong negative correlation between cell oxygenation and WST-1 reagent reduction. In contrast, the very similar MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay, which uses a different tetrazolium salt, showed no sensitivity to the level of oxygen. Unlike MTT, WST-1 reagent is reduced extracellularly through trans-plasma membrane transport (tPMET), thereby suggesting that tPMET is oxygen dependent. We propose that the WST-1 assay can be developed into a sensitive quantitative method to evaluate cell oxygenation in vitro and used to study the role of hypoxia and tPMET in homeostasis and disease (e.g., cancer). At the same time, WST-1 assay should be used cautiously to assess cell viability or proliferation because readings can be affected by certain extrinsic (low atmospheric oxygen or high density culture) or intrinsic (defects in oxygen-sensing pathways) factors.