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At least 127 records · Page 7Linked to original sources
PubMed1988

Color-contrast staining of two different lymphocyte subpopulations: a two-color modification of alkaline phosphatase monoclonal anti-alkaline phosphatase complex technique.

A dual staining method for different human lymphocyte subpopulations with nonoverlapping antigen distribution patterns is described. Cytocentrifuge slide preparations of peripheral blood nonadherant mononuclear cells (NAMNC), bone marrow aspirate or buffy coat smears were fixed in acetone and incubated with a primary mouse monoclonal antibody (MAb) against a lymphocyte antigen (CD8, Ig-light-chain, CD19, CD4) followed by rabbit anti-mouse immunoglobulin (Ig) and the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) complex. After repeating the "bridge" antibody and the APAAP, a red product was developed with fast red TR-naphthol AS-BI phosphate. Following this one-color stain the process was repeated using a different primary mouse MAb against another lymphocyte antigen (CD4, Ig-light chain, CD3, MHCII DR, CD5) and fast blue BB-naphthol AS-MX phosphate at the last step to yield a blue product. Control slides stained by the standard one-color APAAP method with the relevant primary MAb showed that there was no nonspecific labelling and the percent of positive cells in a given test was almost identical. To achieve an intense blue in the second stain for some antigens, e.g., CD4, either the MAb concentration had to be increased or two different MAbs recognizing differing epitopes of the same antigen, e.g., T1 and UCHT2 for CD5, were applied. Any change of red to purple at the site of the first stain after 15 min exposure to the blue-yielding AP substrate is due to residual AP activity of the first stain rather than to crossbinding of immunoreagents.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1986

Ontogenic and phylogenic studies of intestinal, hepatic, and placental alkaline phosphatases. Evidence that intestinal alkaline phosphatase is a late evolutionary development.

We have studied the intrauterine development of guinea pig and rat organ phosphatases using biochemical, immunologic, and histologic techniques. In all organs tested the features of the adult phosphatase activity were achieved during the second or third gestational phases. In the rat, the tissue-unspecific phosphatase activity was found by immunoprecipitation with antiserum to rat liver phosphatase in all gestational phases in liver and placenta. The high liver activity in the first phase of gestation corresponded to hematopoietic cells. Hepatocyte phosphatase did not appear until the second gestational phase. The tissue-unspecific phosphatase activity was found in the first and second gestational phase in surface intestinal epithelial cells, even after crypt formation occurred. Once phosphatase appeared in enterocytes on villi, only the intestinal-type enzyme was detected. Alkaline phosphatase was measured in the liver and intestine from animals in various phyla. In fish and reptiles, the intestinal activity had the enzymatic characteristics of the tissue-unspecific enzyme. The appearance of intestinal alkaline phosphatase with unique properties and high specific activity is a characteristic of mammals.

Alkaline Phosphatase↗
PubMed1998

Effect of desferrioxamine and deferiprone (L1) on the proliferation of MG-63 bone cells and on phosphatase alkaline activity.

In the present work, we studied: (i) the effect of different doses of desferrioxamine and deferiprone on the proliferation of osteoblasts and the possible role of iron in this; and (ii) the effect of both chelators on the metabolic activity of these cells, using the quantification of alkaline phosphatase as an enzymatic marker of cellular activity or differentiation. Cellular proliferation was investigated at different concentrations of deferiprone and desferrioxamine, and the effect of the addition of iron citrate on this proliferation was measured. The production of alkaline phosphatase after incubation with deferiprone (150 and 300 microM) and desferrioxamine (20 and 100 microM) was also studied. Cellular proliferation was completely inhibited with 100 microM of desferrioxamine and 300 microM of deferiprone. In both cases, this effect was corrected by means of co-incubation with iron citrate. In the second phase, using the same dose that inhibited the proliferation, it was observed that after 24 h, both chelators slightly decreased alkaline phosphatase activity, while at 48 and 96 h they increased alkaline phosphatase activity. These results demonstrate that both desferrioxamine and deferiprone inhibit the proliferation of the osteoblast-like cell line MG-63; the effect seems to be related to the chelation of some fraction of available iron. In spite of the effect on bone cell proliferation, the chelators do not impair cellular activity.

Alkaline Phosphatase↗
PubMed1997

[Analysis of lymphocyte populations with flow cytometry in routine bronchoalveolar lavage diagnosis: comparison of a 3-color method with the alkaline phosphatase anti-alkaline phosphatase immunohistochemistry].

BACKGROUND: Identification of lymphocyte phenotypes in bronchoalveolar lavage (BAL) plays a crucial role in the diagnosis of interstitial lung disease. Cells staining positive for specific monoclonal antibodies may be detected by immunocytochemistry or flow cytometry (FCM). The present study compares a three-colour FCM-approach to a standard APAAP protocol for immunocytochemistry. METHODS: BAL-specimens of 22 patients with various lung diseases were investigated. Inclusion criteria was a lymphocytosis of > 10% of all BAL-cells. After the preparation of cytocentrifuge slides, staining was performed with monoclonal antibodies to CD3, CD4 and CD8 following the APAAP-protocol. FCM-analysis was performed with the following panel of conjugates: CD3-FITC, CD4- or CD8-PE, CD45-perCP. Lymphocytes were gated by their SSC/CD45 characteristics. T-helper and T-suppressor percentages were evaluated by quadrant analysis of CD3/CD4 or CD3/CD8 histograms. RESULTS: With the exception of CD3, where the range of values was quite narrow (10% variance), the correlation between the two methods was excellent (CD4: r = 0.98; CD8: r = 0.99; CD4/CD8: r = 0.96; p < 0.0001). CONCLUSION: Flow cytometry reveals similar results compared to immunocytochemistry in the determination of lymphocyte subsets characterised by CD3, CD4 and CD8 antigens.

Adult↗
PubMed1985

Activity of 5'-nucleotidase, AMP deaminase, adenosine deaminase, acid and alkaline phosphatase and nucleotide pyrophosphatase in human thyroid.

The activity of 5'-nucleotidase, AMP deaminase, adenosine deaminase, acid phosphatase, alkaline phosphatase and nucleotide pyrophosphatase was assayed in human thyroid glands. The 5'-nucleotidase activity was higher than that of AMP deaminase which suggested that AMP undergoes degradation primarily as a result of dephosphorylation in thyroid tissue. A high acid phosphatase activity was noted as compared to that of alkaline phosphatase activity. In toxic goitre the increase in adenosine deaminase and acid phosphatase was observed together with the decrease in pyrophosphatase activity.

5'-Nucleotidase↗
PubMed1983

Leukocyte alkaline phosphatase: another organ-specific alkaline phosphatase.

We have used enzyme specific inhibitors and heat inactivation to distinguish Leukocyte alkaline phosphate (LAP) from other organ-specific alkaline phosphatases as well as to compare LAP from normal granulocytes and leukemic cells with elevated LAP. The heat inactivation and inhibition curves of LAP are quite different from those of other organ-specific alkaline phosphatases. The inhibition curves and heat inactivation characteristics of LAP from normal granulocytes and that obtained from chronic granulocytic leukemia (CGL) blast phase cells with elevated LAP are identical. These data suggest that LAP is distinct from other organ-specific alkaline phosphatases, particularly placental alkaline phosphatase. We also conclude that the LAP present in cells with elevated levels is very similar or identical to that of normal granulocytes.

Alkaline Phosphatase↗
PubMed1994

Why are mammalian alkaline phosphatases much more active than bacterial alkaline phosphatases?

Mammalian alkaline phosphatases are 20-30-fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25-30% absolutely conserved. In the active-site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes. In the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site-specific mutagenesis, genetic and X-ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1992

Alkaline phosphatase and phosphotyrosine phosphatase activities of cultured amniotic cells with trisomy 18.

In cultured amniotic cells from fetuses with Edward's syndrome (trisomy 18), the activities of two protein phosphatases, alkaline phosphatase and phosphotyrosine phosphatase, were measured. Comparison with normal fetal cells showed a different behavior for each enzyme. Alkaline phosphatase was significantly lowered while phosphotyrosine phosphatase remained at normal levels. The interest of these enzyme assays in the screening procedure of this severe chromosome defect is discussed.

Alkaline Phosphatase↗
PubMed1984

Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).

A murine monoclonal antibody specific for calf intestinal alkaline phosphatase has been prepared and used in an unlabeled antibody bridge technique for labeling monoclonal antibodies. This procedure--the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) method--gives excellent immunocytochemical labeling of tissue sections and cell smears, comparable in clarity and intensity to that achieved with immunoperoxidase labeling. If the enzyme label is developed with a naphthol salt as a coupling agent and Fast Red or hexazotized new fuchsin as a capture agent, a vivid red reaction product is obtained which is very easily detected by the human eye. For this reason the APAAP technique was found particularly suitable for labeling cell smears (for both cytoplasmic and surface-membrane antigens) and for detecting low numbers of antigen-bearing cells in a specimen (e.g., carcinoma cells in a malignant effusion). It was found possible to enhance the intensity of the APAAP labeling reaction substantially by repeating the second and third incubation steps (i.e., the unlabelled "bridge" antibody and APAAP complexes). The APAAP technique was superior to immunoperoxidase labeling for staining tissues rich in endogenous peroxidase, and could be used in conjunction with immunoperoxidase methods for double immunoenzymatic staining. The method was also applicable to the detection of antigenic molecules following their electrophoretic transfer from SDS-polyacrylamide gels to nitrocellulose sheets ("immunoblotting").

Alkaline Phosphatase↗
PubMed1984

Studies of the biochemical and immunological properties of human neutrophil alkaline phosphatase with comparison to the established alkaline phosphatase isoenzymes.

A range of affinity column chromatographic procedures and various inhibitors have been used to compare human neutrophil alkaline phosphatase with the three established isoenzymes. The column chromatography studies have clearly distinguished neutrophil alkaline phosphatase from the intestinal isoenzyme. Inhibition studies with L-phenylalanine, L-homoarginine and levamisole have revealed a distinct pattern of inhibition for liver, kidney and neutrophil alkaline phosphatase which is quite different from the pattern shown by placental and intestinal alkaline phosphatase. Immunospecificity experiments with a monoclonal antibody raised to human liver alkaline phosphatase have shown that it cross reacts with alkaline phosphatase from kidney, bone and neutrophil. In all studies, neutrophil alkaline phosphatase has virtually identical properties to that of liver, kidney and bone alkaline phosphatase. This is strong evidence that neutrophil alkaline phosphatase is a product of the same structural gene which codes for the liver/bone/kidney group of human alkaline phosphatases.

Alkaline Phosphatase↗
PubMedDate not supplied

Colpocytograms and maternal serum placental cystine aminopeptidase, tissue cystine aminopeptidase, alkaline phosphatase and heat stable alkaline phosphatase activity in monitoring the last four weeks before delivery in high-risk pregnancy.

On the basis of the results of serial enzyme and cytohormonal assays in the last lunar month of gestation in 232 pergnant patients with high-risk pregnancy, it has been shown that the "at term" and inflammatory smears, which persist for over five days before labor, and post-partum" smears, significantly correlate with abnormal (low or decreasing) results of serum placental cystine aminopeptidase activity and with the pathologic course of pregnancy and labor as well as with the poor neonates' condition. The average serum placental and tissue cystine aminopeptidase determinations were the lowest in women with "post-partum" smears or persisting "at term" smears. These activities reached their highest they temporarily decreased. The oxytocinase activity curves in women with cytolytic smears were similar to those in patients with the "before term" smear patterns, which persisted before delivery. The average oxytocinase activity in women with the inflammatory smears was at first the highest and later it decreased most rapidly of all the groups under consideration. The maternal serum alkaline phosphatase and its heat-stable fraction in pregnant patients with "post-partum" and persisting "at term" smears were at first the lowest and just before labor the highest of all the cytologic pregnancy patterns. Colpocytograms confirmed their high prognostic value when compared with the enzyme tests of placental function.

Alkaline Phosphatase↗
PubMed1981

Inhibition of endogenous tissue alkaline phosphatase with the use of alkaline phosphatase conjugates in immunohistochemistry.

In mammals there are two forms of alkaline phosphatase, one of which is widely distributed in a variety of tissues, and one of which is confined to intestine. Levamisole (1-tetramisole) inhibits the nonintestinal form of the enzyme, but is without effect on the intestinal form. We have exploited this difference by using conjugates made with calf intestinal alkaline phosphatase for immunohistochemical demonstration of H2 antigens in frozen section of mouse tissues. The alkaline phosphatase staining is performed in the presence of 1 mm levamisole, which inhibits the endogenous tissue enzyme without loss of staining by the conjugate. Endogenous enzyme can be inhibited by other means, such as exposure to 20% acetic acid, but labile antigens may be destroyed.

Acetates↗