31 results on '"Manes T"'
Search Results
2. Heterogeneity in alkaline phosphatase isozyme expression in human testicular germ cell tumours: an enzyme-/immunohistochemical and molecular analysis
- Author
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Roelofs, H, Manes, T, Janszen, T, Millán, JL, Oosterhuis, Wolter, Looijenga, LHJ (Leendert), and Pathology
- Published
- 1999
3. Following up on screening tests
- Author
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Goldman, K D, Kloss, S, Manes, T, and Rojewski, M
- Subjects
Adult ,Aged, 80 and over ,Male ,Letter ,Time Factors ,New Jersey ,Data Collection ,Age Factors ,Middle Aged ,Telephone ,Humans ,Mass Screening ,Female ,Attitude to Health ,Health Fairs ,Aged ,Follow-Up Studies - Published
- 1998
4. Kinetic characterization of hypophosphatasia mutations with physiological substrates
- Author
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Di Mauro, S., Manes, T., Hessle, H., Kozlenkov, A., Di Mauro, S., Manes, T., Hessle, H., and Kozlenkov, A.
- Published
- 2002
5. Allelic amino acid substitutions affect the confirmation and immunoreactivity of germ-cell alkaline phosphotase phenotypes
- Author
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Hoylaerts, Marc F., Manes, T., and Millán, J.L.
- Published
- 1992
6. Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene.
- Author
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Millán, J L and Manes, T
- Abstract
A full-length placental alkaline phosphatase (PLAP) cDNA was used to identify and clone the PLAP-like Nagao isozyme gene from human genomic libraries. The entire nucleotide sequence of the gene reveals the existence of 11 exons interrupted by 10 small introns (76-427 base pairs). Putative regulatory sequences have been identified in the promoter regions as well as dispersed in the introns. The deduced amino acid sequence of the Nagao isozyme indicates that the mature molecule is composed of 513 amino acids, of which 12 residues are different from the sequence of PLAP (98% homology). A sequence derived from exon III of the Nagao isozyme gene was used to synthesize a peptide (NH2-Lys-Leu-Gly-Pro-Glu-Thr-Phe-Leu-Ala-COOH) that contains two mutations with respect to the corresponding PLAP sequence. This peptide elicited rabbit polyclonal antibodies that reacted specifically with the seminoma Nagao isozyme but not with PLAP in electrophoretic transfer blots. These results indicate that the tumor, and possibly the normal testis, Nagao isozyme is encoded by a gene referred to as germ-cell alkaline phosphatase gene that differs from the PLAP gene expressed by syncitiotrophoblastic cells.
- Published
- 1988
- Full Text
- View/download PDF
7. Mammalian alkaline phosphatases are allosteric enzymes.
- Author
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Hoylaerts, M F, Manes, T, and Millán, J L
- Abstract
Mammalian alkaline phosphatases (APs) are zinc-containing metalloenzymes encoded by a multigene family and functional as dimeric molecules. Using human placental AP (PLAP) as a paradigm, we have investigated whether the monomers in a given PLAP dimer are subject to cooperativity during catalysis following an allosteric model or act via a half-of-sites model, in which at any time only one single monomer is operative. Wild type and mutant PLAP homodimers and heterodimers were produced by stably transfecting Chinese hamster ovary cells with mutagenized PLAP cDNAs followed by enzyme extraction, purification, and characterization. [Gly429]PLAP manifested negative cooperativity when partially metalated as a consequence of the reduced affinity of the incompletely metalated AP monomers for the substrate. Upon full metalation with Zn2+, however, the negative cooperativity disappeared. To distinguish between an allosteric and a half-of-sites model, a [Gly429]PLAP-[Ser84]PLAP heterodimer was produced by combining monomers displaying high and low sensitivity to the uncompetitive inhibitor L-Leu as well as a [Gly429]PLAP-[Ala92]PLAP heterodimer combining a catalytically active and inactive monomer, respectively. The L-Leu inhibition profile of the [Gly429]PLAP-[Ser84]PLAP heterodimer was intermediate to that for each homodimer as predicted by the allosteric model. Likewise, the [Gly429]PLAP-[Ala92]PLAP heterodimer was catalytically active, confirming that AP monomers act independently of each other. Although heterodimers are structurally asymmetrical, they migrate in starch gels with a smaller than expected weighted electrophoretic mobility, are more stable to heat denaturation than expected, and are more sensitive to L-Leu inhibition than predicted by a strict noncooperative model. We conclude that fully metalated mammalian APs are noncooperative allosteric enzymes but that the stability and catalytic properties of each monomer are controlled by the conformation of the second AP subunit.
- Published
- 1997
8. Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase
- Author
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Hoylaerts, M F, Manes, T, and Millán, J L
- Abstract
Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.
- Published
- 1992
- Full Text
- View/download PDF
9. Unusual conformational effect exerted by Z-DNA upon its neighboring sequences.
- Author
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Kohwi-Shigematsu, T, Manes, T, and Kohwi, Y
- Abstract
Supercoiled plasmid DNA harboring an insert of (dG-dC)16, a sequence known to form Z-DNA upon negative supercoiling, was reacted with chloroacetaldehyde. Chloroacetaldehyde, like bromoacetaldehyde, was found to be a specific probe for detecting unpaired DNA bases in supercoiled plasmid DNA. Under torsional stress (at bacterial superhelical density), chloroacetaldehyde reacted at multiple discrete regions within the neighboring sequences of the (dG-dC)16 insert. When the plasmid population was considered as a whole, the distribution of the chemically reactive bases exhibited a pattern of inversion symmetry with the center of inversion in the middle of the (dG-dC)16 insert. However, when a single supercoiled plasmid molecule was considered, chloroacetaldehyde reacted with only one of the neighboring sequences, either 5' or 3' of the (dG-dC)16 insert, but not with both. The possibility that the supercoiled plasmid DNA is in equilibrium with these two structural forms is discussed.
- Published
- 1987
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10. Genetic complexity, structure, and characterization of highly active bovine intestinal alkaline phosphatases.
- Author
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Manes, T, Hoylaerts, M F, Müller, R, Lottspeich, F, Hölke, W, and Millán, J L
- Abstract
Mammalian alkaline phosphatases (APs) display 10-100-fold higher kcat values than do bacterial APs. To begin uncovering the critical residues that determine the catalytic efficiency of mammalian APs, we have compared the sequence of two bovine intestinal APs, i.e. a moderately active isozyme (bovine intestinal alkaline phosphatase, bIAP I, approximately 3,000 units/mg) previously cloned in our laboratory, and a highly active isozyme (bIAP II, approximately 8, 000 units/mg) of hitherto unknown sequence. An unprecedented level of complexity was revealed for the bovine AP family of genes during our attempts to clone the bIAP II cDNA from cow intestinal RNAs. We cloned and characterized two novel full-length IAP cDNAs (bIAP III and bIAP IV) and obtained partial sequences for three other IAP cDNAs (bIAP V, VI, and VII). Moreover, we identified and partially cloned a gene coding for a second tissue nonspecific AP (TNAP-2). However, the cDNA for bIAP II, appeared unclonable. The sequence of the entire bIAP II isozyme was determined instead by a classical protein sequencing strategy using trypsin, carboxypeptidase, and endoproteinase Lys-C, Asp-N, and Glu-C digestions, as well as cyanogen bromide cleavage and NH2-terminal sequencing. A chimeric bIAP II cDNA was then constructed by ligating wild-type and mutagenized fragments of bIAP I, III, and IV to build a cDNA encoding the identified bIAP II sequence. Expression and enzymatic characterization of the recombinant bIAP I, II, III, and IV isozymes revealed average kcat values of 1800, 5900, 4200, and 6100 s-1, respectively. Comparison of the bIAP I and bIAP II sequences identified 24 amino acid positions as likely candidates to explain differences in kcat. Site-directed mutagenesis and kinetic studies revealed that a G322D mutation in bIAP II reduced its kcat to 1300 s-1, while the converse mutation, i.e. D322G, in bIAP I increased its kcat to 5800 s-1. Other mutations in bIAP II had no effect on its kinetic properties. Our data clearly indicate that residue 322 is the major determinant of the high catalytic turnover in bovine IAPs. This residue is not directly involved in the mechanism of catalysis but is spatially sufficiently close to the active site to influence substrate positioning and hydrolysis of the phosphoenzyme complex.
- Published
- 1998
11. Following up on screening tests.
- Author
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Goldman KD, Kloss S, Manes T, and Rojewski M
- Published
- 1998
12. STATUS OF THE SEGS PLANTS
- Author
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Kearney, D., Price, H., Replogle, I., Manes, T., Costanzo, J., Gilon, Y., and Walzer, S.
- Published
- 1992
- Full Text
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13. Bilateral Failure of Oxidized Zirconium Implants in Total Knee Arthroplasty.
- Author
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Kelly B, Manes T, and Main C
- Abstract
Oxidized zirconium was first introduced in total hip arthroplasty procedures to merge the strengths of metal and ceramic into one prosthetic. The subsequent adoption of oxidized zirconium (oxinium) for total knee arthroplasty is attributed to the theory of causing less wear on the tibial components compared to the alternative, cobalt chromium. However, the superficial layer of the femoral component is occasionally breached, exposing the softer zirconium substrate. Multiple mechanisms leading to zirconium substrate exposure have been explained, including collateral ligament instability and polyethylene wear. Such a failure may lead to damage to the periprosthetic tissues and often requires a revision procedure. In the current case report, we present a case of bilateral total knee arthroplasty with oxidized zirconium components that resulted in catastrophic failure and subsequent revision with hinged knee prostheses., (© 2023 The Authors.)
- Published
- 2023
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14. Fusobacterium necrophorum Bacteremia With Evidence of Cavitary Pulmonary Lesion.
- Author
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Mohiuddin Z, Manes T, and Emerson A
- Abstract
Fusobacterium necrophorum (F. necrophorum) is a gram-negative anaerobic bacterium and a known etiologic agent in Lemierre's syndrome. This rare disease commonly presents with persistent sore throat and dysphagia, which can spread to involve the internal jugular vein. Presented in this report is an interesting case of a patient who presented with a progressively worsening sore throat, dysphagia, and productive cough on admission. Blood cultures were positive for F. necrophorum and computed tomography angiogram (CTA) of the chest detected cavitation in the left lower lobe and a large consolidation within the right lower lobe without evidence of a vascular defect. CT of the neck with IV contrast demonstrated no findings of abnormal vascular structures. This patient was diagnosed with pneumonia secondary to F. necrophorum bacteremia and treated successfully with antibiotics and was discharged home. Clinical suspicion is warranted in patients with worsening symptoms of sore throat and dysphagia, as this rare syndrome may be present., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2021, Mohiuddin et al.)
- Published
- 2021
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15. Alpha(v)beta3 integrin expression up-regulates cdc2, which modulates cell migration.
- Author
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Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, and Languino LR
- Subjects
- CDC2 Protein Kinase antagonists & inhibitors, CDC2 Protein Kinase genetics, Calmodulin-Binding Proteins metabolism, Cyclin B metabolism, Cyclin B2, Gene Expression Regulation, Enzymologic, HeLa Cells, Humans, RNA, Messenger genetics, RNA, Messenger metabolism, Tumor Cells, Cultured, Up-Regulation, CDC2 Protein Kinase metabolism, Cell Movement, Integrin alphaVbeta3 metabolism
- Abstract
The alphavbeta3 integrin has been shown to promote cell migration through activation of intracellular signaling pathways. We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1). We report that alphavbeta3 expression in LNCaP (beta3-LNCaP) prostate cancer cells causes increased cdc2 mRNA levels as evaluated by gene expression analysis, and increased cdc2 protein and kinase activity levels. We provide three lines of evidence that increased levels of cdc2 contribute to a motile phenotype on integrin ligands in different cell types. First, increased levels of cdc2 correlate with more motile phenotypes of cancer cells. Second, ectopic expression of cdc2 increases cell migration, whereas expression of dominant-negative cdc2 inhibits migration. Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion. We also show that cdc2 increases cell migration via specific association with cyclin B2, and we unravel a novel pathway of cell motility that involves, downstream of cdc2, caldesmon. cdc2 and caldesmon are shown here to localize in membrane ruffles in motile cells. These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.
- Published
- 2003
- Full Text
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16. Kinetic characterization of hypophosphatasia mutations with physiological substrates.
- Author
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Di Mauro S, Manes T, Hessle L, Kozlenkov A, Pizauro JM, Hoylaerts MF, and Millán JL
- Subjects
- Alkaline Phosphatase chemistry, Alkaline Phosphatase genetics, Base Sequence, DNA Primers, Humans, Hydrogen-Ion Concentration, Kinetics, Models, Molecular, Pyridoxal Phosphate metabolism, Pyrophosphatases metabolism, Hypophosphatasia genetics, Mutation
- Abstract
We have analyzed 16 missense mutations of the tissue-nonspecific AP (TNAP) gene found in patients with hypophosphatasia. These mutations span the phenotypic spectrum of the disease, from the lethal perinatal/ infantile forms to the less severe adult and odontohypophosphatasia. Site-directed mutagenesis was used to introduce a sequence tag into the TNAP cDNA and eliminate the glycosylphosphatidylinositol (GPI)-anchor recognition sequence to produce a secreted epitope-tagged TNAP (setTNAP). The properties of GPI-anchored TNAP (gpiTNAP) and setTNAP were found comparable. After introducing each single hypophosphatasia mutation, the setTNAP and mutant TNAP cDNAs were expressed in COS-1 cells and the recombinant flagged enzymes were affinity purified. We characterized the kinetic behavior, inhibition, and heat stability properties of each mutant using the artificial substrate p-nitrophenylphosphate (pNPP) at pH 9.8. We also determined the ability of the mutants to metabolize two natural substrates of TNAP, that is, pyridoxal-5'-phosphate (PLP) and inorganic pyrophosphate (PPi), at physiological pH. Six of the mutant enzymes were completely devoid of catalytic activity (R54C, R54P, A94T, R206W, G317D, and V365I), and 10 others (A16V, A115V, A160T, A162T, E174K, E174G, D277A, E281K, D361V, and G439R) showed various levels of residual activity. The A160T substitution was found to decrease the catalytic efficiency of the mutant enzyme toward pNPP to retain normal activity toward PPi and to display increased activity toward PLP. The A162T substitution caused a considerable reduction in the pNPPase, PPiase, and PLPase activities of the mutant enzyme. The D277A mutant was found to maintain high catalytic efficiency toward pNPP as substrate but not against PLP or PPi. Three mutations ( E174G, E174K, and E281K) were found to retain normal or slightly subnormal catalytic efficiency toward pNPP and PPi but not against PLP. Because abnormalities in PLP metabolism have been shown to cause epileptic seizures in mice null for the TNAP gene, these kinetic data help explain the variable expressivity of epileptic seizures in hypophosphatasia patients.
- Published
- 2002
- Full Text
- View/download PDF
17. Function assignment to conserved residues in mammalian alkaline phosphatases.
- Author
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Kozlenkov A, Manes T, Hoylaerts MF, and Millán JL
- Subjects
- Animals, Binding Sites, Conserved Sequence, Cysteine chemistry, Disulfides chemistry, Dose-Response Relationship, Drug, Epitopes, Histidine chemistry, Humans, Kinetics, Leucine chemistry, Ligands, Magnesium chemistry, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Phenylalanine chemistry, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins metabolism, Software, Spectrometry, Fluorescence, Time Factors, Tyrosine chemistry, Zinc chemistry, Alkaline Phosphatase chemistry, Alkaline Phosphatase physiology
- Abstract
We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k(cat) and increased K(m) when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxyl-terminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in k(cat), a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors l-Phe and l-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.
- Published
- 2002
- Full Text
- View/download PDF
18. Integrins and prostate cancer metastases.
- Author
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Fornaro M, Manes T, and Languino LR
- Subjects
- Adenocarcinoma pathology, Cell Movement physiology, Humans, Male, Neoplasm Metastasis, Prostatic Neoplasms pathology, Signal Transduction physiology, Adenocarcinoma metabolism, Integrins physiology, Neoplasm Proteins physiology, Prostatic Neoplasms metabolism
- Abstract
Integrins have emerged as modulators of a variety of cellular functions. They have been implicated in cell migration, survival, normal and aberrant cellular growth, differentiation, gene expression, and modulation of intracellular signal transduction pathways. In this review article, the structural and functional characteristics of integrins, their expression and their potential role in prostate cancer metastases will be discussed.
- Published
- 2001
- Full Text
- View/download PDF
19. Heterogeneity in alkaline phosphatase isozyme expression in human testicular germ cell tumours: An enzyme-/immunohistochemical and molecular analysis.
- Author
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Roelofs H, Manes T, Janszen T, Millán JL, Oosterhuis JW, and Looijenga LH
- Subjects
- Alkaline Phosphatase genetics, Animals, Cricetinae, Gene Expression, Humans, Immunoenzyme Techniques, Isoenzymes genetics, Isoenzymes metabolism, Male, Reverse Transcriptase Polymerase Chain Reaction, Tumor Cells, Cultured, Alkaline Phosphatase metabolism, Biomarkers, Tumor metabolism, Neoplasms, Germ Cell and Embryonal enzymology, Testicular Neoplasms enzymology
- Abstract
In humans, alkaline phosphatases are encoded by one tissue-non-specific alkaline phosphatase (TNAP) gene and three tissue-specific alkaline phosphatase genes, intestinal, placental (PLAP), and germ cell-specific alkaline phosphatase (GCAP). Although the presence of alkaline phosphatases in testicular germ cell tumours (TGCTs) of adolescents and adults has been utilized for both detection and patient monitoring, it is not known in detail which isozymes are expressed. Since alkaline phosphatase is detected in carcinoma in situ (CIS), the common precursor of all TGCTs, it might provide a marker for the early diagnosis of TGCTs. Testicular cancers of germ cell and non-germ cell origin along with testicular parenchyma with and without CIS have been analysed for the expression of the different alkaline phosphatase isozymes. Antibodies to TNAP and PLAP/GCAP showed positivity in CIS, seminoma, and embryonal carcinoma. The heterogeneous staining pattern detected in frozen tissue sections was similar to the pattern found in formalin-fixed, paraffin-embedded material, indicating a biological phenomenon and not a handling artefact. Since PLAP and GCAP cannot be distinguished using immunohistochemistry, the expression of these isozymes was studied at the molecular level using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach, in combination with a primer extension assay. The results show that CIS and seminoma predominantly express GCAP, while in embryonal carcinoma the expression of GCAP versus PLAP varies. Due to the presence of alkaline phosphatase transcripts in normal testicular parenchyma, an RT-PCR-based analysis of alkaline phosphatase is not informative for the early detection of TGCTs in biopsy samples., (Copyright 1999 John Wiley & Sons, Ltd.)
- Published
- 1999
- Full Text
- View/download PDF
20. Allelic amino acid substitutions affect the conformation and immunoreactivity of germ-cell alkaline phosphatase phenotypes.
- Author
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Hoylaerts MF, Manes T, and Millán JL
- Subjects
- Alkaline Phosphatase genetics, Alkaline Phosphatase immunology, Antibodies, Monoclonal, Base Sequence, Binding Sites, Cloning, Molecular, Electrophoresis, Starch Gel, Enzyme Stability, Epitopes immunology, Genotype, Hot Temperature, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Placenta enzymology, Polymorphism, Genetic, Protein Conformation, Alkaline Phosphatase chemistry, Amino Acids chemistry, Germ Cells enzymology, Phenotype
- Abstract
The gene encoding placental alkaline phosphatase (PLAP) displays a well-documented allelic polymorphism. Likewise, different phenotypes exist for the PLAP-related germ-cell alkaline phosphatase (GCAP). We investigated the extent to which various allelic GCAP positions are critical in determining the enzymatic, structural, and immunological properties of GCAP phenotypes. Three homozygous GCAP phenotypes [JEG3, BeWo, and wild-type (wt) GCAP] were analyzed and compared with a "core" GCAP mutant that contains the seven amino acid substitutions that are consistently different between PLAP and GCAP but are common to the three known allelic GCAP genotypes. Although some substitutions could influence the electrophoretic behavior of the phenotypes, the allelic differences did not affect the kinetic properties of GCAP. However, they did affect the immunoreactivity and conformation of the variants as detected with a panel of 18 epitope-mapped monoclonal antibodies (MAbs) to PLAP. The selective immunoreactivity of the PLAP/GCAP-discriminating MAb C2 was critically dependent on the nature of the allelic residues 133 and 361 in GCAP. Residue 133 was also important for the general stability of the molecule because BeWo and wt GCAP, which have Asn133 and Val133, respectively, instead of Met133, showed a consistently reduced heat stability compared to core GCAP and JEG3. Because the core GCAP mutant consistently shows the characteristics of wt GCAP, its use as an antigen should allow the generation of monoclonal antibodies to GCAP that will not cross-react with PLAP and whose immunoreactivity will only marginally be influenced by allelic GCAP variation.
- Published
- 1992
21. Genomic structure and comparison of mouse tissue-specific alkaline phosphatase genes.
- Author
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Manes T, Glade K, Ziomek CA, and Millán JL
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Consensus Sequence, Embryo, Mammalian enzymology, Enzyme Induction, Genes, Glycosylation, Humans, Intestines enzymology, Molecular Sequence Data, Organ Specificity, Pseudogenes, Regulatory Sequences, Nucleic Acid, Sequence Homology, Nucleic Acid, Software, Species Specificity, Alkaline Phosphatase genetics, Isoenzymes genetics, Mice genetics, Multigene Family
- Abstract
A full-length human placental alkaline phosphatase (AP) cDNA was used to identify and clone related genes from mouse genomic libraries. We report the cloning, sequence, and structural comparison of the mouse embryonic and intestinal AP genes and a putative AP pseudogene. All three mouse genes are composed of 11 exons interrupted by 10 small introns (70-261 bp) with an organization analogous to that of the three human tissue-specific AP genes. Introns interrupt the coding sequences at identical positions in all three mouse and human tissue-specific AP genes. The deduced amino acid sequence of the isozymes predicts proproteins of 529, 559, and 466 amino acids for embryonic AP, intestinal AP, and pseudo-AP, respectively. A repetitive sequence inserted in exon XI of the mouse intestinal AP gene codes for a unique stretch of 41 amino acids, 20 of which are threonines. This insertion has disrupted a region recognized as being responsible for phosphatidylinositol anchorage of human placental AP to the cytoplasmic membrane. Phylogenetic analysis indicates that the three mouse AP isozymes form a distinct group separate from the human tissue-specific AP isozymes, suggesting the taxon-specific evolution of the AP genes as opposed to independent evolution of AP genes expressed in specific tissues.
- Published
- 1990
- Full Text
- View/download PDF
22. Two alkaline phosphatase genes are expressed during early development in the mouse embryo.
- Author
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Hahnel AC, Rappolee DA, Millan JL, Manes T, Ziomek CA, Theodosiou NG, Werb Z, Pedersen RA, and Schultz GA
- Subjects
- Alkaline Phosphatase analysis, Animals, Base Sequence, Blastocyst enzymology, Embryo, Mammalian enzymology, Female, Intestines enzymology, Isoenzymes analysis, Male, Mice, Molecular Sequence Data, Polymerase Chain Reaction, Pregnancy, RNA, Messenger analysis, Testis enzymology, Alkaline Phosphatase genetics, Embryonic Development, Germ Cells enzymology, Intestines embryology, Isoenzymes genetics
- Abstract
Alkaline phosphatase (AP) activity is stage specific in mouse embryos and may be associated with compaction and separation of trophectoderm from inner cell mass in preimplantation development. We previously sequenced a cDNA and two mouse AP genes that could contribute to the AP activity in embryos. Oligonucleotide primers were constructed from the three sequences and used in the reverse transcription-polymerase chain reaction technique to establish that two of the three AP isozymes are transcribed during preimplantation development. The predominant transcript (E-AP) is from a gene highly homologous to the human tissue-specific APs, but different from the mouse intestinal AP. Tissue non-specific (TN) AP also is transcribed, but there is approximately 10 times less TN-AP than E-AP transcript. The TN-AP isozyme is the predominant transcript of 7 to 14 day embryos and primordial germ cells. A switch in predominance from E-AP to TN-AP must occur during early postimplantation development. This study establishes a framework for experiments to determine the functions of the two isozymes during preimplantation development.
- Published
- 1990
- Full Text
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23. Use of auxiliary labels to improve patient compliance.
- Author
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Fedder DO, Goldstein M, Manes T, Benko JE, and Shangraw R
- Subjects
- Attitude of Health Personnel, Education, Pharmacy, Continuing, Maryland, Drug Labeling, Patient Compliance, Patient Education as Topic
- Published
- 1979
24. CAT constructs with convenient sites for cloning and generating deletions.
- Author
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Tsonis PA, Manes T, Millan JL, and Goetinck PF
- Subjects
- Base Sequence, Chloramphenicol O-Acetyltransferase, Acetyltransferases genetics, Cloning, Molecular methods, DNA Mutational Analysis methods, Promoter Regions, Genetic
- Published
- 1988
- Full Text
- View/download PDF
25. Rapid phage DNA isolation without the use of enzymes.
- Author
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Tsonis PA and Manes T
- Subjects
- Biotechnology, Enzymes, Bacteriophage lambda analysis, DNA, Viral isolation & purification
- Published
- 1988
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