Placenta Alkaline Phosphatase Research Articles

Neuropilin-1 is uniquely expressed on small syncytiotrophoblast extracellular vesicles but not on medium/large vesicles from preeclampsia and normal placentae

Preeclampsia (PE) is a multisystem progressive hypertensive disorder unique to human pregnancy. The placenta is fundamental to its pathogenesis and releases placental factors as well as extracellular vesicles (small and medium/large syncytiotrophoblast extracellular vesicles (STB-EVs)) as a response to syncytiotrophoblast stress such as tissue factor and plasminogen activator inhibitors 1. Neuropilin 1 (NRP-1) is an anti-angiogenic factor involved in development, angiogenesis, arteriogenesis, and vascular permeability. NRP-1 acts as a co-receptor for growth factors such as vascular endothelial growth factor (VEGF), placenta growth factor (PLGF), and epidermal growth factor (EGF). Given the documented pro and anti-angiogenic roles of STB-EVs, we hypothesized that 1) STB-EVs might express NRP-1; and 2) the expression of NRP-1 might differ between normal and preeclampsia STB-EVs. MethodsWe isolated STB-EVs (both small and medium/large) from PE and NP placentae using the physiologic ex vivo dual lobe perfusion model. The enriched STB-EVs were characterized by Western blot, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA) according to the international society of extracellular vesicles (ISEV) guidelines. We assessed for NRP-1 expression with Western blot (placenta and STB-EVs) and immunohistochemistry (placenta). We performed co-expression analysis for placenta alkaline phosphatase (PLAP – a known STB-EV marker) and NRP-1 with immunoprecipitation followed by Western blot. ResultsWe confirmed NRP-1 expression in NP and PE placenta. We showed that NRP-1 Expression was limited to small syncytiotrophoblast membrane extracellular vesicles (S STB-EVs) but not medium/large STB-EVs and that NRP-1 is co-expressed with PLAP. ConclusionNeuropilin-1 is uniquely expressed on small syncytiotrophoblast extracellular vesicles but not on medium/large vesicles from preeclampsia and normal placentae.

Avoiding Pre-Isolation Step in Exosome Analysis: Direct Isolation and Sensitive Detection of Exosomes Using Gold-Loaded Nanoporous Ferric Oxide Nanozymes.

Most of the current exosome-analysis strategies are time-consuming and largely dependent on commercial extraction kit-based preisolation step, which requires extensive sample manipulations, costly isolation kits, reagents, tedious procedures, and sophisticated equipment and is prone to bias/artifacts. Herein we introduce a simple method for direct isolation and subsequent detection of a specific population of exosomes using an engineered superparamagnetic material with multifunctional properties, namely, gold-loaded ferric oxide nanocubes (Au-NPFe2O3NC). In this method, the Au-NPFe2O3NC were initially functionalized with a generic tetraspanin (exosomes-associated) antibody (i.e., CD63) and dispersed in sample fluids where they work as "dispersible nanocarriers" to capture the bulk population of exosomes. After magnetic collection and purification, Au-NPFe2O3NC-bound exosomes were transferred to the tissue-specific, antibody-modified, screen-printed electrode. As a proof of principle, we used a specific placental marker, placenta alkaline phosphatase (PLAP), to detect exosomes secreted from placental cells. The peroxidase-like activity of Au-NPFe2O3NC was then used to accomplish an enzyme-linked immunosorbent assay (ELISA)-based sensing protocol for naked-eye observation along with UV-visible and electrochemical detection of PLAP-specific exosomes present in placental cell-conditioned media. We demonstrated excellent agreement in analytical performance for the detection of placental cell-derived exosomes (i.e., linear dynamic range, 103-107 exosomes/mL; limit of detection, 103 exosomes/mL; relative standard deviation (%RSD) of <5.5% for n = 3) using with and without commercial "total exosome isolation kit"-based preisolation step. We envisage that this highly sensitive, rapid, and inexpensive assay could be useful in quantifying specific populations of exosomes for various clinical applications, focusing on pregnancy complications.

Comparative observational study of serum neoplastic biomarker and immunohistochemistry in 28 cases of intracranial germ cell tumors

Objective To conduct the comparative observational study of serum neoplastic biomarker and immunohistochemistry in intracranial germ cell tumors (GCTs). Methods A retrospective study was conducted on clinical data of 28 cases of germ cell tumors undergoing surgery from March 2013 to July 2016 at Tsinghua University Yuquan Hospital. Presurgical serum neoplastic biomarkers including α-fetoprotein (AFP), β-human chorionic gonadotropin (β-HCG) and alkaline phosphatase (ALP) were measured and compared with postsurgical immunohistochemistry results of tumor specimens. Results Among the 28 cases of GCTs, germinoma was identified in 5 cases, teratoma in 2 cases, immature teratoma in 5 cases, choriocarcinoma in 1 case and mixed germ cell tumor in 15 cases. AFP was inconsisent in 8/28 cases, being positive in serum biomarker and negative in immunohistochemistry, and included 4 cases of immature teratoma and 4 mixed GCTs. β-HCG was incongruent in 5 cases (positive in serum and negative in immunohistochemistry). Among the 18 cases with pure germinoma component, placenta ALP (PLAP) immunohistochemistry revealed positive results in 15 cases, while serum ALP was significantly increased in only 4 cases and not detected in the remaining 14 cases. Conclusions The treatment and prognosis of intracranial GCTs might be correlated with different neoplastic components. Immunohistochemistry combined with serum biomarker could help demonstrate those neoplastic components more accurately and completely. Key words: Central nervous system; Neoplasms, germ cell and embryonal; Neoplastic biomarker; Immunohistochemistry

Molecular Characterization of Alkaline Phosphatase and PLAP in the Germinal Epithelium of Pb and Se+Zn Treated Adult Sprague Dawley Rats

Aim: The role of alkaline phosphatase in cell metabolism and regulation is essential and cuts across cell proliferation, division, cell death, membrane transport and DNA cleavage. This study addresses the role and importance of alkaline phosphates (ALP) and Placenta Alkaline phosphatase (PLAP) in the cells of germinal epithelium of male Rat testes following induced lead (Pb) toxicity and rejuvenation by Selenium (Se) and Zinc (Zn) treatment. Method: Adult Sprague-Dawley Rats (males) were divided into 4 groups of 15 animals each and were treated as thus; Group 1: Normal saline and serves as the control, Group 2: 100 mg/Kg BW of Pb only, Group 3: 100 mg/Kg BW of Pb and 2.25 mg/Kg each of Se and Zn and group 4 received 2.25 mg.Kg each of Se and Zn only. The duration of treatment was 56 days following which the animals were sacrificed by the 57th day and testes fixed in Bouin’s fluid. Result: Pb induced toxicity could be apoptotic involving ROS activation of NO-dependent apoptotic pathway or necrotic involving a wide range damage by ROS induced lipid peroxidation. Selenium and zinc treatment ameliorated the damage induced by lead toxicity in the germinal epithelium. Conclusion: ALP is involved in intrinsic regulation of DNA cleavage in apoptosis by functioning similar to endonucleases and also regulates membrane transport in perioxidated biomembranes. PLAP expression is minimal as tumorgenesis was not recorded, although Pb treatment showed signs of epithelium of endothelial cancer signalling, which was ameliorated by Se+Zn treatment.

Expression of embryonic stem cell markers in cultured JKT‐1, a cell line derived from a human seminoma

Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (alphaFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2gamma and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results.

ErbB-2 and Met Reciprocally Regulate Cellular Signaling via Plexin-B1

Sema4D-induced activation of plexin-B1 has been reported to evoke different and sometimes opposing cellular responses. The mechanisms underlying the versatility of plexin-B1-mediated effects are not clear. Plexin-B1 can associate with the receptor tyrosine kinases ErbB-2 and Met. Here we show that Sema4D-induced activation and inactivation of RhoA require ErbB-2 and Met, respectively. In breast carcinoma cells, Sema4D can have pro- and anti-migratory effects depending on the presence of ErbB-2 and Met, and the exchange of the two receptor tyrosine kinases is sufficient to convert the cellular response to Sema4D from pro- to anti-migratory and vice versa. This work identifies a novel mechanism by which plexin-mediated signaling can be regulated and explains how Sema4D can exert different biological activities through the differential association of its receptor with ErbB-2 and Met.

Analyzing Somatosensory Axon Projections with the Sensory Neuron-SpecificAdvillinGene

Peripheral sensory neurons detect diverse physical stimuli and transmit the information into the CNS. At present, the genetic tools for specifically studying the development, plasticity, and regeneration of the sensory axon projections are limited. We found that the gene encoding Advillin, an actin binding protein that belongs to the gelsolin superfamily, is expressed almost exclusively in peripheral sensory neurons. We next generated a line of knock-in mice in which the start codon of the Advillin is replaced by the gene encoding human placenta alkaline phosphatase (Avil-hPLAP mice). In heterozygous Avil-hPLAP mice, sensory axons, the exquisite sensory endings, as well as the fine central axonal collaterals can be clearly visualized with a simple alkaline phosphatase staining. Using this mouse line, we found that the development of peripheral target innervation and sensory ending formation is an ordered process with specific timing depending on sensory modalities. This is also true for the in-growth of central axonal collaterals into the brainstem and the spinal cord. Our results demonstrate that Avil-hPLAP mouse is a valuable tool for specifically studying peripheral sensory neurons. Functionally, we found that the regenerative axon growth of Advillin-null sensory neurons is significantly shortened and that deletion of Advillin reduces the plasticity of whisker-related barrelettes patterns in the hindbrain.

Testicular seminoma metastasizing to the parotid gland: Report of a case

Among salivary glands, the parotid gland is the most common site for secondary neoplastic involvement, and carcinomas and melanomas are the two most common secondary tumors. We present a case of testicular seminoma metastasizing to the parotid gland. The patient, who had a remote history of testicular seminoma, was initially seen with a parotid mass. A primary parotid lesion was anticipated, and excisional biopsy was performed. The diagnosis of testicular seminoma metastasizing to the parotid gland was established on the basis of histologic findings and was confirmed by immunohistochemical analysis. Metastatic and primary seminomas have the same morphologic features and cellular composition, and granulomatous reaction is often present in both neoplasms. Extragonadal seminoma often occurs in the midline (presacral, retroperitoneal, anterior mediastinal, and pineal regions); when present in any other locations, the tumor should be considered to be metastatic. Placenta alkaline phosphatase is a useful immunohistochemical stain to confirm the diagnosis, and cytokeratin immunohistonegativity rules out carcinomas or epithelioid sarcomas. To our knowledge, this is the first published case of metastatic testicular seminoma to the parotid gland. Cytomorphologic features of seminoma and the presence of granulomatous giant cells are important diagnostic clues. Two other neoplasms with granulomatous reactions, nasopharyngeal carcinoma and epithelioid sarcoma, should be considered in the differential diagnosis.

Estrogen receptor is significantly associated with the epithelioid variants of renal angiomyolipoma: a clinicopathological and immunohistochemical study of 67 cases.

Perivascular epithelioid cells (PEC) in angiomyolipoma (AML) were recently proposed to be its most common progenitor cells. Histologically, triphasic components were present in various proportions, but were overwhelmingly myogenic in epithelioid variants of AML. Despite histological discrimination, the immunophenotypic profiles between triphasic and epithelioid AML have never been compared. The aim of the present study was to clarify the identity of PEC by using immunoreactivity to estrogen receptor (ER), progesterone receptor (PR), bcl-2 and placenta alkaline phosphatase (PLAP), and to use this information to compare triphasic and epithelioid AML. A total of 33 out of 67 cases of renal angiomyolipoma that underwent surgery were reviewed over the period 1998-2003. Two cases were associated with tuberous sclerosis. Ten patients had other malignant tumors, and three patients had a nodal extension. Immunohistochemistry showed that bcl-2 (59.4%), PLAP (46.9%), HMB-45 (100%) was predominantly localized around vessels. The stem cell markers were absolutely negative in all AML types. The estrogen receptors were positive in 14 cases (42.4%) and the progesterone receptors were positive in five cases. Bcl-2 and both female sex hormone receptors were significantly more frequent in the epithelioid variant of AML than in the triphasic type. Perivascular epithelioid cells express bcl-2, ER, PR and PLAP, and ER could be partly associated with myogenic proliferation.

Biochemical and functional analysis of an adenovirus-based ligand complex for gene transfer.

Ligand-mediated approaches to gene transfer offer an alternative to viral vectors for both in vivo and in vitro applications. Although a significant percentage of the plasmid-based DNA complex is lost to lysosomal degradation following receptor-mediated endocytosis, simultaneous infection with adenovirus has been shown to increase the level of transgene expression [Curiel, Agarwal, Wagner and Cotten (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8850-8854; Wagner, Zatloukal, Cotten, Kirlappos, Mechtler, Curiel and Birnstiel (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6099-6103]. In this study we describe an adenovirus-based ligand complex where the plasmid DNA, polycation-ligand conjugate and adenovirus are contained within a single particle structure. At the core of the transfection particle is a replication-defective recombinant adenovirus encoding a cDNA minigene for human placenta alkaline phosphatase that was chemically modified with poly(L-lysine) (Ad-pLys). Electron microscopy of an adenovirus-based ligand complex formed by successively adding plasmid DNA and an asialo-orosomucoid-poly(L-lysine) conjugate to Ad-pLys revealed structures that appeared as intact viral particles coated with a dense biomolecular layer. Adenovirus-based ligand complexes containing either a luciferase or beta-galactosidase reporter plasmid were shown to efficiently deliver the plasmid transgene to cells that express the hepatic asialoglycoprotein receptor. Furthermore, the poly(L-lysine) modification greatly reduced the infectivity potential of the virus without causing a concomitant loss of augmented gene transfer. As an alternative to infectious virions, incomplete products of viral assembly were also considered as a source for endosomalytic activity. However, these defective virions were unable to significantly enhance plasmid transgene delivery.

Serum levels of human alkaline phosphatase isozymes in relation to blood groups

By use of sensitive immunocatalytic assays, based on isozyme specific monoclonal antibodies, the activities of the three main human alkaline phosphatases were determined in serum. The activities were related to ABO blood groups and secretor phenotypes. The activity of intestinal alkaline phosphatase was found to be strongly correlated with ABO blood groups and secretor phenotypes, while neither the placenta alkaline phosphatase activity nor the tissue unspecific alkaline phosphatase activity demonstrated any dependence on blood groups or secretor phenotypes. Non-secretors, independent of ABO blood groups, demonstrated low activities of intestinal alkaline phosphatase in serum, amounting to approximately 20% of the activities in the secretor groups. Within the secretor group, the lowest activities were observed for blood group A (2.8 ± 1.1 IU/l; mean ± SEM) and the highest for blood groups B and O (14.1 ± 1.1 IU/l and 19.0 ± 2.5 IU/l, respectively). These results confirm that the activities of intestinal alkaline phosphatase in serum have to be related both to ABO blood groups and to secretor phenotypes in order to be informative in clinical contexts.

Tissue-specific and dexamethasone-inducible expression of Alkaline Phosphatase from alternative promoters of the rat Bone/Liver/Kidney/Placenta gene

The rat Bone/Liver/Kidney/Placenta Alkaline Phosphatase (ALP) is transcribed from two alternative promoters spaced over 25 kb apart, resulting in two variant transcripts that are identical in their coding sequence. We investigated the steady-state levels of the two variant transcripts in various rat tissues and cell lines using the polymerase chain reaction (PCR) amplification for RNA phenotyping, RNase protection, and northern blot analysis. Our results demonstrate that ALP transcripts from the upstream promoter are preferentially expressed in calvariae, and are almost exclusively expressed in ROS17 2.8 osteogenic sarcoma cells. In contrast, the downstream promoter is preferentially expressed in kidney. Moreover, the increase in ALP activity and mRNA levels following dexamethasone treatment of ROS17 2.8 cells is correlated with an increase in the level of transcripts from the upstream promoter. Thus, the two alternative promoters of the rat BLKP ALP gene are involved in cell-specific and dexamethasone-inducible regulation of its expression.

Expression of human placenta alkaline phosphatase in placenta during pregnancy

To clarify the expression of PLAP during the course of pregnancy, the amount of PLAP mRNA and its activity in normal placental villi were measured. Both PLAP and its mRNA were found in placentae of as early as 7 weeks of gestation, and they continued to increase throughout pregnancy. But they showed different patterns of increase. The amount of PLAP mRNA began to increase dramatically around 13th week and probably continued to increase gradually until term. PLAP activity per gram of villi showed a gradual increase from around 13th week and a marked increase was observed after about 20th week. PLAP levels in sera from pregnant women were also measured, and they showed a pattern of increase imilar to that of PLAP activity per gram of villi. The continuous increase in the expression of PLAP throughout pregnancy suggests that PLAP may play a role in feto-maternal metabolism and placental differentiation.

Cloning and Analysis of the 5' Region of the Rat Bone/Liver/Kidney/Placenta Alkaline Phosphatase Gene. A dual-function promoter

The rat bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) gene is expressed at high level in these particular tissues and at low levels in many other tissues. To study the mechanisms underlying the complex regulation of the rat BLKP ALP expression, we isolated a genomic clone, containing a 10.5-kb insert, which includes the promoter of the BLKP ALP gene with 2kb of 5' flanking region, its first exon (84bp), and over 7kb of the first intron. The promoter of the rat BLKP ALP displays features of a “housekeeping” gene promoter: an atypical TATA-box (TTCATAA); 3 potential Spl binding sites; high GC content (82% in positions -134 to -14); and a high CpG to GpC ratio (60:89 in the 0.85-kb promotor region), indicating an abundance of potential methylation sites. Likewise, transient transfection of CAT fusion genes into ROS 17/2.8 osteoblast-like cells reveals weak expression from the promoter and proximal 5' flanking sequences, which can be elevated by an SV40E enhancer.The homologous human bone/liver/kidney (BLK) ALP promoter, which demonstrates a similar combination of tissue-specific and housekeeping characteristics, shares close similarity (184bp of 79% similarity excluding gaps) with the rat BLKP ALP promoter. The human placental ALP is encoded by a separate gene and its promoter, on the other hand lacks significant similarity to the rat BLKP ALP promoter despite their common expression in the placenta. This lack of similarity appears to reflect the close evolutionary relationship of the human placental ALP gene to the intestinal ALP gene.Significant sequence similarity was found between the rat and human BLK/BLKP ALP promoters and the human and mouse adenine deaminase promoters, and together they may represent a class of dual-function promoters, allowing both constitutive low-level, and tissuespecific higher levels of expression. A pentanucleotide with the consensus sequence 5'-GGCTC-3' is present in these promoters and in the promoters for the human fibronectin and the human α1(II) pro collagen genes in the region of maximal similarity with the rat BLKP ALP promoter, and in the vicinity of the Sp1-binding sites.

Formation of an alkaline phosphatase-immunoglobulin G complex in human sera

An electrophoretically slow-moving band of alkaline phosphatase (ALP) isoenzyme was found in four patients with chronic liver disease and in one with ulcerative colitis. Immunoelectrophoretic studies revealed that the slow band was a complex containing alkaline phosphatase and an immunoglobulin G of the lambda class. Its molecular weight was approximately 280 000. A complex molecule consisting of one molecule of immunoglobulin G and one molecule of alkaline phosphatase was proposed. The complex was similar to the liver and bone alkaline phosphatases in functional properties. Serum containing the complex was capable of binding liver and bone alkaline phosphatase isoenzymes but not the intestine or placenta alkaline phosphatase isoenzymes from normal controls. The presence of an abnormal immunoglobulin which binds liver and bone alkaline phosphatases appears to be responsible for the development of the complex. In one case of chronic liver disease, the complex disappeared after a few months.

ABO BLOOD GROUPS AND SEX RATIO AT BIRTH

We have shown an interaction between ABO and placenta) alkaline phosphatase (PAP) polymorphisms (Am. J. Hum. Genet. 24, 495, 1972) suggesting that allele P1f1 of PAP partially protects fetuses of blood group B from the damaging effects of antibodies produced by ABO incompatible mothers. On the contrary, ABO compatible fetuses are at a disadvantage, since “some” maternal immunological reaction is necessary for the implantation and maintenance of the zygote in utero. Since XY zygotes are antigenically more dissimilar from their mothers than XX zygotes, we expected a high M/F ratio in newborn infants of group B compatible with their mothers. The study of a consecutive series of 2296 newborns showed a M/F ratio of 1.86 among group B compatible infants. This deviation of the sex ratio from that of the general population (∼ 1.05) is highly significant (P <0.01) and further supports our hypothesis that PAP plays an important role in the maternal-fetal immunoiogical relationship.

Mechanism of action of Zn2+ and Mg2+ on rat placenta alkaline phosphatase. II. Studies on membrane-bound phosphatase in tissue sections and in whole placenta.

Alkaline phosphatase (EC 3.1.3.1) bound to trophoblastic cells in rat placenta is activated by Mg2+ and inhibited by Zn2+ in the same way as is found with partially purified soluble alkaline phosphatase in the same tissue (PetitClerc, C., Delisle, M., Martel, M., Fecteau, C. & Brière, N. (1975) Can. J. Biochem. 53, 1089-1100). In studies done with tissue sections (6-10 micron), it is shown that alkaline phosphatase activity and labelling of active sites by orthophosphate are lost during incubation with ethanolamine at pH 9.0. Addition of Mg2+ causes total recovery of catalytic activity and active sites labelling. Zn2+ displaces and replaces at the Mg2+ binding sites. The affinity for both ions is similar, and dissociation of Zn2+ from the enzyme is a very slow process, even in the presence of Mg2+. The Zn2+-alkaline phosphatase and Mg2+-alkaline phosphatase, which only differ by the ion bound to an apparent modulator site, have the same catalytic activity at pH less than 7.0, but the Zn2+ species has little activity at alkaline pH. Phosphorylation of the enzyme by orthophosphate indicates that with both enzyme species phosphoryl intermediate does not accumulate at alkaline pH. These results suggest that with orthophosphate, the phosphorylation step is rate determining for both enzymes, and that Zn2+ affects this step to a much greater extent. It is proposed that Zn2+ and Mg2+ regulate alkaline phosphatase in rat placenta. The concentration of both ions in maternal serum and placenta suggest that such a mechanism could exist in vivo.

Direct immunoperoxidase staining for regan isoenzyme of alkaline phosphatase in human tumor tissues

Immunoperoxidase staining for Regan isoenzyme of alkaline phosphatase was performed on cryostat sections of five human tumor tisssues. With a direct immunoperoxidase staining for the localization of Regan isoenzyme at the light and electron microscope levels, sections previously fixed with 0.05 M phosphate-buffered 4% paraformaldehyde were reacted with rabbit antisera to human placenta alkaline phosphatase conjugated to horseradish peroxidase. Comparison of conventional histochemistry and immunohistochemistry for Regan isoenzyme indicated that strong specific immunoperoxidase staining appeared on the cell membrane surface, and a diffuse one, in the cytoplasm of lung and colon cancer tissue cells showing L-phenylalanine-sensitive alkaline phosphatase. No immunoperoxidase reaction was obtained in tumor cells showing sensitivity to L-homoarginine or lacking aklaline phosphatase activity.