Ewing sarcoma with very late metastasis in the skull: a case report.

BACKGROUND: Ewing sarcoma is a malignant bone tumor; however, its prognosis has improved since the development of modern chemotherapy. Although Ewing sarcoma outcomes have improved, issues related to late complications, secondary malignant neoplasms, and late recurrence or metastasis have emerged. CASE PRESENTATION: We report a case of Ewing sarcoma that recurred in the occipital bone 21 years after primary tumor treatment. A 45-year-old Japanese woman with a history of Ewing sarcoma 21 years prior, was referred to our hospital due to a severe headache. A tumor was detected in the left occipital bone, and the biopsy revealed Ewing sarcoma. Metastasis was suspected because the patient had been treated for Ewing sarcoma of the left clavicle 21 years prior. There have been several cases of local recurrence or metastasis, occurring 15-20 years after the onset of the initial disease. To our knowledge, very late metastasis of Ewing sarcoma in the skull has not been reported. CONCLUSION: We report a rare case of very late metastasis of Ewing sarcoma in the skull with a review of the literature. Delayed metastasis secondary to Ewing sarcoma can occur in the lung, which is the most common site for metastasis, as well as other regions of the body, such as the cranium.

Persistent Severe Cerebral Edema with Neutrophil Infiltration Following Listeria Meningitis.

A 51-year-old man was admitted to the hospital with a diagnosis of Listeria monocytogenes meningitis. Diffuse cerebral edema appeared after improvement of meningitis with appropriate treatment and worsened for two months. Due to brain herniation, brain tissue leaked through the incision made during the drain insertion in a hydrocephalus surgery. We found pathological evidence of significant neutrophil infiltration with a few lymphocytes without bacterial detection in the degraded brain tissue. The present case indicates that fatal cerebral edema with significant neutrophil infiltration may develop even after appropriate treatment for L. monocytogenes meningitis.

Progesterone is a sperm-releasing factor from the sperm-storage tubules in birds.

Because of the presence of sperm-storage tubules (SST) in the utero-vaginal junction (UVJ) in the oviduct, once ejaculated sperm have entered the female reproductive tract, they can survive for a prolonged time in domestic birds, although the specific mechanisms involved in the sperm uptake into, maintenance within, and controlled release from the SST remain to be elucidated. In this report, we provide evidence that progesterone triggers the release of the resident sperm from the SST in the UVJ. The ultrastructural observation of the SST indicated that the resident sperm are released from the SST around 20 h after oviposition. When laying birds were injected with progesterone, most of the sperm were released from the SST within 1 h of injection. In situ hybridization analyses demonstrated the presence of the transcripts of membrane progestin receptor α in the UVJ, and the translated proteins were detected in the UVJ extracts by Western blotting. Moreover, the number of secretory granules in the SST epithelial cells fluctuates during the ovulatory cycle, and the progesterone administration mimics this phenomena. A binding assay using [(3)H]-progesterone indicated the presence of a high affinity, limited capacity, saturable and single binding site for [(3)H]-progesterone in the membrane fraction of the UVJ, and this receptor did not interact with the synthetic antiprogestin RU486. These results demonstrated for the first time that the progesterone stimulates the release of the resident sperm from the SST and that the release of the sperm might occur via membrane progestin receptor α-mediating signal transduction.

FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-beta signaling.

Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days' culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days' culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-beta signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.

Angiotensin II mimics the hypoxic effect on regulating trophoblast proliferation and differentiation in human placental explant cultures.

Regulation of cytotrophoblast differentiation toward extravillous trophoblasts (EVTs) is critical for establishing successful pregnancy. Previous studies have focused primarily on the factors promoting the differentiation, while inhibitory regulators except hypoxia have been less documented. In this study, to test our hypothesis that angiotensin II (Ang II) would inhibit EVT differentiation, we investigated the effects of Ang II on trophoblast outgrowth and the expression of molecules associated with the proliferation and invasion of trophoblasts using human first trimester villous explant cultures. Ang II increased EVT outgrowth and the number of cells in cell columns. Moreover, Ang II-treated explants exhibited increased Ki67 and integrin alpha5 immunoreactivity in EVTs as well as matrix metalloproteinase-2 activity in the conditioned media, and decreased alpha1 integrin immunoreactivity, which are compatible with the features of the proliferative phenotype EVTs. These effects of Ang II were similar to those of hypoxia (3% O(2)). Ang II stimulated the expression of hypoxia inducible factor-1alpha at both mRNA and protein levels, and also enhanced the expression of plasminogen activator inhibitor-1 (PAI-1). Data presented herein suggest a possible role for Ang II in impairing trophoblast differentiation toward an invasive phenotype, which might be associated with shallow invasion in preeclamptic placentas.

FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-beta2.

Human mesenchymal stem cells (hMSCs) are able to both self-replicate and differentiate into a variety of cell types. Fibroblast growth factor-2 (FGF-2) stimulates the growth of hMSCs in vitro, but its mechanisms have not been clarified yet. In this study, we investigated whether cellular senescence was involved in the stimulation of hMSCs growth by FGF-2 and the expression levels of transforming growth factor-beta1 and -beta2 (TGF-betas). Because hMSCs were induced cellular senescence due to long-term culture, FGF-2 decreased the percentage of senescent cells and suppressed G1 cell growth arrest through the suppression of p21(Cip1), p53, and p16(INK4a) mRNA expression levels. Furthermore, the levels of TGF-betas mRNA expression in hMSCs were increased by long-term culture, but FGF-2 suppressed the increase of TGF-beta2 mRNA expression due to long-term culture. These results suggest that FGF-2 suppresses the hMSCs cellular senescence dependent on the length of culture through down-regulation of TGF-beta2 expression.

[Safety evaluation of tissue engineered medical devices using normal human mesenchymal stem cells].

Several recent studies demonstrated the potential of bioengineering using somatic stem cells in regenerative medicine. Adult human mesenchymal stem cells (hMSCs) derived from bone marrow have the pluripotency to differentiate into cells of mesodermal origin, e.g., bone, cartilage, adipose, and muscle cells; they, therefore, have many potential clinical applications. On the other hand, stem cells possess a self-renewal capability similar to cancer cells. For safety evaluation of tissue engineered medical devices using normal hMSCs, in this study, we investigated the expression levels of several genes that affect cell proliferation in hMSCs during in vitro culture. We focused on the relationship between the hMSC proliferation and their transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and cellular senescence was observed for about 3 months. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. The mRNA expressions of Smad3 increased, but those of c-myc and nucleostemin decreased with the length hMSCs were in in vitro culture. In addition, the expression profiles of the genes which regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.

Changes in expression of genes related to cell proliferation in human mesenchymal stem cells during in vitro culture in comparison with cancer cells.

We investigated the expression levels of several genes related to cell proliferation in human mesenchymal stem cells (hMSCs) during in vitro culture for use in clinical applications. In this study, we focused on the relationship between hMSC proliferation and transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and marked changes in hMSC morphology were not observed in 3 months of in vitro culture. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. There had been no change in the TGFbeta3, TGFbetaRII, and TGFbetaRIII mRNA expressions by the 12th passage from the primary culture (at about 3 months). The mRNA expression of Smad3 increased, but those of c-myc and nucleostemin decreased with the length of hMSC in vitro culture. In addition, the expression profiles of the genes that regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months of in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.

Dual mode bioreactions on polymer nanoparticles covered with phosphorylcholine group.

We investigated the preparation of polymer nanoparticles covered with phosphorylcholine (PC) groups and the immobilization of proteins in order to observe dual mode bioreactions on the nanoparticles. For the surface modification on the nanoparticles, a water-soluble amphiphilic phospholipid polymer with PC groups as a hydrophilic moiety was synthesized. In this polymer, an active ester group, which can immobilize proteins, was introduced. Using the phospholipid polymer as a solubilizer, poly(L-lactic acid) nanoparticles were prepared from its methylene chloride solution in an aqueous medium by the solvent evaporation method. The diameter of the nanoparticles was ca. 200 nm and the surface was covered with the PC groups and active ester groups. Proteins could immobilize on the nanoparticles under mild conditions by the reaction between the active ester group and amino group in the proteins. Both an antibody and enzyme were immobilized on the nanoparticles and bioreactions such as the antigen/antibody reaction and enzymatic reaction were observed. When an antigen was added to the suspension of the nanoparticles, aggregation of the nanoparticles occurred and then they precipitated. Also, the enzymatic reaction proceeded well when the enzyme substrate was added to the suspension. Based on these results, we provided polymer nanoparticles functionalized with both the antibody and enzyme, and the dual mode bioreactions could occur. We concluded that the novel polymer nanoparticles could be used for nano-/micro-scaled diagnostic and medical treatment systems.

Endoplasmic reticulum aminopeptidase-1 mediates leukemia inhibitory factor-induced cell surface human leukocyte antigen-G expression in JEG-3 choriocarcinoma cells.

Maternal immune tolerance is required for extravillous trophoblasts (EVTs) to invade the decidua without rejection. Endoplasmic reticulum aminopeptidase-1 (ERAP1) generates human leukocyte antigen (HLA) class I-adapted antigenic peptides, but its function in trophoblasts lacking classical HLA class I molecules remains undetermined. Leukemia inhibitory factor (LIF) is produced from decidua during the implantation period and plays a necessary role in establishing pregnancy. This study is intended to investigate the location and the function of ERAP1 in trophoblastic cells, focusing on LIF. Immunohistochemistry showed strong ERAP1 expression in cultured EVTs. In choriocarcinoma cell lines used as a model for trophoblasts, ERAP1 was expressed more intensively in JEG-3 than BeWo cells. Immunoblot analysis and immunocytochemistry localized ERAP1 to the endoplasmic reticulum (ER) in JEG-3 cells. Flow cytometry with HLA-G antibody to monitor the supply of antigenic peptides presenting to HLA-G in the ER showed that reducing ERAP1 transcripts by RNA interference did not affect cell surface expression of membrane HLA-G1 (mHLA-G1) in JEG-3 cells under basal conditions. In LIF-treated JEG-3 cells, cell surface mHLA-G1 expression was increased along with ERAP1 protein and promoter activities. In contrast to nonstimulated cells, eliminating ERAP1 from LIF-treated JEG-3 cells reduced the cell surface mHLA-G1 expression and soluble HLA-G1 secretion. This study provides the first evidence showing that ERAP1 is localized in the ER of trophoblasts and is involved in regulating cell surface HLA-G expression in the presence of LIF. Consequently, ERAP1 would function to present antigenic peptides to HLA-G in trophoblasts.

Enzymatic photochemical sensing using luciferase-immobilized polymer nanoparticles covered with artificial cell membrane.

We prepared phospholipid polymer nanoparticles immobilized with luciferase, and the nanoparticles were applied as photochemical sensing nanoparticles. An amphiphilic water-soluble polymer having a phosphorylcholine group was used as an emulsifier and a surface modifier to prepare the nanoparticles. The polymer was composed of three kinds of monomer units, that is, 2-methacryloyloxyethyl phosphorylcholine (MPC) as a hydrophilic and bioinert unit, n-butyl methacrylate as a hydrophobic unit and p-nitrophenyl ester having methacrylate as an enzyme-immobilizing unit. The p-nitrophenyl ester groups to immobilize the proteins were located on the surface of the nanoparticles. Luciferase was immobilized by the reaction between the p-nitrophenyl ester groups and the amino group. The enzymatic reaction on the nanoparticles was followed using a microdialysis system with an optical fiber having a 800 microm diameter in the probe. The nanoparticles conjugated with luciferase reacted with ATP, luciferin and oxygen. It is concluded that the nanoparticles are a promising tool for a photochemical sensing microdiagnostic system.

Gene regulation and physiological function of placental leucine aminopeptidase/oxytocinase during pregnancy.

Human pregnancy serum and placenta have the ability to degrade uterotonic peptide oxytocin (OT). Placental leucine aminopeptidase (P-LAP), which is also called cystine aminopeptidase, is the only membrane aminopeptidase known to functionally degrade OT as oxytocinase (OTase). P-LAP/OTase hydrolyzes several peptides other than OT including vasopressin and angiotensin III. P-LAP/OTase predicted from cDNA sequence is a type II integral membrane protein, which is converted to a soluble form existing in maternal serum by metalloproteases, possibly ADAM (a disintegrin and metalloproteinase) members. P-LAP/OTase activity increases with normal gestation, while decreases in the patients with preterm delivery and severe preeclampsia. In placenta, P-LAP/OTase is predominantly expressed in differentiated trophoblasts, syncytiotrophoblasts. Activator protein-2 (AP-2) and Ikaros transcription factors play significant roles in exerting high promoter activity of P-LAP/OTase in the trophoblastic cells. Moreover, P-LAP/OTase is transcriptionally regulated in a trophoblast-differentiation-dependent fashion via up-regulation of AP-2, putatively AP-2alpha. P-LAP/OTase may be involved in maintaining pregnancy homeostasis via metabolizing peptides such as OT and vasopressin.

Possible involvement of aminopeptidase A in hypertension and renal damage in Dahl salt-sensitive rats.

BACKGROUND: Although aminopeptidase A (APA), which is abundant in the kidneys, is responsible for metabolizing angiotensin II (Ang II), its association with salt sensitivity remains uncertain. We aimed to clarify the involvement of APA in salt-induced hypertension and renal damage. METHODS: Male Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats were fed low-salt (0.3%) or high-salt diet (8%) from 6 weeks of age for 12 weeks. Tail-cuff-measured blood pressure (BP), renal APA activity, renal Ang II levels, histologic renal damage, and APA immunoreactivity were periodically examined. RESULTS: Systolic BP progressively increased only in DS rats given the high-salt diet (DS-8% rats). The DR-8% rats had approximately 3-fold higher renal APA activity than the rats given the low-salt diet (DR-0.3% rats) during the maintenance on the high-salt diet. However, although DS-8% rats also had 2.5-fold higher renal APA activity than DS-0.3% rats at 10 weeks, continuing the high-salt diet afterward suppressed the activity in DS-8% rats below the levels observed in DS-0.3% rats. High-salt diet reduced renal Ang II levels by 30% in DR rats, whereas it showed a small and nonsignificant decrease in DS rats. The number of injured glomeruli was markedly elevated in DS-8% rats after 10 weeks. The APA immunostaining in DS-8% rats was enhanced in glomeruli displaying mild damage, diminished in the severely injured glomeruli, and absent in lesions with hyalinization. CONCLUSIONS: High-salt diet in DS rats increased renal APA activity, although renal injury remained mild, but then reduced it along with the progression of glomerulosclerosis, suggesting that reduced APA activity may be involved in the deterioration of salt-induced hypertension and renal injury.

The keratan sulfate disaccharide Gal(6S03) beta1,4-GlcNAc(6S03) modulates interleukin 12 production by macrophages in murine Thy-1 type autoimmune disease.

It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO(3))-GlcNAc(6-SO(3)) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-gamma. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.

Cell separation in microcanal coated with electrically charged phospholipid polymers.

To separate the cell population in whole blood using microcanal, the surface was covered with a polyion complex (PIC) composed of electrically charged phospholipid polymers. The phospholipids polymers were prepared by the polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate with 3-(methacryloyloxypropyl)-trimethyl ammonium iodide as the cationic unit or potassium 3-methacryloyloxypropyl sulfonate as the anionic unit. The PIC was formed at the solid-liquid interface, that is, first, the cationic polymer was coated on the substrate and an aqueous solution containing the anionic polymer with different concentrations was applied to the polymer-coated substrate. The formation of the PIC was followed using a quartz crystal microbalance (QCM), and the PIC surfaces were analyzed by both zeta-potential measurement and X-ray photoelectron spectroscopic measurement. The surface electrical potential on the PIC was controllable from +40 to -40 mV by increasing the amount of the adsorbed anionic polymer. The PIC surface was prepared in microcanal. The surface electrical potential was sequentially changed. When the whole blood was introduced into the microcanal, the cells adhered on the positively charged surface, but could not adhere to the negatively charged surface. Even when the cells adhere to the surface, the morphology of cells was maintained. This is due to MPC units at the surface, which show a good biocompatibility. These results indicated that the change in the surface electrical potential will be a useful method to separate the cells from whole blood.

Determination of ion-pair formation constants of univalent metal-crown ether complex ions with anions in water using ion-selective electrodes: application of modified determination methods to several salts.

Ion-pair formation constants (mol(-1) dm3 unit), K(MX) for a univalent metal salt (MX) and K(MLX) for its ion-pair complex (ML+X-) with a crown ether (L) in water, were determined at various ionic strengths (I) and 25 degrees C by potentiometry with ion-selective electrodes for MX=NaPic, NaMnO4, NaBPh4, KPic, and KMnO4; and MLX = Na(18C6)Pic, K(18C6)Pic, and Na(18C6)BPh4, where Pic- and 18C6 denote a picrate ion and 18-crown-6 ether, respectively. Equations for analyzing I-dependence of logK(MLX) and logK(MX) were derived and fitted well to the I-dependence using a non-linear regression analysis. The equilibrium constants at I = 0 mol dm(-3), K(MLX) degrees and K(MX) degrees, were simultaneously obtained from the analysis. The experimental values of K(MLX) and K(MX) were only in agreement with the values calculated from K(MLX) degrees and K(MX) degrees , respectively, in the ranges of higher I.

Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cells by overexpression of AP-2alpha.

A previous report demonstrated that AP-2alpha favors the survival of ovarian cancer patients by clinical findings. However, the functional roles of AP-2alpha in human ovarian cancers have not been determined. To clarify the roles, we overexpressed AP-2alpha in SKOV3 human ovarian cancer cells, which originally possess little AP-2alpha. AP-2alpha overexpression changed cell morphology from spindle to epithelioid type and suppressed cell proliferation and invasion, which would be partially correlated with decreased phosphorylation levels of the erbB2, Akt and ERK pathways, increased E-cadherin and reduced pro-matrix metalloproteinase-2 levels. Moreover, nude mice intraperitoneally injected with AP-2alpha-overexpressing cells survived longer than those with neo-transfected cells. The present data represent the first direct evidence that AP-2alpha plays a tumor suppressive role in ovarian cancer.

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells.

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.

ADAMs, a disintegrin and metalloproteinases, mediate shedding of oxytocinase.

Placental leucine aminopeptidase (P-LAP), a type-II transmembrane protease responsible for oxytocin degradation during pregnancy, is converted to a soluble form through proteolytic cleavage. The goal of this study was to determine the nature of the P-LAP secretase activity. The hydroxamic acid-based metalloprotease inhibitors GM6001 and ONO-4817 as well as the TNF-alpha protease inhibitor-2 (TAPI-2) reduced P-LAP release, while tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, which are matrix metalloproteinase inhibitors, had no effect on P-LAP release in Chinese hamster ovary (CHO) cells stably overexpressing P-LAP, thus indicating possible involvement of ADAM (a disintegrin and metalloproteinase) members in P-LAP shedding. Furthermore, overexpression of ADAM9 and ADAM12 increased P-LAP release in P-LAP-CHO transfectants. Immunohistochemical analysis in human placenta demonstrated strong expression of ADAM12 in syncytiotrophoblasts, while little expression of ADAM9 was detected throughout the placenta. Our results suggest ADAM members, at least including ADAM12, are involved in P-LAP shedding in human placenta.