Identification of 33 polymorphisms in the adipocyte-derived leucine aminopeptidase (ALAP) gene and possible association with hypertension

Adipocyte-derived leucine aminopeptidase (ALAP) inactivates angiotensin II and/or generates bradykinin in the kidney, suggesting a possible role for ALAP in the regulation of blood pressure. We considered the hypothesis that genomic variants of the ALAP gene are associated with hypertension or individual variations in blood pressure. We screened for mutations in the ALAP gene in 48 unrelated Japanese individuals and identified 33 polymorphisms including 15 novel polymorphisms. We then performed a two-stage analysis. In the first stage, the eight missense polymorphisms were evaluated for associations with blood pressure in 96 apparently healthy individuals. In the second stage, only the most promising polymorphisms were evaluated for association with essential hypertension in 143 hypertensive and 348 normotensive subjects. Among the eight missense polymorphisms, the Ile276Met and Lys528Arg polymorphisms showed significant association with blood pressure. Subsequent analysis confirmed association between the Lys528Arg polymorphism and essential hypertension. The estimated odds ratio for essential hypertension was 2.3 for presence of the Arg allele at codon 528, in comparison with presence of the Lys/Lys genotype (P = 0.004). These findings support involvement of ALAP in the regulation of blood pressure.     

Expression of c-myc and c-fms oncogenes in trophoblastic cells in hydatidiform mole and normal human placenta.

AIMS: To compare the expression of c-myc and c-fms proto-oncogenes in the placenta and hydatidiform mole. METHODS: Twelve hydatidiform moles and six induced abortion cases were collected. c-myc and c-fms proto-oncogene expression was analysed by northern blot hybridisation and immunohistochemical staining. RESULTS: The results of northern blot hybridisation analysis showed that c-fms was expressed more strongly in hydatidiform moles compared with normal placenta of similar gestational age. Moreover, c-fms mRNA concentrations increased with more advanced gestational age in moles but not in normal placentas. c-myc expression was very low in hydatidiform moles and normal placentas. Both oncogenes, however, had no direct correlation with the clinical course of the molar pregnancies. CONCLUSION: The difference in c-fms expression between hydatidiform moles and normal placentas suggests that c-fms may have a role in the development of molar pregnancies.     

Expression of leucine aminopeptidase 3 (LAP3) correlates with prognosis and malignant development of human hepatocellular carcinoma (HCC)

Leucine aminopeptidases (LAPs) were associated with tumor cell proliferation, invasion and/or angiogenesis. LAP3 is one important member of this family. However, its clinical significance and biological function in hepatocellular carcinoma (HCC) remains unknown. In the present study, we demonstrated that LAP3 expression was significantly up-regulated in HCC tissues as well as cells and was closely correlated with lower differentiation, positive lymph node metastasis and high Ki-67 expression, indicating a poor prognosis. Then cell viability assays, flow cytometry assays, wound-healing assays and matrigel invasion assays were performed to demonstrate that LAP3 promoted HCC cells proliferation by regulating G1/S checkpoint in cell cycle and advanced HCC cells migration. Furthermore, we discovered that knockdown LAP3 will enhance the sensitivity of HCC cells to cisplatin, thus promoting the cell death of HCC cells. Collectively, our results indicated that up-regulated expression of LAP3 might contribute to the proliferation and metastasis of HCC. Our data gains greater insight into the cancer-promoting role of LAP3 and its functions in HCC cells, possibly providing potential therapeutic strategies for clinical trials.     

Comparison between leucyl aminopeptidase and pseudo leucine aminopeptidase activities in sera

The highest activities of leucyl aminopeptidase(LAP, cytosol aminopeptidase, EC 3.4.11.1) in sera have been found in patients with acute hepatitis(Kanno et al., Am J Clin Path, 82: 700-705, 1984). I observed inpatients with very high activities of LAP and alcohol dehydrogenase(AD) in sera. However, only slight elevations of serum pseudo leucine aminopeptidase(PLA), that is, membrane alanyl aminopeptidase(MAA, microsomal aminopeptidase, EC 3.4.11.2) activities for hydrolysis of leucyl-4-nitroanilide were observed in these patients. They were patients in critical care unit with ischemia caused by a cardiopulmonary arrest, multiple trauma, acute myocardial infarction or operation. Therefore, we should measure LAP activities in sera rather than PLA(MAA) activities in these patients.     

Expression of functional chemokine receptors of human placental cells

PROBLEM: Chemokine receptors of placental trophoblasts possibly act as co-receptors or alternative receptors of maternal fetal infection by HIV. To clarify their possible expression and the physiological roles of chemokines on human placentae, we studied chemokine chemokine receptor expression and the effects of exogenous chemokines on choriocarcinoma cell lines. MATERIALS AND METHODS: Placental samples were obtained from 13 placentae of various gestational ages. Villous tissue was mechanically dissected from samples. Trophoblasts were enriched by anti-human chorionic gonadotropin (hCG)-coated magnetic beads. Human choriocarcinoma cell lines (JAR, BeWo, JEG-3) were maintained in RPMI 1640 media supplemented with 10% FCS. Expression of chemokine receptors was studied by RT-PCR. The effects of MIP-1alpha, RANTES, MCP-1 on hCG production were estimated by EIA. Effects of chemokines on proliferation of choriocarcinoma cell lines were examined by MTT assay. RESULTS: We observed mRNA expression of CCR-1, 2, 3, 4, 5 and CXCR-1, 2, 4 in 1st trimester placental villi, CCR-I, 2, 4 and CXCR-1, 2. 4 in 2nd trimester placental villi, CCR-1, 2, 4 and CXCR-4 in 3rd trimester placental villi. Using MACS enriched trophoblasts, we observed identical results. A choriocarcinoma cell line BeWo expressed CCR-1, 3, 4 and CXCR-1, 2, 4 while JEG-3 and JAR expressed CCR-1, 3, 4, 5 and CXCR-1, 2, 4. Expression of the CCR-5 and CXCR-4 protein in choriocarcinoma cell lines and MACS-enriched trophoblats were confirmed by flow cytometry. Chemokine MCP-3, MIP-1alpha, RANTES mRNA were expressed by the 1st, 2nd and 3rd trimester placental samples and the three choriocarcinoma cell lines examined. MCP-1 was expressed by 1st and 2nd trimester placental villi. Administration of chemokines up-regulated proliferation (10(-1) - 10 ng/mL) and hCG production (10(-1) - 10(-2)ng/ mL) of the three choriocarcinoma cell lines examined. CONCLUSIONS: Our results suggest possible roles of chemokines/chemokine receptors on placental physiology and their involvement in HIV transmission as alternative receptors.     

Urinary excretion of leucine aminopeptidase in pregnancy

This paper describes enzyme studies in normal and abnormal pregnancy. Urinary leucine aminopeptidase (L.A.P.) excretion remained relatively low throughout normal single pregnancy. Urinary L.A.P. excretion was, however, raised towards term in four out of five cases of multiple pregnancy, but in one patient the predelivery urinary L.A.P. was not raised, and the second twin in this case died shortly after delivery with gross congenital abnormalities.Urinary L.A.P. was also investigated in ;high-risk' patients. One such patient had excessive loss of this enzyme throughout pregnancy, and in the discussion it is suggested that this could be due to excessive loss of oxytocinase in the urine.Patients with toxaemia were assessed on the basis of foetal survival and the maximum 24-hour pre-delivery urinary levels of leucine aminopeptidase. This urinary value could not be used to predict foetal outcome, but rose to over 120 mg. beta-naphthylamine per 24 hours in the presence of frank proteinuria. If intrauterine death occurred, the urinary L.A.P. value fell gradually. Urinary L.A.P. was also elevated in essential hypertension towards term, but in these patients there was no gross proteinuria.     

Absence of Y chromosome in human placental site trophoblastic tumor.

Placental site trophoblastic tumor is a neoplasm of extravillous intermediate trophoblast at the implantation site, preceded in the majority of cases by a female gestational event. Our pilot investigation suggested that the development of this tumor might require a paternally derived X chromosome and the absence of a Y chromosome. Twenty cases of placental site trophoblastic tumor were included in this study. Genotyping at 15 polymorphic loci and one sex determination locus was performed by multiplex PCR followed by capillary electrophoresis. X chromosome polymorphisms were determined by PCR amplification of exon 1 of the human androgen receptor gene using primers flanking the polymorphic CAG repeats within this region. Genotyping at 15 polymorphic loci was informative and paternal alleles were present in all tumors, confirming the trophoblastic origin of the tumors. The presence of an X chromosome and the absence of a Y chromosome were observed in all tumors. Among 13 cases in which analysis of the X chromosome polymorphism was informative, all but one demonstrated at least two X alleles and seven cases showed one identifiable paternal X allele. These results confirm a unique pathogenetic mechanism in placental site trophoblastic tumor, involving an exclusion of the Y chromosome from the genome and, therefore, a tumor arising from the trophectoderm of a female conceptus. As epigenetic regulations of imprinting during X chromosome inactivation are of significant biological implications, placental site trophoblastic tumor may provide an important model for studying the sex chromosome biology and the proliferative advantage conferred by the paternal X chromosome.     

Expression of metalloprotease insulin-degrading enzyme insulysin in normal and malignant human tissues

Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3.4.22.11), a thiol metalloendopeptidase, is involved in intracellular degradation of insulin, thereby inhibiting its translocation and accumulation to the nucleus. Recently, protein expression of IDE has been demonstrated in the epithelial ducts of normal breast and breast cancer tissue. Utilizing four different antibodies generated against different epitopes of the IDE molecule, we performed Western blot analysis and immunohistochemical staining on several normal human tissues, on a plethora of tumor cell lines of different tissue origin, and on malignant breast and ovarian tissue. Applying the four IDE-directed antibodies, we demonstrated IDE expression at the protein level, by means of immunoblotting and immunocytochemistry, in each of the tumor cell lines analyzed. Insulin-degrading enzyme protein expression was found in normal tissues of the kidney, liver, lung, brain, breast and skeletal muscle, as well as in breast and ovarian cancer tissues. Immunohistochemical visualization of IDE indicated cytoplasmic localization of IDE in each of the cell lines and tissues assessed. In conclusion, we performed for the first time a wide-ranging survey on IDE protein expression in normal and malignant tissues and cells thus extending our knowledge on the cellular and tissue distribution of IDE, an enzyme which to date has mainly been studied in connection with Alzheimer's disease and diabetes but not in cancer.     

Functional analysis of leucine aminopeptidase in Caenorhabditis elegans

To investigate the function of the enzyme leucine aminopeptidase in nematodes, a Caenorhabditis elegans leucine aminopeptidase gene identified in the genome sequence was functionally analysed by transfection of a leucine aminopeptidase beta-galactosidase reporter construct and characterisation of a null mutant. The leucine aminopeptidase transgene is expressed along the length of the gut, and immunolocalisation shows the enzyme in the buccal cavity, pharynx, anterior gut and rectum. It is constitutively expressed as seen by analysis of cDNAs constructed from mRNAs of nematodes taken at 2 h intervals through the life-cycle; and by western blot analysis of protein from the same set of nematodes. Leucine aminopeptidase null mutants had a slower growth rate and delayed onset of egg-laying. We suggest that in C. elegans, leucine aminopeptidase is a digestive enzyme.     

Comparative cytochemical study of dipeptidyl aminopeptidase (DAP) II and IV in normal and malignant haemic cells.

Modified cytochemical methods were used to show dipeptidyl aminopeptidases (DAP) II and IV in peripheral blood buffy coat preparations and bone marrow smears. In 23 normal buffy coats both enzymes were confined to lymphocytes. DAP II was found in T and B lymphocytes (about 80%) while DAP IV was restricted to T lymphocytes only (around 46%). In 11 normal bone marrows DAP II was found in 53% of the lymphocytes, as well as in plasma cells, macrophages, and occasional myeloblasts. DAP IV was found only in lymphocytes (around 32%). DAP II activity, but not DAP IV activity, was present in all of the mast cells in a case of systemic mastocytosis. Whereas DAP II was found, to a variable extent, in leukaemic myeloblasts, monoblasts, proerythroblasts, and in megakaryoblasts in 52 cases of acute myeloid leukaemia, DAP IV was not shown. Variable positivity to DAP II and DAP IV was found in the lymphoblasts in seven cases of acute lymphoblastic leukaemia, in 14 cases of B chronic lymphocytic leukaemia, and in three cases of non-Hodgkin's lymphoma. DAP II activity was variable compared with DAP IV activity, which was constantly reduced. Virtually all of the myeloma cells (96%) all of the myeloma cells (96%) in five cases of multiple myeloma and two cases of plasma cell leukaemia were DAP II positive and DAP IV negative. In 10 cases of hairy cell leukaemia most hairy cells were positive to DAP II (74%) with no demonstrable DAP IV activity. In a single case of Sézary's syndrome around 90% of the helper T cells were positive to DAP II with no DAP IV activity.     

晚期正常妊娠和子痫前期胎盘中组织蛋白酶B的表达

目的探讨组织蛋白酶B(cathepsin B)在晚期妊娠胎盘绒毛的表达及在子痫前期发病中可能的作用。方法分别取正常足月胎盘36例和子痫前期胎盘30例,免疫组化方法检测胎盘绒毛cathepsin B的表达情况,同时观察胎盘组织的病理变化。结果 cathepsin B在晚期妊娠胎盘绒毛细胞滋养细胞的细胞质中有高表达。正常足月胎盘cathepsin B表达阴性(-)1例(2.7%),弱阳性(+)16例(43.2%),阳性(～)20例(54.1%);子痫前期胎盘cathepsin B表达阴性(-)5例(16.7%),弱阳性(+)16例(53.3%),阳性(～)9例(30.0%),cathepsin B在子痫前期胎盘阴性率表达较正常足月胎盘高,而阳性率表达较低,两组比较差异有显著性(P0.05);结论cathepsin B在晚期妊娠胎盘中仍然有高表达,cathepsin B在正常足月妊娠和子痫前期胎盘表达的差异提示cathepsin B不仅参与了正常妊娠的维持,cathepsin B表达降低导致的滋养细胞侵袭能力下降,可能与子痫前期的发病有关。     

Paternal X-chromosome inactivation in human trophoblastic cells

Dosage compensation for X-chromosome-linked genes between male and female mammals occurs by inactivation of one of the two X chromosomes in the female. In somatic cells, either the paternal or the maternal X chromosome is randomly inactivated in a given cell. In contrast, in the extra-embryonic tissues of mice, the paternally-derived X chromosome is preferentially inactivated. The evidence for paternal X-chromosome inactivation in humans is controversial and remains to be clarified. In this study, we have developed a sensitive polymerase chain reaction (PCR) technique to investigate the methylation pattern of the X-linked androgen receptor (AR) gene. The 5' CpG island of this gene is methylated on the inactive X chromosome and hypomethylated on the active X chromosome in somatic cells. The paternal and the maternal alleles of the AR gene may be distinguished by a polymorphism in the number of CAG triplet repeats within the CpG island. As a source of human extra-embryonic tissue, we used chorionic villus (CV) samples from female conceptuses of 10-12 weeks gestation. From a tiny branch of a CV sample, two distinct cell lineages, the trophoblastic and mesodermal lineages, were dissected apart by trypsin digestion and micromanipulation and DNA was extracted separately from these purified tissues. Digestion of the DNA with the methylation-sensitive restriction enzyme, Hpall, followed by PCR amplification revealed that the paternal allele is preferentially methylated in trophoblastic cells, but not in mesodermal cells. These results strongly suggest that the paternal X chromosome is preferentially inactivated in the human extra-embryonic tissues early in development.