A study of various properties of beta-galactocerebrosidase from human chorion using synthetic fluorescent substrates

The properties of beta-galactocerebrosidase from human chorionic villi, cultured chorionic villi and cultured skin fibroblasts were compared, using 6-hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranoside (HMGaL) as substrate. The effects of bile salt and Triton X-100 on beta-galactocerebrosidase were examined. It was shown that optimization of the HMGaL assay system requires the presence of pure sodium taurocholate and Triton X-100 at concentrations of 4.5 mM and 0.28 mM, respectively. The optimal pH value was found to be equal to 4.5-5.0; Km for the substrate was 0.03 mM. A comparison of beta-galactocerebrosidase from chorionic villi and cultured chorionic villi with the enzyme from skin fibroblasts revealed the similarity of some properties of these enzymes. The experimental results suggest that HMGaL can be used as a substrate for the identification of chorionic villi beta-galactocerebrosidase in an early prenatal diagnosis of Krabbe's disease.     

Branching enzyme activity of cultured amniocytes and chorionic villi: prenatal testing for type IV glycogen storage disease.

Although type IV glycogen storage disease (Andersen disease; McKusick 23250) is considered to be a rare, autosomally recessive disorder, of the more than 600 patients with glycogenosis identified in our laboratory by enzymatic assays, 6% have been shown to be deficient in the glycogen branching enzyme. Most of the 38 patients with type IV glycogen storage disease who are known to us have succumbed at a very early age, with the exception of one male teenager, an apparently healthy 7-year-old male, and several 5-year-old patients. Fourteen pregnancies at risk for branching enzyme deficiency have been monitored using cultured amniotic fluid cells, and four additional pregnancies have been screened using cultured chorionic villi. Essentially no branching enzyme activity was detectable in eight samples (amniocytes); activities within the control range were found in five samples (three amniocyte and two chorionic villi samples); and five samples appeared to have been derived from carriers. In two of the cases lacking branching enzyme activity, in which the pregnancies were terminated and fibroblasts were successfully cultured from the aborted fetuses, no branching enzyme activity was found. Another fetus, which was predicted by antenatal assay to be affected, was carried to term. Skin fibroblasts from this baby were deficient in branching enzyme. Pregnancies at risk for glycogen storage disease due to the deficiency of branching enzyme can be successfully monitored using either cultured chorionic villi or amniocytes.     

Methodological approaches to immunohistochemical demonstration of beta-hexosaminidase in human placental and renal tissue with monoclonal antibodies

The lysosomal enzyme, beta-hexosaminidase (Hex), was studied in full-term human placentas and in renal tissue using monoclonal antibodies raised against Hex purified from human placentas. The immunohistochemical reaction for Hex was pronounced in trophoblastic cells and macrophages of the basal plate and the smooth chorion, but was faint or negative in the amnion as well as in the syncytiotrophoblast and Hofbauer cells of the chorionic villi. The maternal decidual cells of the basal plate were negative. Biochemical enzyme analysis showed the highest activity in basal plate cells (containing trophoblast, decidual cells, macrophages and neutrophils) and a low activity in the chorionic villi. Placental tissue was less positive with monoclonal antibodies specific for Hex A, compared with antibodies reacting with both Hex A and Hex B. Epithelial cells of the renal proximal tubules were positive to the same degree with antibodies recognizing both Hex A and Hex B as well as those recognizing only Hex A.     

Estrogen synthetase (aromatase) in cultured human term placental cells and neoplastic human trophoblast

Estrogen synthetase (aromatase) is present in large amounts in human term placenta. However, the localization of aromatase within the cellular structure of the placental villus is obscure. By immunocytochemical techniques using antibodies that separately recognize each component of the aromatase cytochrome P-450 enzyme system, the fraction of term placental trophoblast cells in primary culture expressing each aromatase component antigen increased from 20% in fresh mononucleated cells to about 65% for multinucleated giant cells after 72 h. In contrast, about 80% of human choriocarcinoma cells in continuous culture (JAr line) expressed each aromatase component antigen. The fraction of trophoblast cells in primary culture containing human chorionic gonadotropin increased from about 14% in fresh mononucleated cells to about 45% after 72 h and was about 30% in the choriocarcinoma cells. Fibroblast cells in culture, derived from trypsin-treated placental villi, contained aromatase activity, albeit much lower than term placental trophoblast cells. Aromatase specific activity in these placental fibroblasts did not change following growth with dibutyryl cAMP plus theophylline for 72 h.     

Pyruvate carboxylase activity in chorionic villi: possibility of application to prenatal diagnosis

Pyruvate carboxylase (PC) activity was assayed in 27 chorionic villi samples (CVS) obtained at 9-12 weeks of gestation. The kinetic properties of the CVS enzyme were similar to those of liver PC; more than 75% of PC activity was recovered in the mitochondrial fraction of CVS. Apparent Km for pyruvate, ATP, acetyl CoA and HCO3- in the presence of saturation concentrations of the other reactants, were 0.3, 0.44, 0.015 and 6.0 mmol/l, respectively. The optimum pH was 7.5-8.0. The activity of PC in CVS was 3.2 +/- 0.3 nmol/min/mg protein, which is severalfold higher than that of amniotic fluid fibroblasts.     

Chorionic DNA analysis for the prenatal diagnosis of familial hypercholesterolaemia

Prenatal diagnosis for familial hypercholesterolaemia (FH) was performed by using restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene on chorionic villi DNA taken during the 10th week of pregnancy. Both parents were FH heterozygotes and had previously had a healthy son and an FH homozygous son. Two RFLPs were informative in this family and revealed that the fetus was unaffected by FH. At birth the child was found to have an LDL cholesterol level of 30 mg/dl and a normal LDL receptor activity in cultured umbilical cord fibroblasts. RFLP analysis on chorionic villi DNA is highly recommended for all heterozygous FH couples in whom the LDL receptor gene mutation/s is/are still to be characterized.     

First-trimester prenatal diagnosis of Tay-Sachs disease.

The prenatal diagnosis of Tay-Sachs disease was made in two at-risk fetuses by the analysis of chorionic villi obtained at 9 and 11 menstrual weeks, respectively. The diagnoses were based on the absence of beta-hexosaminidase A activity as determined by: (1) specific enzyme assays, (2) anion-exchange chromatography, and (3) cellulose acetate gel electrophoresis. The enzymatic diagnoses were confirmed on fetal tissue as well as by ultrastructural demonstration of the first-trimester fetal neuropathology. Optimal assay conditions for beta-hexosaminidase A in chorionic villi were established for the prenatal diagnosis of Tay-Sachs disease. Importantly, it was noted that a small amount of decidua or maternal blood could lead to misdiagnosis. Thus, extreme care must be taken in the preparation of chorionic villi for Tay-Sachs as well as other prenatal metabolic diagnoses.     

Rapid prenatal testing for human beta-glucuronidase deficiency (MPS VII)

Prenatal diagnosis for the lysosomal storage disorders is typically achieved by enzymatic analysis of the relevant lysosomal enzyme in cultured amniocytes or chorionic villi. While prenatal diagnosis of some genetic diseases can be done by analysis of pertinent metabolites in amniotic fluid, there are few data regarding prenatal diagnosis of lysosomal disorders by enzyme analysis of amniotic fluid. Prenatal diagnosis by enzyme analysis of amniotic fluid has the potential advantage of providing a more rapid prenatal test result. In this study we describe an assay for the prenatal diagnosis of the mucopolysaccharidosis beta-glucuronidase deficiency (MPS VII; MIM #253220) using amniotic fluid and we confirm its reliability in detecting an affected fetus in an at-risk pregnancy by enzyme analysis of cultured amniocytes and fetal fibroblasts. Because MPS VII is rare and few instances of prenatal diagnosis for this and nearly all other lysosomal disorders have been accomplished by enzyme analysis of amniotic fluid, confirmation of results obtained from enzyme analysis of amniotic fluid should be carried out by enzyme or mutation analysis using cultured amniocytes or chorionic villus specimens.     

Beta-mannosidase (EC 3.2.1.25) activity during and following germination of tomato (Lycopersicon esculentum Mill.) seeds. Purification,cloning and characterization

Beta-mannosidase, a high-salt-soluble enzyme, increases in activity in seeds of tomato prior to the completion of germination. This increase occurs in both the lateral and micropylar endosperm and becomes more evident during post-germinative seedling growth. The beta-mannosidase activity profile is similar to that of endo beta-mannanase although it is the first to increase in the lateral endosperm. Tomato seed beta-mannosidase was purified to homogeneity and its cDNA (LeMside1) obtained by 3'-RACE PCR using oligonucleotide sequences based on four peptide sequences obtained from the purified enzyme. The derived amino acid sequence of the tomato beta-mannosidase shows the enzyme is a member of the Glycosyl Hydrolases Family 1 (GHF1) but has a very low sequence identity with that of beta-mannosidases from non-plant sources; no other plant sequence for the enzyme is known. There appears to be only one gene encoding beta-mannosidase in tomato, the sequence of which has been determined (LeMSide2). Its expression occurs first in the micropylar endosperm, and then declines after germination. This is followed by an increase in its expression in the lateral endosperm, which precedes that of the gene for endo beta-mannanase. Expression of the beta-mannosidase gene increases appreciably in the growing seedling embryo. With this report, the cloning of all three of the enzymes involved in galactomannan mobilization (endo beta-mannanase, alpha-galactosidase and beta-mannosidase) in tomato seeds has now been achieved.     

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased (P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.     

Biochemical Characterization of a Lysosomal Protease Deficient in Classical Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) and Development of an Enzyme-Based Assay for Diagnosis and Exclusion of LINCL in HUman Specimens and Animal Models

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progressive and fatal neurodegenerative disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant sequence similarity to prokaryotic pepstatin-insensitive acid proteases. We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological samples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fibroblasts, and amniocytes). The enzyme has a pH optimum of 3.5 and is rapidly inactivated at neutral pH. A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease). A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indicates that the assay can distinguish homozygotes, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing. Analysis of archival specimens indicates that several specimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2. Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2. In addition, several animals with NCL-like neurodegenerative symptoms [mutant strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate. Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity.     

The relationship between beta-mannosidase and endo-beta-mannanase activities in tomato seeds during and following germination: a comparison of seed populations and individual seeds

beta-Mannosidase and endo-beta-mannanase are involved in the mobilization of the mannan-containing cell walls of the tomato seed endosperm. The activities of both enzymes increase in a similar temporal manner in the micropylar and lateral endosperm during and following germination. This increase in enzyme activities in the micropylar endosperm is not markedly reduced in seeds imbibed in abscisic acid although, in the lateral endosperm, endo-beta-mannanase activity is more suppressed by this inhibitor than is the activity of beta-mannosidase. Gibberellin-deficient (gib-1) mutants of tomato do not germinate unless imbibed in gibberellin; low beta-mannosidase activity, and no endo-beta-mannanase activity is present in seeds imbibed in water, but both enzymes increase strongly in activity in the seeds imbibed in the growth regulator. For production of full activity of both beta-mannosidase and endo-beta-mannanase in the endosperm, this tissue must be in contact with the embryo for at least the first 6 h of imbibition, which is indicative of a stimulus diffusing from the embryo to the endosperm during this time. These results suggest some correlation between the activities of beta-mannosidase and endo-beta-mannanase, particularly in the micropylar endosperm, in populations of tomato seeds imbibed in water, abscisic acid and gibberellin. However, when individual micropylar endosperm parts are used to examine the effect of the growth regulators and of imbibition in water on the production of the two enzymes, it is apparent that within these individual seed parts there may be large differences in the amount of enzyme activity present. Micropylar endosperms with high endo-beta-mannanase activity do not necessarily have high beta-mannosidase activity, and vice versa, which is indicative of a lack of co-ordination of the activities of these two enzymes within individuals of a population.     

A study of the heterogenous structure of guinea pig lysosomal beta-mannosidase using a polyclonal antibody

Lysosomal beta-mannosidase (EC 3.2.11.25) has a functional size of 120-150 kDa, but the enzyme purified from guinea pig liver (GPL) reportedly gave a single band corresponding to a molecular mass of 110 kDa. In order to investigate the subunit structure and tissue-specific expression of beta-mannosidase, we prepared a polyclonal antibody against GPL beta-mannosidase in rabbits which immunoprecipitated beta-mannosidase activity, free from other lysosomal hydrolase activity. Following storage at -20 degrees C and SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, a sample of purified GPL beta-mannosidase gave a major Coomassie blue staining band at 97 kDa. This was confirmed by Western blot analysis, which also revealed a faster moving 37 kDa protein. In contrast, Western blot analysis of fresh GPL homogenate prepared in the presence of proteinase inhibitors showed a major band at 150 kDa. Upon freezing and thawing, we observed immunoreactive bands at 120 and 20 kDa and finally, immunoreactive bands at 97, 37 and 20 kDa. The formation of the 97, 37 and 20 kDa forms from the 150 kDa species was accelerated by an n-butanol/ether extraction of the associated lipids, suggesting some tight hydrophobic association of these subunits. In contrast to liver, both fresh and freeze-thawed preparations of guinea pig kidney (GPK) yielded only the 97, 37 and 20 kDa subunit forms confirming that these are the major beta-mannosidase subunits. Endo-F treatment converted both the liver and kidney 97 kDa into a 91 kDa form and the 37 kDa form into a 35 kDa form, whereas the 20 kDa form was unaffected. Total beta-mannosidase activity, as measured with the synthetic substrate 4MU-beta-mannoside was unaffected by dissociation of the 150 form into the 97, 37 and 20 kDa subunits, suggesting that these are the functional forms of the enzyme rather than proteolytic degradation products.     

Implications of the placental structure compatibility for interspecies embryo transfer

Comparative histological features of the chorionic villi in placental cotyledons of the common eland (Taurotragus oryx ) and bongo (Boocercus euryceros ) antelopes and okapi (Okapia johnstoni ) and giraffe (Giraffa camelopardalis ) were examined. The chorionic villi in both antelope species showed only moderate branching and/or surface corrugation and their cross-sections were polygonal to oval. The close similarity in the structure of cotyledons has been apparently a contributing factor for success in mutual interspecies embryo transfer. The chorionic villi in okapi and giraffe had very different structures. In okapi the villi on cross-section were round and filled with thin connective tissue. They showed minimal branching and surface corrugation. In giraffe the villi showed extensive surface corrugation, had multiple fine branches, and were filled with a more dense connective tissue. Prospect for materno-fetal compatibility in mutual embryo transfer between these species is guarded.     

229例流产绒毛染色体核型分析及两种长期培养方法比较

目的:探讨早期自然流产绒毛染色体核型分析在自然流产病因检测中的应用价值,并比较两种长期培养方法的差别。方法:选择孕早期自然流产的孕妇229例,在无菌条件下,从宫腔内取出绒毛,同时或单独经胰酶消化法与切碎贴壁法进行细胞培养,传代之后常规进行G显带,在显微镜下做核型分析。结果:229例流产胎儿绒毛,培养成功206例,成功率为89.96%。异常核型105例,异常率为50.97%,数目异常者101例,占异常核型的96.19%,以16三体最为多见。胰酶消化法的培养成功率及收获时间都显著优于直接贴壁法,差异具有统计学意义(P0.05)。结论:自然流产绒毛染色体核型分析对流产查因具有实用价值。胰酶消化法较切碎贴壁法对流产绒毛长期培养及染色体核型分析更具实用性。     

Hydrolytic properties of a beta-mannosidase purified from Aspergillus niger.

A beta-mannosidase was purified to homogeneity from the culture filtrate of Aspergillus niger. A specific activity of 500 nkat mg-1 and a 53-fold purification was achieved using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The isolated enzyme has an isoelectric point of 5.0 and appears to be a dimer composed of two 135-kDa subunits. It is a glycoprotein and contains 17% N-linked carbohydrate by weight. Maximal activity was observed at pH 2.4 5.0 and at 70 degrees C. The beta-mannosidase hydrolyzed beta-1,4-linked manno-oligosaccharides of degree of polymerization (DP) 2-6 and also released mannose from polymeric ivory nut mannan and galactomannan. The Km and Vmax values for p-nitrophenyl-beta-D-mannopyranoside were 0.30 mM and 500 nkat mg-1, respectively. Hydrolysis of D-galactose substituted manno-oligosaccharides showed that the beta-mannosidase was able to cleave up to, but not beyond, a side group. An internal peptide sequence of 15 amino acids was highly similar to that of an Aspergillus aculeatus beta-mannosidase belonging to family 2 of glycosyl hydrolases.     

Features of the histotopography of proteins in human chorionic and placental tissues at different stages of normal and pathologic development

Contents of total, basic and acidic proteins have been studied in the human chorionic tunic at successive stages of a normal embryogenesis and at certain types of pathological pregnancy by means of histochemical methods. According to the staining degree, there is certain heterogeneity of the villi at various differentiation levels. An essential decrease in intensity of histochemical reactions is revealed in the chorionic villi at stillbirth and tetania gravidatum. The peculiarities in histotopography of the proteins and aminoacids studied reflect principle histochemical and structural reconstructions of the chorionic villi at various stages of normal and pathological pregnancy.     

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.