Germ cell expression of placental alkaline phosphatase in male pseudohermaphroditism

We have reviewed testes removed from 14 individuals with male pseudohermaphroditism (13 with androgen insensitivity and one with 17 alpha-hydroxylase deficiency) and have studied staining for placental alkaline phosphatase in germ cells. Placental alkaline phosphatase positivity was identified in fetal and premature neonatal controls but not in cryptorchid males or normal autopsy control material from boys older than 6 months. It was present in autopsy testes younger than 6 months and cases of androgen insensitivity in boys younger than 8 months, suggesting immaturity. It was also expressed in two patients with male pseudohermaphroditism aged 5 years and 14 years, both of whom had intra-tubular germ cell neoplasia by morphological criteria. Our study confirms the use of placental alkaline phosphatase as a marker of germ cell neoplasia in this specific group who are at high risk of malignancy.     

Heat stability of human placental alkaline phosphatase

Alkaline phosphatase prepared from human placentae shows greater resistance to heat inactivation than any other known alkaline phosphatase of human origin. In the presence of magnesium this enzyme may be heated at 70°C. for 30 minutes without loss of activity whereas other human alkaline phosphatases lose most of their activity on being heated at 56°C. for this period of time. This heat stability is seen in freshly prepared enzyme, in alcohol-fractionated and freeze-dried material, and in the sera of individuals into whom placental alkaline phosphatase has been infused. The clinical implications of our observations are briefly indicated.     

Kinetic aspects of human placental alkaline phosphatase enzyme membrane.

The crosslinking of alkaline phosphatase of human placenta with human serum albumin has been optimized. During the physico-chemical characterization of this immobilized biocatalyst, special attention was paid to attributes such as the irreversibility of the enzyme support bonding, the stability of the catalytic activity, and the effects of pH and temperature on this activity. Regarding stability, patterns of denaturation are proposed, to account for inactivation curves over time and under storage/operation conditions. These patterns, in some cases, indicate the existence of different populations of immobilized enzyme molecules, with a different degree of sensitivity to denaturation. The activity vs pH profiles are clearly modified by the immobilization process. This is because the pH of the free homogeneous solution, measurable with a pH-meter, differs from the real pH of the immediate microenvironment of the immobilized enzyme molecules due to the effects of proton accumulation in the microenvironment (in the reaction catalysed by alkaline phosphatase, protons are produced), to limitations to the free diffusion of H+ and to the possible partition effects of H+ due to polar interactions with residues or molecules of the enzyme membrane. In the experimental working conditions, the apparent optimum temperatures are centered at 40 degrees C, inactivation (thermal denaturation) occurring above this temperature. In the temperature range 10-40 degrees C, the kinetic control over the overall activity of the immobilized enzyme was observed, causing the Arrhenius profiles to be linear.     

Serum placental type alkaline phosphatase in cigarette smokers

By means of enzyme immunoassays based on two monoclonal antibodies with specificities for distinct forms of placental type alkaline phosphatase (Pl-ALP), together with L-leucine inhibition studies, it has been possible to distinguish the Nagao type carcinoplacental enzyme from other placental type alkaline phosphatases. This approach has shown that it is the Nagao type (placental like) enzyme which is detectable in small amounts in the plasma of healthy individuals, particularly cigarette smokers.     

An alternate origin for the placental isoenzyme of alkaline phosphatase.

By means of an electrofocusing technique, the serum specimens of pregnant women routinely revealed three bands of alkaline phosphatase (ALP) that were released by placental syncytiotrophoblast, germ cell (ovary), and fetal intestine at pl of 4.73, 4.65, and 4.55, respectively. When defining reference ranges for ostensibly healthy persons of all decades of age and investigating patients with elevated total ALP activity, we found the isoenzyme at pl 4.73 to be above the established reference range in some nonpregnant women and in men. Evidence suggests that T lymphocytes from whole blood or in vivo tissue were the origin of this pl 4.73 ALP band; the isoenzyme being released during cell lysis, in cellular turmoil from any cause, or when active cell division occurs. A designation of lymphocyte ALP is proposed for this ALP band that is observed in a broad group of diseases associated with abnormal lymphocyte function.     

Role of placental alkaline phosphatase in the interaction between human placental trophoblast and Trypanosoma cruzi.

Congenital Chagas disease, due to the intracellular parasite Trypanosoma cruzi, is associated with premature labor, miscarriage, and placentitis. Human enzyme placental alkaline phosphatase (PLAP) (EC 3.1.3.1.) is membrane-anchored through glycosylphosphatidylinositol (GPI). PLAP is present in plasma in late pregnancy, 36 to 40 weeks; there are lower levels in maternal Chagas disease. Infants born to such mothers may have congenital Chagas disease. Human placental villi (PV) were treated with phospholipase-C (PL-C) and then cultured with T. cruzi to determine the effect of the parasites on PLAP activity as an in vitro model. There is less PLAP activity after treatment by PL-C and during culture with T. cruzi. Pretreatment of PV with PL-C before culture with T. cruzi yielded essentially normal specific activity of PLAP and prevented or greatly reduced infective penetration of villi by parasites. The results are consistent with a pathogenetic role for placental alkaline phosphatase in congenital Chagas disease. Receptor activation of membrane attachment to PLAP may be a device used by T. cruzi to enable parasite invasion of human trophoblast.     

Determination of serum activity of placental alkaline phosphatase]

The authors have developed an enzymatic method for the measurement of serum placental alkaline phosphatase (PLAP) based on the hydrolysis of paranitrophenyl phosphate into para-nitrophenol. The specificity for the isoenzyme involved is achieved by means of its characteristics thermostability. After one hour incubation at 60 degrees C, PLAP still maintains its full activity, while other isoenzymes are completely inactivated. The sensitivity of the method was improved (less than 1 U/l) by optimizing parameters such as the volume of specimen, the nature of the buffer (2-methyl-amino-ethanol), the reaction time and the pH. Analysis of the method performances has revealed a lower detection limit of 0.12 U/l, a linearity from 0 to 50 U/l, a precision lower than 10% for most of the analytical range and a complete enzyme recovery. The reference range was estimated from 0 U/l to 0.33 U/l in a group of 125 non-smokers without any cancerous disease.     

Measurement of placental alkaline phosphatase activity in benign and malignant pleural effusions.

The usefulness of placental alkaline phosphatase (PLAP) as a diagnostic marker of malignancy was assessed in pleural fluid from 60 patients with effusions. Pleural fluid PLAP activities were measured by an enzyme linked immunoassay (ELISA) using the two monoclonal antibodies H17E2 and H317. Similar values were found in groups of patients with primary bronchial tumours (n = 12), secondary malignancies (n = 23), and "benign" conditions (n = 25). The highest values were found in a small subgroup of patients with metastatic ovarian carcinoma. However, the production of this enzyme by normal lung makes the measurement of PLAP in pleural fluid unhelpful as a diagnostic aid to distinguish "benign" from malignant effusions.     

Kinetic aspects of human placental alkaline phosphatase enzyme membrane

The crosslinking of alkaline phosphatase of human placenta with human serum albumin has been optimized. During the physico-chemical characterization of this immobilized biocatalyst, special attention was paid to attributes such as the irreversibility of the enzyme support bonding, the stability of the catalytic activity, and the effects of pH and temperature on this activity. Regarding stability, patterns of denaturation are proposed, to account for inactivation curves over time and under storage/operation conditions. These patterns, in some cases, indicate the existence of different populations of immobilized enzyme molecules, with a different degree of sensitivity to denaturation. The activity vs pH profiles are clearly modified by the immobilization process. This is because the pH of the free homogeneous solution, measurable with a pH-meter, differs from the real pH of the immediate microenvironment of the immobilized enzyme molecules due to the effects of proton accumulation in the microenvironment (in the reaction catalysed by alkaline phosphatase, protons are produced), to limitations to the free diffusion of H⁺ and to the possible partition effects of H⁺ due to polar interactions with residues or molecules of the enzyme membrane. In the experimental working conditions, the apparent optimum temperatures are centered at 40°C, inactivation (thermal denaturation) occurring above this temperature. In the temperature range 10-40°C, the kinetic control over the overall activity of the immobilized enzyme was observed, causing the Arrhenius profiles to be linear.     

Further studies on the genetics of placental alkaline phosphatase

1. Six common and nine rare electrophoretically distinct phenotypes of human placental alkaline phosphatase are described. 2. Their incidence has been determined in nearly 600 placentae derived from single births in an ‘ English’ population, and in placentae from more than 500 twin births. Smaller numbers from Negro and Indian populations have also been studied. 3. Strong evidence supporting the hypothesis that these phenotypes are determined by three common and six rare alleles at an autosomal locus is provided by: (a) The characteristics of the electrophoretic patterns observed in the different phenotypes. (b) The agreement of the observed numbers of the various phenotypes in the different populations studied with the numbers expected, assuming a Hardy-Weinberg equilibrium. (c) Detailed sib-pair analysis of the phenotypes in the pairs of placentae obtained from more than 400 dizygotic twins. These data also show that the placental phenotype is determined by the genotype of the foetus. 4. The results on the Negro population indicate that the gene frequencies are very different from those in the ‘English’ population. 5. The effect of neuraminidase on the electrophoretic mobility of placental alkaline phosphatase suggests that there may be eight available sialic acid residues per enzyme molecule. This appears to be the same for all the common phenotypes. 6. A model for the structure of placental alkaline phosphatase is suggested which will account for the triple-banded pattern characteristic of heterozygotes and also for the relative intensities of the three bands seen in different heterozygotes.     

Translocation of alkaline phosphatase during the activation of B cells

The association of alkaline phosphatase (ALPase) with the cytoskeleton in lymphoid cells was investigated. Extracting cells with non-ionic detergents such as Triton, we determined that ALPase is present in the cytoskeletal fraction in fully differentiated B lymphocytes, X63 myeloma cells and Sp2/O hybridoma cells. During the course of B-lymphocyte activation, the ALPase shifted from a soluble to a Triton-insoluble form. Changes in the phosphorylation of Triton-insoluble proteins with molecular weights of 120, 100, 90, 75, 34 and 31 kDa were detected, coinciding with the appearance of the ALPase in this fraction. The possible role of ALPase in the differentiation of B cells is discussed.     

人类胎盘碱性磷酸酶在脲变性时构象与活力的变化

目的：研究脲对人胎盘碱性磷酸酶构象与活力的影响。方法：用分光法测定不同浓度脲溶液中的紫外差光谱及活力;荧光法测其荧光光谱。结果：低浓度脲（〈２．０ｍｏｌ／Ｌ）,酶的差光谱在２６２ｎｍ出现负峰,荧光强度下降,发射峰位蓝移;脲浓度为０．５ｍｏｌ／Ｌ时,酶活力仍维持在原有水平,此后随脲浓度增加,酶活力下降,继续提高脲浓度。差光谱在２７６ｎｍ出现正峰;荧光强度回升,发射峰位红移;酶活力迅速下降,当脲浓度高     

Origin and structural evolution of the early proliferating oval cells in rat liver

We have analyzed the histological changes in rat liver after 2-acetylaminofluorene (AAF) administration. The data demonstrate that AAF-induced oval cells were preferentially generated by proliferation of the terminal biliary ductules that we suggest constitute the primary hepatic stem cell niche. The oval cells formed ductular structures, representing an extension of the canals of Hering. This histological organization provides continuous bile drainage of the hepatocytes and uninterrupted blood flow in the sinusoids. The oval cell ductules are surrounded by a continuous basement membrane that is intermittently disrupted by processes of stellate cells that form direct cell-cell contact with the oval cells. Although both AAF treatment and bile duct ligation results in proliferation of biliary epithelial cells, the mechanism(s) responsible for the proliferation of the biliary epithelium seems to differ in the two models. In contrast to the biliary proliferation stimulated by bile ligation, AAF-induced oval cell proliferation as well as the capacity of these cells to differentiate into hepatocytes, bile epithelial cells and possibly other cell lineages can be blocked by administration of dexamethasone.