Immunoglobin-binding proteins in human placenta]

Phenomena of the binding of poor-soluble placenta proteins (PSPP) with pregnant women sera IgG as well as placenta blood IgG were studied. PSPP were extracted from the placenta tissue, washed out from soluble proteins, by the use of 3M KCl solution containing 0.005 M PMSF. PSPP were separated by the use of two-dimensional isoelectrofocusing and SDS-PAG electrophoresis and more than 30 different polypeptides were visualized. Having used various ELISA procedures with pregnant women sera IgG, placenta blood IgG as well as its Fab and Fc-fragments we have shown that both the receptor-type and the antigen-antibody-like interaction of PSPP took place. Both the polypeptide compositions and the isoelectrofocusing points ranges of the antigen-antibody-like interacting IgG-binding PSPP were determined by the use of the peroxidase conjugated Fab-fragments of the placenta blood IgG.     

Endotoxin-neutralizing antimicrobial proteins of the human placenta

Microbial colonization and infection of placental tissues often lead to adverse pregnancy outcomes such as preterm birth, a leading cause of neonatal morbidity and mortality. The fetal membranes of the placenta, a physical and active barrier to microbial invasion, encapsulate the fetus and secure its intrauterine environment. To examine the innate defense system of the human placenta, antimicrobial peptides were isolated from the fetal membranes of human placenta and characterized biochemically. Two salt-resistant antimicrobial host proteins were purified to homogeneity using heparin-affinity and reversed-phase HPLC. Characterization of these proteins revealed that they are identical to histones H2A and H2B. Histones H2A and H2B showed dose-dependent inhibition of the endotoxin activity of LPS and inhibited this activity by binding to and therefore blocking both the core and lipid A moieties of LPS. Consistent with a role for histones in the establishment of placental innate defense, histones H2A and H2B were highly expressed in the cytoplasm of syncytiotrophoblasts and amnion cells, where the histone proteins were localized mainly to the epithelial surface. Furthermore, culturing of amnion-derived WISH cells led to the constitutive release of histone H2B, and histones H2A and H2B contribute to bactericidal activity of amniotic fluid. Our studies suggest that histones H2A and H2B may endow the epithelium of the placenta with an antimicrobial and endotoxin-neutralizing barrier against microorganisms that invade this immune-privileged site.     

Thermodynamic effects of the hydrophobic surfactant proteins on the early adsorption of pulmonary surfactant

We determined the influence of the two hydrophobic proteins, SP-B and SP-C, on the thermodynamic barriers that limit adsorption of pulmonary surfactant to the air-water interface. We compared the temperature and concentration dependence of adsorption, measured by monitoring surface tension, between calf lung surfactant extract (CLSE) and the complete set of neutral and phospholipids (N&PL) without the proteins. Three stages generally characterized the various adsorption isotherms: an initial delay during which surface tension remained constant, a fall in surface tension at decreasing rates, and, for experiments that reached approximately 40 mN/m, a late acceleration of the fall in surface tension to approximately 25 mN/m. For the initial change in surface tension, the surfactant proteins accelerated adsorption for CLSE relative to N&PL by more than ten-fold, reducing the Gibbs free energy of transition (DeltaG(O)) from 119 to 112 kJ/mole. For the lipids alone in N&PL, the enthalpy of transition (DeltaH(O), 54 kJ/mole) and entropy (-T. DeltaS, 65 kJ/mole at 37 degrees C) made roughly equal contributions to DeltaG(O). The proteins in CLSE had little effect on -T. DeltaS(O) (68 kJ/mole), but lowered DeltaG(O) for CLSE by reducing DeltaH(O) (44 kJ/mole). Models of the detailed mechanisms by which the proteins facilitate adsorption must meet these thermodynamic constraints.     

The proteins of human lung surfactant

Human pulmonary surfactant was purified from bronchoalveolar lavage of patients. The proteins present in surfactant were analyzed by SDS-polyacrylamide gel electrophoresis into serum and non-serum components. One non-serum surfactant protein (Mr = 43 000) was then identified in the 100 000 X g supernatant of a lung homogenate on the basis of phospholipid binding. This lung protein was purified and partially characterized. The presence of 3-methyl histidine and reaction in Western blot analysis with antibody against chicken muscle actin both strongly suggested that the 43 000 Da protein of human surfactant is indeed cytoplasmic actin. It is proposed that this surfactant protein is involved in the secretion and not necessarily in the function of surfactant.     

Small GTP-binding proteins in the nuclei of human placenta.

Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [alpha(32)P]-GTP. Prior to this study the number of GTP-binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP-binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [alpha(32)P]-GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [alpha(32)P]-GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP-binding proteins. The GTP-binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP-binding proteins in the development and physiology of the placenta.     

Ultracytochemical localization of guanylate cyclase in early human placenta

Previous biochemical and cytochemical studies have indicated that in human term placenta the enzyme guanylate cyclase (GC) is associated mostly with the cytosolic fraction of homogenates and localized on the syncytiotrophoblast microvillous border. In the present study we have shown cytochemically the GC particulate form in early human placenta using guanylyl-imidodiphosphate [Gpp(NH)p] as substrate and NaN3 as activator. In samples of placental villi taken from the 6th to 12th week of pregnancy, the GC reaction product was always found on the apposing Langhans cytotrophoblast and syncytiotrophoblast plasma membranes. Furthermore, GC was present on cells in mitosis of the Langhans cytotrophoblast. From the 11th week GC was also visible on basal plasma membranes of Langhans cytotrophoblast and on endothelial cells of fetal capillaries. In samples of human term placenta GC was detectable on the syncytiotrophoblast microvillous border. This suggests a shift of enzyme localization during pregnancy.     

Small GTP‐binding proteins in the nuclei of human placenta

Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [α³²P]‐GTP. Prior to this study the number of GTP‐binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP‐binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [α³²P]‐GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [α³²P]‐GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP‐binding proteins. The GTP‐binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP‐binding proteins in the development and physiology of the placenta. J. Cell. Biochem. 84: 100–107, 2002. © 2001 Wiley‐Liss, Inc.     

Expression and localization of surfactant proteins in human nasal epithelium

Surfactant proteins (SPs), designated SP-A, SP-B, SP-C, and SP-D, play an important role in surfactant metabolism and host defense mechanisms in the lung. This study investigates expression of the different SP types in human nasal mucosa and cultured normal human nasal epithelial (NHNE) cells and whether the expression of SP mRNA is influenced by the degree of mucociliary differentiation. RT-PCR was performed with mRNA from cultured NHNE cells and nasal mucosa. Immunohistochemical staining for SPs was performed on nasal mucosa specimens. Western blot analysis was performed on cell lysates from cultured NHNE cells. SP-A2, SP-B, and SP-D mRNAs were expressed in normal NHNE cells and human nasal mucosa. SPs were localized in ciliated cells of the surface epithelium and serous acini of the submucosal glands. SP-A, SP-B, and SP-D proteins were expressed in cultured NHNE cells. The degree of mucociliary differentiation influenced expression of the SP gene. We demonstrate that SP-A, SP-B, and SP-D are expressed in human nasal mucosa and cultured NHNE cells. Further study of the functional role of SPs in the upper airway is required.     

Secretion of human chorionic gonadotrophin by the early placenta in Vitro.

The secretion in vitro of HCG and proteins was studied in fragments of placenta from women in the first trimester of pregnancy by a pulse-chase system. A 10-min pulse with [3H]leucine was used. It was concluded that the approximate half-time of release of HCG was 150 min. Proteins precipitable with trichloroacetic acid had a bi-exponential pattern, the half-times of release being 100 and 270 min. These rates of release indicate that the HCG produced by the early placenta was rapidly passed into the circulation rather than stored.     

In the absence of antibody IgGsorb precipitates human placenta phosphotyrosine-containing proteins

Lectin-purified human placenta plasma membrane proteins were phosphorylated in vitro. Mixing the reaction mixture with IgGsorb and incubation of the resultant pellet with p-nitrophenyl phosphate demonstrated the presence of phosphorylated-insulin receptor beta-subunit and a phosphorylated-180 kDa protein in acrylamide gel electrophoresis. The same two proteins were detected in the electrophoretic analyses of anti-phosphotyrosine immunoprecipitated phosphorylation reaction mixtures. In the absence of antibody, the amount of phosphorprotein in the IgGsorb pellet was dependent on the amount of IgGsorb added. IgGsorb did not precipitate 125I-labeled lectin-purified human placenta protein. Further, 10 mM O-phosphotyrosine completely inhibited the precipitation of phosphorylated human placenta proteins. These data suggest that IgGsorb specifically bound and precipitated phosphotyrosine-containing proteins in soluble human placenta plasma membranes.     

Lung surfactant in subacute pulmonary disease

Pulmonary surfactant is a surface active material composed of both lipids and proteins that is produced by alveolar type II pneumocytes. Abnormalities of surfactant in the immature lung or in the acutely inflamed mature lung are well described. However, in a variety of subacute diseases of the mature lung, abnormalities of lung surfactant may also be of importance. These diseases include chronic obstructive pulmonary disease, asthma, cystic fibrosis, interstitial lung disease, pneumonia, and alveolar proteinosis. Understanding of the mechanisms that disturb the lung surfactant system may lead to novel rational therapies for these diseases.     

细胞色素P450scc在人早期胎盘中的表达（简报）

人妊娠期间,胎盘合成大量的类固醇激素,与妊娠的启动、维持、分娩以及胎儿的发育均存在密切的关系。阐明胎盘类固醇激素特别是孕酮合成与分泌的调节机制对于寻找理想的生育调控技术和生殖保健方法具有重要的意义。因此,胎盘类固醇激素合成与分泌的调节向来是生殖生物学与妇产科学领域所关注的焦点问题之一,