Characterization of a nuclear protein conferring brefeldin A resistance in Schizosaccharomyces pombe.

The fungal metabolite brefeldin A disrupts protein secretion and causes the redistribution of the Golgi complex to the endoplasmic reticulum. Previously we isolated six genes that, when present in multiple copies, confer brefeldin A resistance to wild type Schizosaccharomyces pombe. Here we describe the characterization of one of these genes, hba1. This gene encodes an essential protein that shares homology with the mammalian protein RanBP1 and the protein encoded by the Saccharomyces cerevisiae gene YRB1 and contains a peptide motif present in several proteins found within the nuclear pore complex. The protein encoded by hba1 is localized to the nucleus, and it was determined that this protein is phosphorylated in vivo. The characterization of hba1 thus demonstrates a novel mechanism of drug resistance in S. pombe.

Ran-binding protein-1 is an essential component of the Ran/RCC1 molecular switch system in budding yeast.

We have performed a screen for genes that affect chromosome stability when overexpressed in the budding yeast Saccharomyces cerevisiae. Two of the genes recovered in the screen, CST17 and CST20, share a number of phenotypic properties, suggesting their involvement in the same cellular process. DNA sequence analysis of these genes revealed that they encode components of the Ran/RCC1 molecular switch system: CST17 is Ran itself (Ras-like nuclear protein) and CST20 is a novel yeast protein with a high degree of similarity to mammalian RanBP1, which is known to interact with Ran-GTP in vitro. We demonstrate that the CST20 protein can interact with Ran-GTP in vitro under similar conditions, indicating that it is the functional yeast homolog of mammalian RanBP1. The results of immunoprecipitation experiments show that the two yeast proteins form a complex in vivo. Deletion of the gene encoding RanBP1 revealed that it is essential for viability, as are Ran and RCC1. Similar phenotypic consequences of overproduction of either Ran or RanBP1 indicate that the latter protein is a functional component of the Ran/RCC1 molecular switch system, which is implicated in the control of a number of nuclear functions. Our finding that overproduction of two components of this system results in mitotic chromosome nondisjunction and sensitivity to an anti-microtubule drug benomyl suggest their involvement in mitosis as well. Thus RanBP1 is a functional component of a highly conserved molecular system that affects diverse cellular processes. The availability of this gene in S. cerevisiae provides a genetic system for the analysis of RanBP1 function in vivo.

Specific binding of Synechococcus sp. strain PCC 7942 proteins to the enhancer element of psbAII required for high-light-induced expression.

The psbAII gene of the cyanobacterium Synechococcus sp. strain PCC 7942 is a member of a three-gene family that encodes the D1 protein of the photosystem II reaction center. Transcription of psbAII is rapidly induced when the light intensity reaching the culture increases from 125 microE.m-2.s-1 (low light) to 750 microE.m-2.s-1 (high light). The DNA segment upstream of psbAII that corresponds to the untranslated leader of its major transcript has enhancer activity and confers high-light induction. We show that one or more soluble proteins from PCC 7942 specifically bind to this region of psbAII (designated the enhancer element). In vivo footprinting showed protein binding to the enhancer element in high-light-exposed cell samples but not in those maintained at low light, even though in vitro mobility shifts were detectable with extracts from low- or high-light-grown cells. When 12 bp were deleted from the psbAII enhancer element, protein binding was impaired and high-light induction of both transcriptional and translational psbAII-lacZ reporters was significantly reduced. This finding indicates that protein binding to this region is required for high-light induction of psbAII. The mutant element also showed impaired enhancer activity when combined with a heterologous promoter.

Epitopic heterogeneity of human 3 beta-hydroxysteroid dehydrogenase in villous and extravillous human trophoblast.

A new monoclonal antibody (FDO26G) is described which was raised against purified human 3 beta-hydroxysteroid dehydrogenase type I (3 beta-HSD type I). FDO26G reacted strongly with villous syncytiotrophoblast, weakly with some trophoblast cells in chorion laeve, and not at all with extravillous trophoblast in cytotrophoblast cell islands and decidual trophoblast. All these types of trophoblast reacted strongly with monoclonal antibody FDO161G, which has previously been shown to react with 3 beta-HSD type I and, like FDO26G, reacts strongly with adrenal cells. Mapping experiments using a combination of lacZ fusion polypeptides and synthetic peptides located the FDO26G epitope to residues 354-366 at the C-terminal end of the molecule, a sequence that is identical in the type I and type II forms of the enzyme. The epitope contains a consensus for a casein kinase-II site with serine 359 as the candidate phosphorylation site. This suggested that the lack of reactivity of FDO26G to 3 beta-HSD in extravillous trophoblast might be due to phosphorylation at serine 359. Peptide 354-366 was synthesized with phosphoserine at residue 359 and its binding to FDO26G was compared with that of the unphosphorylated peptide. FDO26G bound the phosphopeptide at least as strongly as the unphosphorylated peptide. It is concluded that the lack of staining of extravillous trophoblast by FDO26G is due to the presence of a different sequence at residues 354-366 and that a hitherto unidentified third isoform of human 3 beta-HSD is expressed in these cells.

A morphologic study of trophoblast isolated from peripheral blood of pregnant women.

OBJECTIVE: Our purpose was to evaluate the morphologic characteristics of cells separated from peripheral maternal blood with immunomagnetic beads coated with trophoblast-reactive monoclonal antibodies of restricted specificity. STUDY DESIGN: Blood samples were collected from 14 pregnant women at 9 to 12 or 16 to 18 weeks' gestation. Immunomagnetic beads coated with trophoblast-reactive monoclonal antibodies were used to isolate cells from the blood. The isolated cells were then fixed, embedded, and sectioned to enable morphologic identification. RESULTS: Multinucleate cells identical to syncytiotrophoblast sprouts were identified in 12 of 14 samples. CONCLUSION: Multinucleate syncytiotrophoblast can be isolated from the peripheral blood of normal pregnant women in both the first and second trimesters of pregnancy. The isolated trophoblast may allow prenatal genetic diagnosis by a noninvasive technique.

Nucleotide sequence of psbB from Synechococcus sp. strain PCC 7942.

The nucleotide sequence was determined for the Synechococcus sp. strain PCC 7942 psbB gene, which encodes the CP-47 protein of Photosystem II. The derived amino-acid sequence is highly conserved with those from other cyanobacterial and chloroplast psbB sequences. Transcript mapping experiments indicated two psbB transcription start sites in Synechococcus.

Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase.

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.

Isolation of fetal trophoblast cells from peripheral blood of pregnant women.

Fetal trophoblast cells were isolated from maternal peripheral blood by means of murine monoclonal antibodies of high specificity and affinity for human syncytiotrophoblast and nonvillous cytotrophoblast cells. The cells were isolated in sufficient numbers to allow polymerase chain reaction (PCR) amplification of the Y-chromosome-specific DNA sequence from the peripheral blood of thirteen pregnant women. The fetal sex predicted by PCR analysis of the isolated trophoblast cells accorded with that ascertained by karyotyping of chorionic villus samples in eleven of twelve women studied in early pregnancy and with the sex of the baby on delivery in one woman studied at 34 weeks' gestation. Isolation of these fetal cells could allow noninvasive diagnosis of a wide range of inherited disorders.

Phase I clinical trial of a World Health Organisation birth control vaccine.

A birth control vaccine incorporating a synthetic peptide antigen representing the aminoacid sequence 109-145 of the C-terminal region of the beta subunit of human chorionic gonadotropin (hCG-beta) was submitted to a phase 1 clinical trial. Thirty surgically sterilised female volunteers, divided into five equal groups for different vaccine doses, received two intramuscular injections six weeks apart. Over a six-month follow-up there were no important adverse reactions, and potentially contraceptive levels of antibodies to hCG developed in all subjects. In the highest vaccine dose group, the results gave promise of a contraceptive effect of six months' duration.

PIP: A Phase I clinical trial of the immunogenicity and safety of a vaccine against the C-terminal region of the beta subunit of human chorionic gonadotropin (hCG-B) demonstrated a dose-related immune response. The antigen was a synthetic peptide of the C 109-145 region of hCG-B, conjugated to diphtheria toxoid, and administered in a water-soluble synthetic adjuvant in a saline-oil emulsion. This vaccine had been previously tested for toxicity in laboratory animals and for immunogenicity, safety and contraceptive effectiveness in baboons. 30 previously sterilized women were given 2 injections 6 weeks apart, ranging from 50 to 1000 mcg of the antigen. Each woman tested free of HLA B27 antigen and reacted negative to the diphtheria toxoid skin test. Based on calculated contraceptive antibody binding level of 0.52 nmol/l, all subjects mounted an effective antibody response for at least 6 months. 2 subjects in the group given 1000 mcg who were followed for 9 and 10 months maintained this level of antibody. 12 women showed an anamnestic response to diphtheria toxoid, while 8 did not. The only adverse reactions were mild, transient pain at the injection site. Several women who received unstable adjuvant experienced more severe myalgia. Menstrual changes appeared in 5 subjects: early menopause in 1, spotting in 3 and menorrhagia in 1 woman. Only transient positive findings were seen in some sera screened for autoantibodies. This preliminary trial indicates that anti-hCG vaccine is a hopeful reversible contraceptive.

Corticosteroid binding globulin in normotensive and hypertensive human pregnancy.

Corticosteroid binding globulin (CBG) concentrations in maternal plasma have been measured throughout pregnancy in a series of 100 singleton pregnancies in 89 normotensive women. Plasma CBG concentrations were monitored also in 10 women with essential or renovascular hypertension. Plasma albumin, cortisol and oestriol were measured concurrently. Plasma CBG increased two and a half to three times during pregnancy. In those women who developed hypertension in pregnancy (mean arterial pressure greater than 107 mmHg), the plasma CBG concentrations were significantly lower than in those who remained normotensive. In women who developed hypertension, the CBG either failed to increase at the same rate as in normal pregnancies or the level fell before the appearance of hypertension. The earlier the onset of hypertension, the greater the decline in CBG. In all subjects, the CBG concentration at 34-36 weeks gestation was directly related to the birthweight of the infant. Plasma cortisol levels were depressed in hypertension relative to that in the normotensive women. Whilst plasma albumin levels decreased at least 30% in most women during pregnancy, the fall tended to be less in hypertensive women, but there was marked overlap between patient groups. Plasma oestriol concentrations were depressed only in the very severely affected cases. It is suggested the CBG concentration is a further reflection of the metabolic abnormalities associated with hypertension in pregnancy, and that it can be used as a marker to identify and monitor those patients at risk.

Identification of extra-villous trophoblast cells in human decidua using an apparently unique murine monoclonal antibody to trophoblast.

Murine monoclonal antibodies were raised to human first trimester trophoblast cells. Eleven antibodies reacted with first trimester trophoblast, tested by immunoperoxidase staining on frozen sections, but only one had apparent specificity for trophoblast after examining reactivities with a panel of other cells and tissues. This antibody, designated FD0161G, bound selectively to syncytiotrophoblast and non-villous trophoblast in first trimester and term placentae. Villous cytotrophoblast was negative. This was clearly demonstrated on freeze-dried, paraffin embedded tissue sections which have superior architecture to frozen sections. FD0161G reacted with extra-villous trophoblast cells in human decidua which are also delineated by a monoclonal anti-cytokeratin antibody. Unlike the latter, however, FD0161G did not react with decidual glands. Thus FD0161G could be used as a specific probe for extra-villous trophoblast in decidual tissue.

Cell surface antigens of human trophoblast: definition of an apparently unique system with a monoclonal antibody.

An epitope with apparent specificity for the surface of human syncytiotrophoblast was defined by a murine monoclonal antibody, FDO46B (IgG1, kappa). The epitope was predominantly expressed during the first trimester of pregnancy. Binding was detected on frozen tissue sections and on cultured trophoblast by the immunoperoxidase technique. It was also detected on the surface of a small percentage (less than 10%) of cultured choriocarcinoma cells (JEG-3). A panel of human tissues was negative, as were normal and malignant human lymphocytes. The antigen bearing the FDO46B epitope was still expressed by trophoblast after culture in the presence of tunicamycin, indicating that it is possibly protein in nature. This antigen may have potential utility as a target for a contraceptive vaccine.

Monoclonal antibody production by hybridoma growth in Freund's adjuvant primed mice.

Incomplete Freund's adjuvant was used as a priming agent prior to the injection of hybridoma cells in mice to expand monoclonal antibody production. Two hybridoma cell lines, FDO28B (IgG1) and FDO31C (IgM), which produce monoclonal antibodies reactive towards human placenta, were used. Monoclonal antibody was detected in the ascites fluids by agarose gel electrophoresis. It was found that the time interval between adjuvant priming and cell injection could be reduced to 1 day, allowing collection of ascites fluid containing monoclonal antibody within 2 weeks of priming. In addition, as few as 1 X 10(5) hybridoma cells were needed to collect approximately 5-7 ml of ascites fluid containing antibody detectable by gel electrophoresis. Thus priming with incomplete Freund's adjuvant enables production of large amounts of monoclonal antibody in a short time using a low number of hybridoma cells.

Identification of an SP-1-like protein in non-pregnancy serum: isolation using a monoclonal antibody.

Human SP-1, a glycoprotein synthesized by the placenta during pregnancy, was shown to exist as polymers in maternal serum by a rapid passive transfer immunoblotting technique following conventional agarose electrophoresis. Moreover, the SP-1 polymers in serum were shown to dissociate into one main component upon treatment with 8 M urea prior to electrophoresis. However, an unexpected observation was the existence of an SP-1-like immunoreactive species in male serum with the sensitive immunoblotting technique. This SP-1-like protein in male serum had similar properties to its placentally derived counterpart in pregnancy serum, namely a propensity for complex formation and a reduced electrophoretic mobility following neuraminidase treatment. The relationship between the two SP-1 proteins was demonstrated by isolating them from their respective sera using an immobilized monoclonal antibody raised to purified SP-1 from pregnancy serum. Immunoblotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the existence of two major SP-1 species in pregnancy serum. These two SP-1 species had apparent molecular weights of 68,000 and 64,000. In addition, there were two minor bands at 35,000 and 32,000. These smaller SP-1 species did not appear to be subunits of the larger entities since they were detectable in the absence of reducing conditions. SDS-PAGE and immunoblotting showed that the immunoaffinity-purified SP-1 species from male and pregnancy sera had similar, but not identical, molecular weights.

Polymerization of human transcortin in plasma.

Using radial immunodiffusion and antiserum raised against purified transcortin, polymers of native transcortin have been identified in plasma. They are characterized by multiple precipitin bands, the size of which diminishes on dilution, consistent with reversibility of this form of polymer. Spectrophotometric scans have been used to study the time course of polymerization in purified transcortin, in which dilution reversibility can be demonstrated also, and in which disaggregation may be produced by addition of sodium dodecyl sulphate and dithiothreitol, but apparently not by cortisol.

Purification and characterization of human transcortin.

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.

Enterohepatic circulation of prednisolone in rats.

In the present study human equivalent doses of the synthetic glucocorticoid, prednisolone, were administered intravenously into male rats. The prednisolone was rapidly cleared from blood and accumulated in the liver, from where it and metabolized prednisolone was excreted into the small intestine after a short time. By cannulation of the common bile duct with serial collection of bile it was shown that liver was able to handle relatively large doses of the steroid. There was good evidence of an enterohepatic circulation of steroid with relationships of time and content of prednisolone in liver and small intestine.