Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium.

Members of the Wnt family of signaling molecules are important in cell specification and epithelial-mesenchymal interactions, and targeted gene deletion of Wnt-7a in mice results in complete absence of uterine glands and infertility. To assess potential roles of the Wnt family in human endometrium, an endocrine-responsive tissue, we investigated in the proliferative and secretory phases of the menstrual cycle, endometrial expression of several Wnt ligands (Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, and Wnt-8b), receptors [Frizzled (Fz)-6 and low-density lipoprotein receptor-related protein (LRP)-6], inhibitors [FrpHE and Dickkopf (Dkk)-1], and downstream effectors (Dishevelled-1, glycogen synthase kinase-3beta, and beta-catenin) by RT-PCR, real-time PCR and in situ hybridization. No significant menstrual cycle dependence of the Wnt ligands (except Wnt-3), receptors, or downstream effectors, was observed. Wnt-3 increased 4.7-fold in proliferative compared with secretory endometrium (P < 0.05). However, both inhibitors showed dramatic changes during the cycle, with 22.2-fold down-regulation (P < 0.05) of FrpHE and 234.3-fold up-regulation (P < 0.001) of Dkk-1 in the secretory, compared with the proliferative phase. In situ hybridization revealed cell-specific expression of different Wnt family genes in human endometrium. Wnt-7a was exclusively expressed in the luminal epithelium, and Fz-6 and beta-catenin were expressed in both epithelium and stroma, without any apparent change during the cycle. Both FrpHE and Dkk-1 expression were restricted to the stroma, during the proliferative and secretory phase, respectively. These unique expression patterns of Wnt family genes in different cell types of endometrium and the differential regulation of the inhibitors during the proliferative and secretory phase of the menstrual cycle strongly suggest functions for a Wnt signaling dialog between epithelial and stromal components in human endometrium. Also, they underscore the likely importance of this family during endometrial development, differentiation and implantation.

Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility.

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.

Global gene profiling in human endometrium during the window of implantation.

Implantation in humans is a complex process that is temporally and spatially restricted. Over the past decade, using a one-by-one approach, several genes and gene products that may participate in this process have been identified in secretory phase endometrium. Herein, we have investigated global gene expression during the window of implantation (peak E2 and progesterone levels) in well characterized human endometrial biopsies timed to the LH surge, compared with the late proliferative phase (peak E2 level) of the menstrual cycle. Tissues were processed for poly(A(+)) RNA and hybridization of chemically fragmented, biotinylated cRNAs on high density oligonucleotide microarrays, screening for 12,686 genes and expressed sequence tags. After data normalization, mean values were obtained for gene readouts and fold ratios were derived comparing genes up- and down-regulated in the window of implantation vs. the late proliferative phase. Nonparametric testing revealed 156 significantly (P < 0.05) up-regulated genes and 377 significantly down-regulated genes in the implantation window. Up-regulated genes included those for cholesterol trafficking and transport [apolipoprotein (Apo)E being the most induced gene, 100-fold], prostaglandin (PG) biosynthesis (PLA2) and action (PGE2 receptor), proteoglycan synthesis (glucuronyltransferase), secretory proteins [glycodelin, mammaglobin, Dickkopf-1 (Dkk-1, a Wnt inhibitor)], IGF binding protein (IGFBP), and TGF-beta superfamilies, signal transduction, extracellular matrix components (osteopontin, laminin), neurotransmitter synthesis (monoamine oxidase) and receptors (gamma aminobutyric acid A receptor pi subunit), numerous immune modulators, detoxification genes (metallothioneins), and genes involved in water and ion transport [e.g. Clostridia Perfringens Enterotoxin (CPE) 1 receptor (CPE1-R) and K(+) ion channel], among others. Down-regulated genes included intestinal trefoil factor (ITF) [the most repressed gene (50-fold)], matrilysin, members of the G protein-coupled receptor signaling pathway, frizzled-related protein (FrpHE, a Wnt antagonist), transcription factors, TGF-beta signaling pathway members, immune modulators (major histocompatibility complex class II subunits), and other cellular functions. Validation of select genes was conducted by Northern analysis and RT-PCR using RNA from endometrial biopsies obtained in the proliferative phase and the implantation window and by RT-PCR using RNA from cultured endometrial epithelial and stromal cells. These approaches confirmed up-regulation of genes corresponding to IGFBP-1, glycodelin, CPE1-R, Dkk-1, mammaglobin, and ApoD and down-regulation for PR membrane component 1, FrpHE, matrilysin, and ITF, as with the microarray data. Cultured endometrial epithelial cells were found to express mRNAs for glycodelin, CPE-1R, Dkk-1, the gamma aminobutyric acid A receptor pi subunit, mammaglobin, matrilysin, ITF and PR membrane component 1. The expression of IGFBP-1, CPE1-R, Dkk-1, and ApoD mRNAs increased upon decidualization of stromal cells in vitro with progesterone after E2 priming, whereas FrpHE decreased, consistent with the microarray results. Overall, the data demonstrate numerous genes and gene families not heretofore recognized in human endometrium or associated with the implantation process. Reassuringly, several gene products, known to be differentially expressed in the implantation window or in secretory endometrium, were verified, and the striking regulation of select secretory proteins, water and ion channels, signaling molecules, and immune modulators underscores the important roles of these systems in endometrial development and endometrial-embryonic interactions. In addition, the current study validates using high density oligonucleotide microarray technology to investigate global changes in gene expression in human endometrium.

Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology.

A prerequisite for implantation in humans is differentiation (decidualization) of stromal cells in the endometrium, believed to be stimulated by progesterone (P) and/or cAMP. In the current study, advances in microarray technology have allowed us to investigate genes differentially expressed in human endometrial stromal cells decidualized in vitro in response to P or cAMP, compared to nondecidualized cells. Endometrial stromal cells were isolated from endometrial biopsy tissue and cultured without steroid hormones, with 1 microM P (after E2 priming), or 1 mM 8-bromo-cAMP. Total RNA was isolated and reverse transcribed to synthesize 32P-labeled cDNA probes using primers corresponding to genes represented on the Clontech Human Atlas cDNA Expression Array. After hybridization, signals were quantified by phosphor imaging densitometry and were normalized to GAPDH and ubiquitin. Of the 588 genes screened, marked upregulation was observed of cytokines, growth factors, nuclear transcription factors, members of the cyclin family, and mediators of the cAMP signal transduction pathway. Additional mRNAs expressed unexpectedly and regulated by P and cAMP, include the insulin receptor, some neurotransmitter receptors, neuromodulators, the FSH receptor, inhibin/activin betaA subunit, inhibin alpha, and TNF-related apoptosis-inducing ligand (TRAIL). Expression of previously unrecognized genes regulated in decidualized human endometrial stromal cells suggests mechanisms not yet appreciated in the endometrium during decidualization. In addition, marked upregulation of cytokines, chemokines, growth factors, apoptosis modulators, and their receptors in decidualized stromal cells supports a major role for paracrine interactions between the stroma and other endogenous and transient cell populations within the endometrium and during early pregnancy.

Allelic variants of the follistatin gene in polycystic ovary syndrome.

In an earlier study of 37 candidate genes for polycystic ovary syndrome (PCOS), the strongest evidence for genetic linkage was found with the region of the follistatin gene. We have now carried out studies to detect variation in the follistatin gene and assess its relevance to PCOS. By sequencing the gene in 85 members of 19 families of PCOS patients, we found sequence variants at 17 sites. Of these, 16 sites have variants that are too rare to make a major contribution to susceptibility; the only common variant is a single base pair change in the last exon at a site that is not translated. In our sample of 249 families, the evidence for linkage between PCOS and this variant is weak. We also examined the expression of the follistatin gene; messenger RNA levels in cultured fibroblasts from PCOS and control women did not differ appreciably. We conclude that contributions to the etiology of PCOS from the follistatin gene, if any, are likely to be small.

Full- or half-cycle treatment of severe premenstrual syndrome with a serotonergic antidepressant.

Evidence that serotonergic antidepressants are effective for treating premenstrual syndrome (PMS) raises the question of whether dosing only in the symptomatic premenstrual phase is effective for this disorder. This preliminary randomized, double-blind study compared the responses to half-cycle or full-cycle dosing of sertraline in 31 patients who completed a preceding double-blind, short-term treatment trial. The subjects fulfilled criteria for severe PMS when they entered the preceding controlled trial. At the end of the short-term treatment trial, the double-blind was not broken; both improved and unimproved subjects were randomized in a double-blind fashion to receive either full-cycle or half-cycle sertraline in the 3-month extension study. Results showed that the total premenstrual scores from the Penn Daily Symptom Report (DSR) were lower in the half-cycle dosing group in each of the 3 treatment months but did not differ with statistical significance from the full-cycle dosing group. Further analysis of the 17 DSR items showed that mood swings, nervous tension, feeling out of control, and confusion were significantly lower (p < 0.05) at endpoint in the half-cycle dosing group. Overall, subjects who improved in prior treatment remained improved; approximately half the subjects who were unimproved at entry into the extension study improved, regardless of the dosing regimen. The results add support to other preliminary reports of efficacy of serotonergic antidepressants administered premenstrually and indicate the clinical importance of determining an optimal dose/benefit ratio of serotonergic antidepressants for PMS patients.

Presence of immunoreactive corticotropin-releasing hormone in human endometrium.

Immunoreactive CRH (IrCRH) is produced locally in experimentally induced and spontaneous inflammation. Where it exerts autocrine or paracrine proinflammatory effects. In addition, CRH is secreted by the human placenta, rat Leydig cells, and rat and human ovaries, where it may participate in the inflammatory processes of ovulation and luteolysis, and/or the regulation of steroidogenesis. Finally, CRH is secreted in vitro by cultured human epithelial and decidualized stromal endometrial cells. To investigate the presence of CRH in human endometrium in vivo, we examined this tissue immunohistochemically and by extraction/RIA using a polyclonal, highly specific antirat/human CRH antibody. Endometrial biopsies from 33 women, aged 23-43 yr (median age, 33.5 yr), were performed by linear endometrial curettage for diagnostic purposes at different stages of the cycle. Intense IrCRH staining was localized in the cytoplasm of cells of the endometrial glands in all samples examined. IrCRH was also found in endometrial stromal cells exhibiting decidual reaction and in local immune accessory cells. The mobility of the endometrial IrCRH molecule was similar to that of r/hCRH in reverse phase high pressure liquid chromatography. The presence of CRH in the endometrium, and more specifically in the glandular epithelium during the proliferative and secretory phases of the menstrual cycle together with its known proinflammatory properties, suggest that this neuropeptide might participate in the inflammatory-like phenomena of endometrial physiology, such as menstrual shedding, surface epithelium repair, and/or implantation of the blastocyst. The presence of CRH in decidualized stromal cells is in accordance with its previously reported production by in vitro decidualized cultured endometrial stromal cells as well as by the placental decidua.

Randomized trial of long-term diuretic therapy for infants with oxygen-dependent bronchopulmonary dysplasia.

STUDY OBJECTIVE: To determine whether long-term oral diuretic therapy would improve the pulmonary function of preterm infants with bronchopulmonary dysplasia. DESIGN: Randomized, double-blind, placebo-controlled study. SETTING: Level III intensive care nursery. INTERVENTION: We randomly selected 43 stable patients with oxygen-dependent bronchopulmonary dysplasia to receive either orally administered spironolactone and chlorothiazide or placebo. These drugs were continued until the patients no longer required supplemental oxygen. Both groups received furosemide as needed. MEASUREMENTS AND RESULTS: Each infant had pulmonary function tests at study entry, 4 weeks after study entry, 1 week and 8 weeks after being weaned to room air and off study drugs, and at 1 year of corrected age. Pulmonary function tests include dynamic pulmonary compliance, airway resistance, thoracic gas volume, and maximal expiratory flow at functional residual capacity; most of the infants had functional residual capacity measured. Between the first and second pulmonary function tests (while the infants were receiving diuretic or placebo), the infants in the diuretic group had a significant improvement in dynamic pulmonary compliance (46%; p < 0.001) and airway resistance (31%; p < 0.05); there were no changes in compliance or resistance in the placebo group. Although patients in both the diuretic and the placebo groups required progressively less supplemental oxygen, by 4 weeks after study entry the patients in the diuretic group needed less supplemental oxygen than did those in the placebo group (p < 0.01). There were no significant differences in results of serial pulmonary function tests in either group after discontinuation of diuretic therapy. Despite the significant differences in pulmonary function between the two groups, there was no significant difference between them in the total number of days that supplemental oxygen was required. Significantly more infantsin the placebo group received more than 10 doses of furosemide on an as-needed basis. CONCLUSIONS: Long-term diuretic therapy in stable infants with oxygen-dependent bronchopulmonary dysplasia, after extubation, improves their pulmonary function and decreases their fractional inspired oxygen requirement, but does not decrease the number of days that they require supplemental oxygen. The improvement in pulmonary function associated with diuretic therapy is not maintained after treatment is discontinued.

Synaptic plasticity in the rat striatum following chronic haloperidol treatment.

Administration of the dopamine (DA) antagonist haloperidol leads to the development of behavioral hypersensitivity as well as enhanced neuronal growth when striatal extracts from these animals are incubated with mesencephalic cultures. For determining if alterations in neuronal growth also occur in vivo, the ultrastructure of the neuropil in the dorsolateral quadrant of the striatum from rats treated (24 days) with haloperidol (1.25 mg/kg) was examined by electron microscopy. Haloperidol-treated rats developed statistically significant behavioral hypersensitivity relative to vehicle-treated controls (p < 0.01). Evaluation of the neuropil revealed that haloperidol treatment enhanced, relative to vehicle-treated controls, the overall number of synaptic boutons by 9% (p < 0.01). The number of perforated synaptic profiles as well as the number of double synapses was increased by 20 and 50%, respectively, although this increase was not statistically significant. The number of myelinated axons remained unchanged, while the number of dendritic spines was increased by 21% (p < 0.05). These data suggest that chronic haloperidol treatment enhanced the growth and possible sprouting of presynaptic neurons and also induced postsynaptic plastic changes. These ultrastructural changes may contribute in part to hypersensitivity behaviors.

A radiographic method for estimating lung volumes in sick infants.

Estimation of lung volumes by conventional methods in sick infants is technically difficult and is the subject of controversy. In this study, we compared both thoracic gas volume (TGV), measured with an infant whole body plethysmograph, and functional residual capacity (FRC), determined by the nitrogen washout technique, to planimetric measurements of anteroposterior chest radiographs in 26 infants with bronchopulmonary dysplasia (BPD). The tidal volume (TV) of each patient was added to TGV and FRC because these were measured at the end of expiration whereas chest radiographs were taken at the end of spontaneous inspiration. The regression equations expressing the relationships between TGV and right+left lung field areas [A+B], and between FRC and lung areas are expressed as follows: [TGV+TV](mL) = 3.3 mL/cm2 x [A+B] cm2 + 24 mL and [FRC+TV](mL) = 3.5 mL/cm2 x [A+B] cm2 - 13.5 mL. Correlation coefficients of 0.9 and 0.7 for TGV and FRC, respectively, suggest a stronger correlation between TGV and lung areas than between FRC and lung areas. Lung areas measured by planimetry correlate closely with physiological measurement of lung volumes. We conclude that the planimetric method is an inexpensive and reliable technique for estimating lung volumes in young infants with BPD when chest radiographs are available.

E-cadherin expression during the differentiation of human trophoblasts.

The morphologic and functional differentiation of human trophoblast cells culminates in the formation of the terminally differentiated multinucleated syncytial trophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form syncytia, recapitulating the in vivo process. In the present studies, we investigated the expression of the Ca(2+)-dependent cell adhesion molecule (CAM), E-cadherin, during the morphologic differentiation of trophoblastic cells. Cytotrophoblasts were isolated from human chorionic villi, and JEG-3 and BeWo choriocarcinoma cells, cytotrophoblastic cell lines which under standard culture conditions are not fusion competent, were obtained by dispersion of ongoing cultures. Cultures were terminated at timed intervals and E-cadherin was analyzed by immunocytochemistry and electron microscopy using specific antibodies. In addition, E-cadherin expression was investigated by western and northern blotting. During the aggregation of cytotrophoblasts, E-cadherin was localized on the cell surface at points of cell-cell contact and could not be demonstrated following cellular fusion. In contrast, it remained on the surface of aggregated JEG-3 and BeWo cells throughout the duration of culture. Western blot analysis revealed a time-dependent increase in E-cadherin (120 x 10(3) Mr) which coincided with maximal aggregate formation at 24 h in both normal cytotrophoblasts and JEG-3 cells. A marked reduction of E-cadherin in fusing cytotrophoblasts was subsequently observed as syncytial trophoblasts became the predominant cellular form in culture. In agreement with the immunohistochemical observations, there was no change in E-cadherin levels in the non-fusing JEG-3 cells. Northern blotting demonstrated a significant reduction in the 4.5 kb transcript in fusion-competent cells over the 96 h of culture. Exposure of the normally non-fusing BeWo cells to 1.5 mM 8-bromo cyclic AMP induced cellular fusion and syncytium formation. This process was accompanied by a disappearance of E-cadherin from the cell surface as assessed by immunocytochemistry and western blotting and a parallel reduction in the abundance of the E-cadherin mRNA. Immunoneutralization experiments using an antiserum directed against the extracellular domain of cadherins inhibited syncytium formation in normal trophoblasts compared to an antiserum against the E-cadherin cytoplasmic tail, which had no effect upon aggregation and fusion of these cells. We conclude that E-cadherin exists in a dynamic state in fusion-competent cytotrophoblasts and that down regulation of its gene expression coincides with cellular fusion. In addition, this process appears to be cyclic AMP-mediated in BeWo choriocarcinoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Dopamine distribution and behavioral alterations resulting from dopamine infusion into the brain of the lesioned rat.

In an effort to verify the "dopamine secretion hypothesis" as the mechanism responsible for the antiparkinsonian efficacy of adrenal medullary transplants into the brain, the effects of dopamine infusion into the brains of rats with unilateral substantia nigra lesions were examined. The apomorphine-induced rotation, characteristic of this animal model, was diminished after 7 days of continuous dopamine infusion (10 micrograms/hr) into the ipsilateral striatum, whereas intraventricular infusion was without effect. Chromatographic analysis of the dopamine distribution after 10 days of infusion into either region revealed that ipsilateral delivery of dopamine did not result in contralateral increases in dopamine content. Examination of the adjacent striatum following ipsilateral intraventricular delivery indicated that dopamine had only penetrated 1 mm. Even with intrastriatal delivery, there were still parts of the infused striatum which had below-normal levels of dopamine. The fact that striatal tissue presents a significant barrier to the penetration of dopamine is discussed in relation to adrenal medullary and fetal nigral transplants.

2-hydroxyestradiol enhanced progesterone production by porcine granulosa cells: dependence on de novo cholesterol synthesis and stimulation of cholesterol side-chain cleavage activity and cytochrome P450scc messenger ribonucleic acid levels.

2-Hydroxyestradiol (2-OH-E2) stimulates progestin secretion by granulosa cells, but the intracellular locus of the stimulatory effect has not been clarified. The objectives of the present studies were to 1) determine the role of de novo sterol synthesis in the effect of 2-OH-E2 on progestin biosynthesis, and 2) examine the effects of 2-OH-E2 on cholesterol side-chain cleavage (SCC) activity and the level of messenger RNA (mRNA) for P450scc. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase with lovastatin (5 micrograms/ml) or mevinolin (5 micrograms/ml) reduced FSH- and 2-OH-E2-stimulated (but not E2-stimulated) progesterone production. Mevalonate (20 mM) enhanced basal progesterone production and reversed the inhibitory effect of lovastatin but did not affect progesterone biosynthesis in the presence of 2-OH-E2. As an index of the activity of cholesterol SCC enzyme, granulosa cells were exposed to 25-hydroxycholesterol (10 micrograms/ml) for 24 h and progesterone secretion monitored. Conversion of 25-hydroxycholesterol into progesterone was stimulated 2- to 3-fold by maximally effective concentrations of 2-OH-E2, E2, and FSH. 2-OH-E2 and/or E2 further enhanced 25-hydroxycholesterol conversion in the presence of FSH, LH, and epinephrine. Aminoglutethimide, an inhibitor of SCC, reduced 2-OH-E2- and 2-OH-E2 plus FSH-stimulated progesterone production by 97% and 95%, respectively. 2-OH-E2 also increased basal (by 2 to 3-fold) and FSH-stimulated (to 3.5-fold of FSH-treated controls) levels of mRNA for cytochrome P450scc. Collectively, our studies support the hypothesis that 2-OH-E2-enhanced progesterone biosynthesis by porcine granulosa cells is dependent on de novo cholesterol synthesis and is associated with increased levels of the mRNA encoding cytochrome P-450scc, which leads to increases in basal and gonadotropin-induced SCC activity.

Clozapine fails to prevent the development of haloperidol-induced behavioral hypersensitivity in a cotreatment paradigm.

We have previously established that chronic cotreatments involving antimuscarinic agents and haloperidol attenuate the development of behavioral hypersensitivity without affecting dopamine receptor proliferation. The antipsychotic agent clozapine also has significant antimuscarinic activity and was coadministered with haloperidol in rats for 2 months to determine if it would similarly attenuate the development of hypersensitivity. Clozapine or chlorpromazine cotreatment, unlike thioridazine cotreatment, did not attenuate the development of haloperidol-induced behavioral hypersensitivity. Clozapine or thioridazine cotreatment also failed to prevent the development of haloperidol-induced D2 receptor proliferation, whereas chlorpromazine cotreatment enhanced D2 receptor proliferation relative to haloperidol-treated animals. Alterations in dopamine biochemistry in the striatum or nucleus accumbens could not explain this dissociation between behavioral hypersensitivity and dopamine receptor proliferation. It is therefore hypothesized that dopamine receptor proliferation is permissive for behavioral hypersensitivity and that factors in addition to alterations in dopamine function contribute to the expression of dopamine hypersensitivity states.

Cloning and sequence of cDNA for human placental cytokeratin 8. Regulation of the mRNA in trophoblastic cells by cAMP.

A 1735 bp cDNA for human placental cytokeratin 8 is described which encompasses the entire coding sequence as well as 33 and 250 base pairs of 5'- and 3'-untranslated region, respectively. The level of cytokeratin 8 mRNA in various fetal tissues and placentae of different gestational ages was determined as were the effects of 8-bromo-cAMP on cytokeratin 8 mRNA in primary cultures of cytotrophoblasts and JEG-3 choriocarcinoma cells. Cytokeratin 8 mRNA was abundant in fetal small intestine, placenta, pancreas, lung, liver, and kidney. Levels of cytokeratin 8 mRNA in placenta increased slightly during pregnancy. 8-Bromo-cAMP suppressed cytokeratin 8 mRNA in primary cultures of cytotrophoblasts, whereas the cAMP analog increased mRNA levels in JEG-3 cells, revealing differential regulation of this mRNA in normal and transformed trophoblastic cells.

Dopaminergic alterations in cotreatments attenuating haloperidol-induced hypersensitivity.

Chronic treatment of the laboratory rat with haloperidol results in an increased stereotypic behavioral response to subsequent dopamine agonist challenge. This behavioral hypersensitivity (BH) is thought to reflect an increase in DA receptor number following chronic pharmacologic denervation. Using a cotreatment strategy, we demonstrate here that a variety of agents can attenuate or prevent the development of BH when administered chronically with haloperidol. Cotreatment with lithium and amantadine prevented the changes in DA biochemistry as well as the proliferation of DA receptors normally associated with chronic haloperidol treatment. Cotreatment with thioridazine or scopolamine did alter the changes in DA biochemistry normally associated with haloperidol treatment, but failed to attenuate the DA receptor proliferation. Taken together, these data suggest that mechanisms in addition to DA biochemical and receptor changes participate in the development and subsequent expression of BH. DA receptor proliferation must, therefore, be considered permissive to the development of BH.

Effect of total parenteral nutrition with intravenous fat on lipids and high density lipoprotein heterogeneity in neonates.

Plasma lipid concentrations and high density lipoprotein (HDL) subclass distributions were evaluated in 22 newborn infants nourished with intravenous (iv)-fat. The majority of infants were premature with respiratory distress syndrome. Based on baseline (prior to iv-fat) HDL subclass profiles determined by gradient gel electrophoresis (GGE), infants fell into two classes, one with two or more pronounced peaks within the normal HDL spectrum (group I, 17 subjects) and the other with highly unusual HDL distribution (group II, five subjects). Total plasma cholesterol increased in both groups during low and high fat intravenous feeding. HDL-cholesterol, however, did not change with iv-fat where mean values for groups I and II at baseline, iv-low fat and -high fat were: group I, 31.2 +/- 7.1, 30.0 +/- 8.8, and 36.6 +/- 16.7 mg/dl, respectively; and group II, 20.0 +/- 7.8, 20.2 +/- 7.4, and 19.8 +/- 8.8 mg/dl, respectively. Unlike HDL-cholesterol levels that remained constant with iv-fat, apolipoprotein (apo) AI concentrations increased significantly: group I, 73.0 +/- 11.0, 88.3 +/- 15.9, and 93.1 +/- 21.9 mg/dl, respectively; and group II, 31.8 +/- 10.5, 41.0 +/- 12.8, and 59.3 +/- 18.5 mg/dl, respectively. In group I infants, iv-fat is associated with an increase in larger-sized particles, particularly in the (HDL2b)gge range; in group II there is an increase in (HDL3b)gge and (HDL3c)gge components and a disappearance of particles that fall outside of the size range of normal HDL. In both groups, enteral feeding is associated with a further normalization of HDL subclass distribution. The aberrant GGE profiles and very low apoAI levels of group II infants at baseline were associated with unusual HDL morphology determined by electron microscopy where discoidal structures were prominent. With iv-fat, discoidal particles decline in number while normal spherical structures increase. Prevalence of discoidal HDL at baseline was associated with low concentrations of lecithin:cholesterol acyltransferase (LCAT) (1.12 +/- 0.5 micrograms/ml); with iv-fat this enzyme rose to 1.61 +/- 0.18 micrograms/ml. Increased LCAT is associated with the normalization of HDL morphology. It is likely that iv-fat improves the nutritional status of premature infants, thereby stimulating increased liver synthesis of important proteins, including apoAI and LCAT, associated with HDL metabolism.