Infant colic and feeding difficulties.

AIMS: To examine the relation between colic and feeding difficulties and their impact on parental functioning for a primarily clinic referred sample. METHODS: Forty three infants (and their mothers) were enrolled between 6 and 8 weeks of age. Infants were divided into two groups, colic (n = 19) and comparison (n = 24), based on a modified Wessel rule of three criteria for colic. Families were assessed at two visits; one occurred in the laboratory and one occurred in a paediatric radiology office. Outcome measures included the clinical assessment of infant oral motor skills, behavioural observation of mother-infant feeding interactions, maternal questionnaires on infant crying, sleeping and feeding behaviours, and the occurrence of gastro-oesophageal reflux (GOR) in the infants using abdominal ultrasound. RESULTS: Infants in the colic group displayed more difficulties with feeding; including disorganised feeding behaviours, less rhythmic nutritive and non-nutritive sucking, more discomfort following feedings, and lower responsiveness during feeding interactions. Infants in the colic group also had more evidence of GOR based on the number of reflux episodes on abdominal ultrasound as well as maternal report of reflux. Mothers in the colic group reported higher levels of parenting stress. CONCLUSIONS: Results provide the first systematic evidence of feeding problems in a subgroup of infants with colic. Data also illustrate the impact of these difficulties on parental and infant functioning. The association between feeding difficulties and colic suggests the potential for ongoing regulatory problems in infants presenting with clinically significant colic symptoms.

Evidence for estrogenic contamination of the MAPK inhibitor PD98059.

PD98059 blocks phosphorylation and activation of MAPK proteins, ERK1 and ERK2. In the course of examining the effect of PD98059 on estrogen-induced transcription of reporter genes in a human breast cancer cell line and in yeast, we found that two of four different batches of PD98059 produced estrogenic effects in a dose-dependent manner. In a competitive binding assay, these preparations of PD98059 displaced radiolabeled estradiol from ER alpha. Furthermore, in the yeast assay, addition of a coactivator protein, AIB1, enhanced the transcriptional effect of PD98059, indicating that it induces receptor-coactivator interactions. Although concentrations of PD98059 required to activate ER alpha in these experimental systems are 10(4)- to 10(5) higher than the concentration of estradiol required to do the same, the concentrations required to block MAPK activation are well above those which would produce maximal estrogenic effects. Thus, when PD98059 is used in estrogen-responsive cells, contaminating estrogenic activity may confound interpretation of experimental results.

Expression and subcellular localization of the cyclin-dependent kinase inhibitor p27(Kip1) in epithelial ovarian cancer.

OBJECTIVE: To determine if p27(Kip1) expression was altered in epithelial ovarian cancers as compared to normal ovarian surface epithelial (NOSE) cells and to determine if subcellular localization of p27(Kip1) was an important feature. METHODS: Thirteen tumor samples (1 Stage IC [early] and 12 Stage III/IV [advanced]) from patients with epithelial ovarian cancer and five NOSE samples were evaluated. Samples were surgically dissected to obtain an enriched population (90%) of cancer cells. The level of p27(Kip1) protein expression was determined by Western blot analysis. Actin was used as a loading control, and results were quantified by scanning densitometry using the ratio of the p27(Kip1) signal to the actin signal for comparison. To evaluate the subcellular localization of p27(Kip1), immunocytochemical staining was performed. Clinical pathological parameters were correlated to nuclear p27(Kip1) staining to establish if any association existed. RESULTS: When comparing the expression of p27(Kip1) between NOSE and ovarian cancer samples, only 2 of 13 ovarian cancer samples had altered p27(Kip1) expression. No correlation was found between the expression level of p27(Kip1) on Western blot and clinical pathological correlates. While no correlation between expression level of p27(Kip1) and subcellular localization was found, decreased nuclear staining (1+) was associated with shorter survivals using the log-rank test (P < 0.001). More importantly, in all tumor samples examined under the microscope, no nuclear p27(Kip1) staining was noted in cells that were undergoing mitosis. CONCLUSIONS: p27(Kip1) protein degradation may not be modified in ovarian cancer cells undergoing mitosis. Altered expression of p27(Kip1) is not an overwhelming feature in certain epithelial ovarian cancers. Decreased nuclear staining of p27(Kip1) is associated with poor survival in some epithelial ovarian cancers.

DNA methylation and ovarian cancer. I. Analysis of CpG island hypermethylation in human ovarian cancer using differential methylation hybridization.

OBJECTIVE: The aim of this study was to examine CpG island methylation patterns in ovarian cancer and determine whether epigenetic information can be related to clinical data of patients. CpG island (CpGI) hypermethylation is commonly associated with cancer progression, but little is currently known about the role of methylation in ovarian cancer. METHODS: Differential methylation hybridization (DMH) analysis at 742 loci was performed to determine methylation signatures for 20 primary epithelial ovarian carcinomas (Stages II, III, and IV adenocarcinomas, serous papillary), 6 ovarian cancer cell lines, and normal ovarian surface epithelial cells. RESULTS: Between 23 and 108 methylated CpGIs were seen in the ovarian carcinomas. Fewer (P < 0.05) methylated CpGIs were observed in the ovarian cancer cell lines; however, a number of CpGIs were commonly hypermethylated in both the cell lines and the tumor samples. A methylation signature, consisting of frequently (P < 0.05) methylated CpGIs, was determined for the samples. The observed pattern of methylation in ovarian cancers included several (11) CpGI tags that were previously reported to be hypermethylated in human breast cancer. CONCLUSIONS: Epigenetic signatures in ovarian cancer were determined using DMH. This proof-of-concept study lays the foundation for genome-wide screening of methylation to examine epigenotype-phenotype relationships in ovarian cancer.

DNA methylation in ovarian cancer. II. Expression of DNA methyltransferases in ovarian cancer cell lines and normal ovarian epithelial cells.

OBJECTIVE: The aim of this study was to investigate whether expression of the enzymes that catalyze cytosine CpG island methylation, DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b is altered in human ovarian cancer. Aberrations in DNA methylation are common in cancer and have important roles in tumor initiation and progression. Tumors that display frequent and concurrent inactivation of multiple genes by methylation are designated as having a CpG Island methylator phenotype, or CIMP. To date, colon, gastric, and most recently ovarian cancers meet the CIMP criteria for cancer. We hypothesized that altered expression of DNA methyltransferases can result in hypermethylation events seen in CIMP cancers. METHODS: DNMT1, DNMT3a, and DNMT3b mRNA levels in eight ovarian cancer cells lines (Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5) were compared to DNMT expression in normal ovarian surface epithelial cells using semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: In HeyA8 and HeyC2 ovarian cancer cells, DNMT1 expression levels were up to threefold higher (P < 0.05) than in normal ovarian surface epithelial cells. SK-OV-3 and PA-1 displayed increased DNMT3b expression (P < 0.05) compared to normal ovarian surface epithelial cells. Transcript levels for DNMT3a, however, were similar in cancer and normal ovarian cells. CONCLUSIONS: We observed differential expression of the DNMT genes in some ovarian cancer cell lines and conclude that alterations in DNMT expression might contribute to the CIMP phenotype in ovarian cancer. However, based on the lack of aberrant DNMT expression in some of the cancer cell lines examined, we further suggest that another mechanism(s), in addition to DNMT overexpression, accounts for methylation anomalies commonly observed in ovarian cancer.

Effects of the xenoestrogen bisphenol A on expression of vascular endothelial growth factor (VEGF) in the rat.

Bisphenol-A (BPA) is used to produce polymers for production of polycarbonate and epoxy resins that are used in food containers and dental appliances. BPA binds to estrogen receptors and induces estrogenic activity in a number of biological systems. We recently reported that although Fisher 344 (F344) and Sprague-Dawley (S-D) rat strains exhibit different sensitivities to BPA at the level of vaginal epithelial cell proliferation, there was no difference in immediate early proto-oncogene expression between the two animal strains. In the present study we investigated the effects of BPA on expression of another estrogen-target gene, vascular endothelial growth factor (VEGF), in the uterus, vagina, and pituitary of F344 and S-D rats. Adult rats were ovariectomized and treated with BPA by intraperitoneal injection at concentrations of 0.02 to 150 mg/kg body wt. Expression of VEGF was monitored by RNase protection assay at 2 hr after treatment. There was a significant effect of dose of BPA on the type of VEGF isoform expressed in the uterus, vagina, and pituitary. BPA induced greater (P < 0.01) levels of VEGF164 and VEGF120+188 than VEGF110 levels. The lowest BPA dose that had a significant (P< 0.05) effect on VEGF expression compared with vehicle treatment was 37.5 mg/kg body wt.; dose-response curves did not differ between strains. This is the first report that the primary response of the uterus, vagina, and pituitary to BPA includes rapid induction of VEGF expression. Due to the capacity of VEGF to engage pleiotropic signaling pathways in other cellular systems, we suggest that modulation of VEFG may play a role in establishing the response of estrogen-target organs to estrogenic xenobiotics.

Estrogen receptor beta in the sheep ovary during the estrous cycle and early pregnancy.

Objectives were to sequence and examine the expression of the estrogen receptor beta (ERbeta) in the sheep ovary. The sequence of the ovine ERbeta (oERbeta) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERbeta contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERbeta. In addition, an oERbeta isoform having a 139-base pair deletion (oERbeta1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERbeta protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERbeta protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERbeta was detected in the theca interna. Relative steady-state amounts of oERbeta mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERbeta mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERbeta to oERbeta1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERbeta1. Results indicate that the oERbeta is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERbeta might regulate estrogen actions during early CL development in sheep.

Understanding enantioselective processes: a laboratory rat model for alpha-hexachlorocyclohexane accumulation.

Since cyclodextrin gas chromatography columns became popular for chiral separations, many researchers have noticed high enantiomeric ratios [ER: (+)-enantiomer/(-)-enantiomer] for alpha-HCH in the brains of wildlife. This investigation used the laboratory rat as a model for these phenomena. Rats were either pretreated with phenobarbital (PB) or left untreated and then dosed with alpha-HCH. Animals were sacrificed after 1 or 24 h. The ER averaged 0.95 +/- 0.01 in blood, 1.29 +/- 0.02 in fat, and 0.77 +/- 0.004 in liver. ERs in brain ranged from 2.8 +/- 0.5 to 13.5 +/- 0.4. Both the tissue concentration distribution and the ERs agree well with those previously reported in wildlife. To determine whether high brain ERs were due to enantioselective metabolism or transport through the blood-brain barrier, alpha-HCH exposed brain and liver tissue slices were compared. Concentrations in the brain slices did not decrease with PB pretreatment but did decrease in the liver slices. Enantiomeric ratios in the brain slices averaged 1.11 +/- 0.02 and were 0.76 +/- 0.03 in liver slices for the PB pretreated rats. These data indicate that the enantioselective metabolism of alpha-HCH by the brain is not the mechanism responsible for high ERs in this tissue.

Expression of protein kinase C isozymes in nonpregnant and pregnant human myometrium.

OBJECTIVE: The aim of this study was to compare the distributions of protein kinase C isozymes in human nonpregnant and pregnant myometrial tissues and primary cell cultures. STUDY DESIGN: Myometrial tissues were obtained at hysterectomy from nonpregnant women and at cesarean delivery from women both before and during early labor at term. Western immunoblot analysis was performed on homogenates of myometrial tissues and primary cell cultures with monoclonal antibodies specific for protein kinase C isozymes. Redistribution and translocation of protein kinase C were examined by means of immunocytochemical methods. RESULTS: Nonpregnant myometrial tissues contained protein kinase C isozymes alpha, gamma, delta, mu, iota, and zeta but not beta(1), beta(2), theta, or epsilon. Pregnant myometrial tissues both before and during early labor contained the same protein kinase C isozymes and also beta(1) and beta(2). The protein kinase C isozyme distribution in primary myometrial cell cultures was identical to that in the myometrial tissues. Protein kinase C redistribution and translocation were demonstrated in these cultured myometrial cells. CONCLUSION: Both human myometrial tissues and primary cell cultures expressed a broad range of protein kinase C isozymes. Protein kinase C isozymes beta(1) and beta(2) were absent in nonpregnant myometrium but were induced during pregnancy. Labor at term did not alter protein kinase C isozyme expression.

Environmentally relevant xenoestrogen tissue concentrations correlated to biological responses in mice.

The effects of xenoestrogens have been extensively studied in rodents, generally under single, high-dose conditions. Using a continuous-release, low-dose system in ovariectomized mice, we correlated the estrogenic end points of uterine epithelial height (UEH) and vaginal epithelial thickness (VET) with concentrations of two organochlorine pesticide isomers in fat and blood. Silastic capsules containing a range of doses of either ss-hexachlorocyclohexane (ss-HCH) or o, p'-dichlorodiphenyltrichloroethane (o,p'-DDT) were implanted subcutaneously, and animals were killed after 1 week. Average blood levels achieved by the various doses were 4.2-620 ng/mL for o,p'-DDT and 5.0-300 ng/mL for ss-HCH. Fat concentrations of o,p'-DDT and ss-HCH correlated linearly to blood levels (o,p'-DDT, r(2) = 0.94; ss-HCH, r(2) = 0.83). Fat concentrations (nanograms per gram of tissue) were higher than blood concentrations (nanograms per milliliter) by 90 +/- 5- and 120 +/- 9-fold (mean +/- SE) for o, p'-DDT and ss-HCH, respectively. The VET ranged from 12 +/- 0.9 microm in controls to 114 +/- 8 microm in treated animals, and was correlated to blood levels of either treatment compound. The UEH ranged from an average of 7.7 +/- 0.3 microm in controls to 26 +/- 2 microm in high-dose o,p'-DDT-treated animals. The UEH was also correlated with ss-HCH concentration, but it plateaued at approximately 11 microm at the highest doses. The lowest blood concentrations that produced statistically significant increases in VET or UEH were 18 +/- 2 ng/mL o,p'-DDT and 42 +/- 4 ng/mL ss-HCH. These values are within the same order of magnitude of blood concentrations found in some human subjects from the general population, suggesting that human blood concentrations of these organochlorines may reach estrogenic levels.

Immunohistochemical detection of estrogen receptor alpha in male rat spinal cord during development.

The alpha subtype of the estrogen receptor (ERalpha) is present in nociceptive and parasympathetic regions of the adult rat spinal cord. The pattern of ERalpha expression in the rat spinal cord during development, however, is unknown. We used a polyclonal antibody (ER-21) to examine the expression of ERalpha in male rat lumbosacral spinal cords at embryonic day (E) 17, E21 (the day before birth), postnatal day (P) 1 (the day of birth), P8, P17, P21, and P36. At E17, ERalpha immunoreactivity (ERalpha-ir) was observed predominantly in ependymal cells. Perinatally, ERalpha-ir was also present in neurons in dorsal root ganglia and in fibers capping and within laminae I and II. By P8, ERalpha-ir was absent in ependymal cells, but ERalpha-ir fibers were dense in laminae I and II and in sympathetic and parasympathetic areas. ERalpha-ir was also present in neurons in the dorsal horns. To determine whether ERalpha-ir fibers in laminae I and II were processes of spinal neurons or primary afferents, dorsal rhizotomies were performed on P17 and P21 animals. Unilateral transection of the lumbosacral dorsal roots virtually eliminated ERalpha-ir fibers in the ipsilateral superficial laminae, demonstrating that the majority of ERalpha-ir fibers in these laminae were primary afferents. We show for the first time that ERalpha-ir is present in neurons and fibers of male prenatal and postnatal spinal cord. The presence of ERalpha in neuronal nuclei and processes may reflect diverse roles and novel mechanisms of action for 17 beta-estradiol in development of spinal sensory and autonomic circuitry.

Expression of estrogen receptor coactivators in the rat uterus.

Nuclear receptor coactivators associate in a ligand-dependent manner with estrogen receptors (ER) and other nuclear receptors, and they enhance ligand-dependent transcriptional activation. This study examined basal coactivator expression in rat uterus to investigate if expression of these genes is regulated by estradiol-17 beta or tamoxifen. Ovariectomized mature and immature rats were injected with estradiol-17 beta, tamoxifen, or vehicle (i.e., sesame oil) alone. Uteri were collected and analyzed for changes in coactivator mRNA expression using Northern blot and in situ hybridization analyses. Constitutive uterine mRNA expression of switch protein for antagonist (SPA), SRC-1, GRIP1, RAC3, RIP140, and p300 mRNAs was observed in control uteri, and treatment with ER ligands did not alter coactivator mRNA levels. The data suggest that expression of these coactivator genes is not sensitive to estradiol or tamoxifen in the rat uterus. No cell type-specific pattern of expression was apparent in uterine sections from mature and immature rats; however, silver grains were more abundant in luminal and glandular epithelial cells compared with the stroma and myometrium, indicating that coactivator mRNA levels vary among the uterine compartments. Thus, to our knowledge, we show for the first time that there is constitutive expression of several uterine nuclear receptor coactivators in a physiological setting that remains insensitive to estrogenic regulation. Furthermore, we speculate that higher constitutive levels of coactivator expression in glandular and luminal epithelial cells may be associated with increased hormonal responsiveness by these uterine compartments.

Strain differences in vaginal responses to the xenoestrogen bisphenol A.

Bisphenol A (BPA) is the monomer component of polycarbonate plastics and epoxy resins; human exposure derives from leachate in foodstuffs packaged in certain plastics or from epoxy-based dental appliances. BPA stimulates prolactin secretion in Fischer 344 (F344) rats but not in Sprague-Dawley (S-D) rats. The present studies were performed to determine if another classic estrogen target tissue, the rat vagina, responds to BPA in a strain-specific manner. In F344 rats BPA increased DNA synthesis in vaginal epithelium with a median effective dose (ED(50)) of 37.5 mg/kg body weight; DNA synthesis was not stimulated in S-D rats by any dose tested. Clearance of (3)H-BPA from blood followed the same time course in both strains of rats, with a half-life of 90 min. Scatchard analysis of [(3)H]estradiol binding showed no strain differences in concentration or affinity of the vaginal estrogen receptor. BPA increased the level of mRNA for the immediate early gene, c-fos, with similar dose-response curves in both rat strains. Thus, F344 and S-D rats exhibit differences in sensitivity to BPA at the level of cell proliferation in the vaginal epithelium. However, metabolic clearance of BPA and the early events that lead to the proliferative response, receptor-ligand interaction and induction of immediate early genes, show no strain differences. These observations suggest that differences in intermediate effects must account for the difference in sensitivity of the proliferative response to the xenoestrogen. Furthermore, these results point to the need for caution in choosing a suitable end point and animal model when seeking to test the estrogenic effects of xenobiotics.

Expression of fos and jun proto-oncogenes in benign versus malignant human uterine tissue.

OBJECTIVE: The objective of this study was to evaluate expression of fos and jun proto-oncogenes in benign human uterine tissue compared with malignant uterine tissue. METHODS: Forty-two endometrial tissue specimens were obtained at the time of hysterectomy. Tissue samples from different phases of the menstrual cycle and from postmenopausal patients were stained using immunohistochemical methods to detect Fos and Jun proteins, estrogen and progesterone receptor status, and Ki67 (detects a nuclear antigen associated with proliferating cells). Tissue was examined microscopically for nuclear staining in endometrial epithelium and stroma. The endometrium was based on the patient's last menstrual period, pathologic dating, and proliferative versus nonproliferative status as determined by Ki67. Benign and malignant specimens were subjected to Northern blot analysis to evaluate levels of expression of c-fos, c-jun, and jun-B mRNA. The pattern of c-fos mRNA expression in malignant samples was further evaluated using in situ hybridization. RESULTS: In proliferative, secretory, postmenopausal, and progesterone-influenced, uterine specimens immunohistochemically stained and examined, the endometrial and stromal nuclei stained for both Fos and Jun in varying intensities. However, no pattern was found in the variation of intensity according to the phase of the endometrium. Similarly, in malignant and benign endometrial tissue examined by Northern blot and in situ hybridization analyses, expression of proto-oncogene mRNAs was readily detectable, but no statistical correlation between type of tissue examined, grade of adenocarcinoma, and stage of endometrial cancer was found in this study. CONCLUSIONS: In rodent models, control of uterine cell proliferation is related to change in expression of fos and jun proto-oncogenes. Our results indicate that hormonal control is likely to be different in human endometrium and probably involves genes other than the proto-oncogenes under study. Expression of Fos and Jun do not correlate with endometrial cancer stage and grade.

Effect of estradiol on estrogen receptor expression in rat uterine cell types.

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.

Protective effects of estrogen in a rat model of age-related cataracts.

Women have a higher incidence of cataracts, and epidemiologic data suggest that the increased risk may be caused by a lack of estrogen in postmenopausal years. We have examined the effects of estrogen on methylnitrosourea (MNU)-induced cataractogenesis in Sprague-Dawley rats. Animals were ovariectomized, injected with MNU, and treated with estradiol or estrone by a continuous-release, subcutaneous Silastic implant, or they received an empty Silastic implant (no hormone). In the no-hormone group, rats developed opaque lenses approximately 6 months after MNU treatment. By 8 months, 74% (14/19) of the no-hormone rats had evident opacity in one or both eyes by simple gross inspection; 58% (22/38) of the eyes in this group were opaque. Estradiol or estrone treatment reduced the incidence of cataractous eyes to 12% or 25%, respectively. Lenses were examined under a dissecting microscope for light transmission. The lenses of the group treated with no hormone had light transmission of 26% +/- 9.2%, whereas lenses from the estradiol-treated animals had light transmission of 72% +/- 5.8%. Histological examination revealed that the anterior cortices of the opaque lenses were disrupted and showed the hallmark signs of age-related cataracts; in addition, some eyes that appeared clear by macroscopic examination showed the early histologic signs of cataractogenesis. It was demonstrated with reverse transcription-PCR that lens cells express both alpha and beta types of estrogen receptor, suggesting that the protective effects of the hormones may be a direct, receptor-mediated phenomenon. Thus, the MNU-treated, ovariectomized rat serves as a model for age-related cataractogenesis, and observation of a clear protective effect of estrogens in this system supports the implications of epidemiologic data.

Evaluating the effects of endocrine disruptors on endocrine function during development.

The major concerns with endocrine disruptors in the environment are based mostly on effects that have been observed on the developing embryo and fetus. The focus of the present manuscript is on disruption of three hormonal systems: estrogens, androgens, and thyroid hormones. These three hormonal systems have been well characterized with regard to their roles in normal development, and their actions during development are known to be perturbed by endocrine-disrupting chemicals. During development, organs are especially sensitive to low concentrations of the sex steroids and thyroid hormones. Changes induced by exposure to these hormones during development are often irreversible, in contrast with the reversible changes induced by transient hormone exposure in the adult. Although it is known that there are differences in embryonic/fetal/neonatal versus adult endocrine responses, minimal experimental information is available to aid in characterizing the risk of endocrine disruptors with regard to a number of issues. Issues discussed here include the hypothesis of greater sensitivity of embryos/fetuses to endocrine disruptors, irreversible consequences of exposure before maturation of homeostatic systems and during periods of genetic imprinting, and quantitative information related to the shape of the dose-response curve for specific developmental phenomena.

Transforming growth factor-beta differentially inhibits epithelial ovarian carcinoma cells from primary and metastatic isolates without up-regulation of p21WAF1.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is known to inhibit primary epithelial ovarian carcinoma cells. The mechanism by which this inhibitory response is achieved is poorly understood. Furthermore, whether this response is consistent in cells from metastatic sites compared with the primary site cells is unknown. The authors wanted to determine whether TGF-beta differentially inhibited ovarian carcinoma cells from primary tumor sites compared with metastatic sites and to establish whether this response was associated with up-regulation of p21WAF1 or overexpression of p53. METHODS: Tumor cells were purified from primary and metastatic sites in five patients with advanced epithelial ovarian carcinoma. TGF-beta effect at concentrations of 10, 1, and 0.1 ng/mL was determined by tritiated thymidine incorporation assay. Expression of p21WAF1 was determined by Northern and slot blot analysis. p53 was detected by immunocytochemistry. RESULTS: Metastatic tumor isolates were more responsive to the inhibitory effect of TGF-beta compared with their corresponding primary tumor isolates at 0.1 ng/mL. Increasing TGF-beta concentration conferred no additional inhibitory effect on the metastatic isolates; however, a dose-related phenomenon was observed in primary tumor isolates. p21WAF1 mRNA was up-regulated in only 2 of 10 primary and metastatic isolates. There was no correlation between TGF-beta responsiveness, p21WAF1 up-regulation, and p53 overexpression. CONCLUSIONS: Differential inhibition was observed between primary and metastatic tumor isolates. p21WAF1 up-regulation and p53 overexpression were not major modulators in TGF-beta regulation of primary and metastatic tumor growth in early passaged ovarian carcinoma cells.

The design of an optimized portable artificial kidney system using recirculation and regeneration of dialysate.

This paper is intended as an overview of the research carried out at Coventry University in the design of a portable artificial kidney system. It was seen that the key to the problem was the reduction in dialysate volume, and so it was decided to develop a prototype that would utilize the regeneration and recirculation of a small volume of dialysate. A prototype system has been produced and used to simulate a dialysis session. Activated carbon was used as a sorbent for the regeneration of the dialysate, circulating in a closed loop. For the purpose of this work, the adsorption of urea was investigated as this is, volumetrically, the major solute to be removed. Peltier effect cooling was used to vary the dialysate temperature down to 2 degrees C, as activated carbon will adsorb greater amounts of urea at lower temperatures. A series of tests was then carried out to investigate the effect of dialysate temperature, flowrate and volume on the amount of urea that could be dialysed. From the experimental results, a model of the system was derived, which made it possible to determine the implications of different operating conditions on the overall mass and size of a portable dialysis system. The output of this model was then used to establish a design specification and produce an optimum design solution for the system.

A comparative tribological study of the wear of composite cushion cups in a physiological hip joint simulator.

A composite cushion acetabular cup for a total hip replacement has been designed and developed jointly by Leeds University and DePuy International. In order to assess the long-term performance of this novel design, two sets of simulator tests of more than 4 million cycles duration have been carried out with the cushion bearings using the Leeds PA hip joint simulator with bovine serum as the lubricant. The results of these simulator tests were compared to the results from a previously reported study that used 32 mm ultrahigh molecular weight polyethylene (UHMWPE) acetabular cups. Under a physiological walking cycle simulation, with continuous cyclic motion and loading, the composite cushion cups produced negligible wear compared to a volumetric wear rate of 32 mm3 per million cycles for the conventional UHMWPE acetabular cups. This study has demonstrated for the first time the beneficial effects of fluid film lubrication in reducing wear in composite cushion acetabular cups.