bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in Growth Hormone Secretory Dynamics in Adolescent Girls with Anorexia Nervosa and Effects on Bone Metabolism
Authors: Misra Madhusmita , Miller Karen K. , Bjornson Jennifer , Hackman Annie , Aggarwal Avichal , Chung Joyce , Ott Melissa , Herzog David B. , Johnson Michael L. , Klibanski Anne
Published: December 2003

Anorexia nervosa (AN) is a disorder that is increasing in frequency in adolescents, and the age of onset is often in the prepubertal years, potentially affecting the development of peak bone mass and linear growth. The GH-IGF-I axis plays an important role in bone formation, and alterations in GH secretory patterns have been described in adult women with AN. However, GH secretory dynamics in adolescents with AN have not been described, and the effects of alterations in GH secretory patterns and GH concentration on bone metabolism in AN are not known. We examined patterns of GH secretion by deconvolutional analysis, and GH concentration by Cluster analysis, in adolescent girls with AN (n = 22) and controls (n = 20) of comparable bone age and pubertal stage. We also examined the roles of cortisol, leptin, and estradiol in the regulation of GH secretion and concentration, and the relationship of GH secretory patterns and concentration to bone metabolism. Basal GH secretion and secretory pulse number in adolescent girls with AN were increased compared with control values (P = 0.03 and 0.007, respectively), and increased disorderliness of GH secretion (approximate entropy) was found in AN (P = 0.004). Mean and nadir GH concentrations and total area under the concentration curve were increased (P = 0.03, 0.002, and 0.03, respectively), and IGF-I levels were decreased (P = 0.0002) in girls with AN compared with healthy adolescent girls. IGF-I levels correlated negatively with nadir GH concentrations (r = −0.35; P = 0.02). Serum cortisol levels were higher in girls with AN than in controls (P < 0.0001) and correlated inversely with IGF-I (r = −0.58; P = 0.0001) and weakly with GH concentration (area under the concentration curve; r = −0.43; P = 0.05). A strong inverse relationship between markers of nutritional status (body mass index, fat mass, and leptin) and basal and pulsatile GH secretion, and mean and nadir GH concentrations was observed. GH concentration predicted levels of all markers of bone formation and a marker of bone resorption (N-telopeptide) in healthy controls, but not in AN. We demonstrate increases in basal GH secretion, number of secretory bursts, and GH concentration in adolescents with AN compared with controls, accompanied by low IGF-I levels. These data are consistent with the hypothesis that an acquired GH resistance occurs in this undernourished group. We also demonstrate that GH secretion and concentration are nutritionally regulated, and that the effects of nutrition exceed the effects of cortisol on GH concentration. Acquired GH resistance may play a role in the osteopenia and decreased peak bone mass frequently associated with AN.
Local Androgen Inactivation in Abdominal Visceral Adipose Tissue
Authors: Blouin Karine , Richard Christian , Bélanger Chantal , Dupont Pierre , Daris Marleen , Laberge Philippe , Luu-The Van , Tchernof André
Published: December 2003

We examined the expression and activity of two enzymes from the aldoketoreductase (AKR) family 1C, namely type 5 17β-hydroxysteroid dehydrogenase (17β-HSD-5, AKR1C3) and type 3 3α-hydroxysteroid dehydrogenase (3α-HSD-3, AKR1C2) in female sc and omental adipose tissue and in preadipocyte primary cultures. 17β-HSD-5 preferentially synthesizes testosterone from the inactive adrenal precursor androstenedione, whereas 3α-HSD-3 inactivates dihydrotestosterone. mRNAs of both enzymes were detected in adipose tissue from the omental and sc compartments. Real-time PCR quantification indicated a 3-fold higher 3α-HSD-3 expression compared with 17β-HSD-5, and the expression of both enzymes tended to be higher in the sc vs. the omental depot. Accordingly, dose-response and time-course experiments performed in preadipocyte primary cultures indicated that 3α-HSD activity was higher than 17β-HSD activity (13-fold maximum velocity difference). We measured 3α-HSD activity in omental and sc adipose tissue samples of 32 women for whom body composition and body fat distribution were evaluated by dual-energy x-ray absorptiometry and CT, respectively. We found that androgen inactivation in omental adipose tissue through 3α-HSD activity was significantly higher in women with elevated vs. low visceral adipose tissue accumulation (1.7-fold difference; P < 0.05). Moreover, omental adipose tissue 3α-HSD activity was positively and significantly associated with CT-measured visceral adipose tissue (r = 0.43; P < 0.02) and omental adipocyte diameter (r = 0.42; P < 0.02). These results indicate that local androgen inactivation is a predominant reaction in female abdominal adipose tissue, with the greatest conversion rates observed in the presence of abdominal visceral obesity. Increased androgen inactivation in omental adipose tissue of abdominally obese women may impact locally on the regulation of adipocyte metabolism.
Increased Expression of the 25-Hydroxyvitamin D3-1α-Hydroxylase Gene in Alveolar Macrophages of Patients with Lung Cancer
Authors: Yokomura Koushi , Suda Takafumi , Sasaki Shigekazu , Inui Naoki , Chida Kingo , Nakamura Hirotoshi
Published: December 2003

25-Hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) plays a central role in calcium metabolism by synthesizing the active hormone 1α,25-dihydroxyvitamin D3 in the kidney. Its increased expression in the extrarenal tissues has been found in alveolar macrophages in sarcoidosis but not in any other pathological conditions. We found that 1α-hydroxylase-mRNA in alveolar macrophages measured by semiquantitative RT-PCR was 2-fold greater in patients with lung cancer than in control subjects (0.61 ± 0.20 vs. 0.34 ± 0.11, respectively; P < 0.0001). When the clinical stages of lung cancer were divided into early (stage IA–IIIA) and advanced (stage IIIB and IV) and the expression of 1α-hydroxylase gene was compared among the control, early, and advanced groups, the advanced group showed the highest expression, followed by the early group, then the control group (0.34 ± 0.11, 0.52 ± 0.11, and 0.69 ± 0.23 for control, early, and advanced groups, respectively; P < 0.0001). The 1α-hydroxylase-mRNA level was well correlated with serum 1α,25-dihydroxylase D3 concentration and the 1α,25-dihydroxylase D3 to 25-hydroxyvitamin D3 ratio, but none of the findings related to calcium metabolism among the patients with lung cancer. Increased local production of 1α,25-dihydroxyvitamin D3 may be associated with the pathological conditions, such as immunosuppression, in lung cancer.
Quality of Life, Psychosocial Well-Being, and Sexual Satisfaction in Women with Polycystic Ovary Syndrome
Authors: Elsenbruch Sigrid , Hahn Susanne , Kowalsky Daniela , Öffner Alexandra H. , Schedlowski Manfred , Mann Klaus , Janssen Onno E.
Published: December 2003

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by chronic anovulation and hyperandrogenism. PCOS is one of the leading causes of infertility and manifests with hirsutism, acne, and obesity. To investigate its impact on health-related quality of life and sexuality, 50 women with PCOS and 50 controls were evaluated with standardized questionnaires (36-item short-form health survey, symptom checklist revised, and life satisfaction questionnaire). The impact of hirsutism, obesity, and infertility was assessed using five-point rating scales, and sexual satisfaction was analyzed with visual analog scales. Patients showed greater psychological disturbances on the symptom checklist revised dimensions, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, aggression, and psychoticism, along with a lower degree of life satisfaction in the life satisfaction questionnaire scales health, self, and sex. Health-related quality of life measured with the 36-item short-form health survey revealed significantly decreased scores for physical role function, bodily pain, vitality, social function, emotional role function, and mental health in patients with PCOS. Although patients had the same partner status and frequency of sexual intercourse, they were significantly less satisfied with their sex life and found themselves less attractive. Most of the differences were not affected by correction for body weight. In conclusion, PCOS causes a major reduction in the quality of life and severely limits sexual satisfaction.
Effects of Hypo- and Hyperthyroidism on Noradrenergic Activity and Glycerol Concentrations in Human Subcutaneous Abdominal Adipose Tissue Assessed with Microdialysis
Authors: Haluzik Martin , Nedvidkova Jara , Bartak Vladimir , Dostalova Ivana , Vlcek Petr , Racek Pavel , Taus Michal , Svacina Stepan , Alesci Salvatore , Pacak Karel
Published: December 2003

Thyroid hormones play a major role in lipid metabolism. However, whether they directly affect lipolysis locally in the adipose tissue remains unknown. Therefore, we measured abdominal sc adipose tissue norepinephrine (NE), basal, and isoprenaline-stimulated lipolysis in 12 hypothyroid patients (HYPO), six hyperthyroid patients (HYPER), and 12 healthy controls by in vivo microdialysis.

Adipose tissue NE was decreased in HYPO and increased in HYPER compared with controls (90.4 ± 2.9 and 458.0 ± 69.1 vs. 294.9 ± 19.5 pmol/liter, P < 0.01). Similarly, basal lipolysis, assessed by glycerol assay, was lower in HYPO and higher in HYPER than in controls (88.2 ± 9.9 and 566.0 ± 42.0 vs. 214.3 ± 5.1 μmol/liter P < 0.01). The relative magnitude of isoprenaline-induced glycerol increase was smaller in HYPO (39 ± 19.4%, P < 0.05 vs. basal) and higher in HYPER (277 ± 30.4%, P < 0.01) than in controls (117 ± 5.6%, P < 0.01). The corresponding changes in NE after isoprenaline stimulation were as follows: 120 ± 9.2% (P < 0.05), 503 ± 113% (P < 0.01), and 267 ± 17.2 (P < 0.01).

In summary, by affecting local NE levels and adrenergic postreceptor signaling, thyroid hormones may influence the lipolysis rate in the abdominal sc adipose tissue.
Low Vitamin D and High Parathyroid Hormone Levels as Determinants of Loss of Muscle Strength and Muscle Mass (Sarcopenia): The Longitudinal Aging Study Amsterdam
Authors: Visser Marjolein , Deeg Dorly J. H. , Lips Paul
Published: December 2003

The age-related change in hormone concentrations has been hypothesized to play a role in the loss of muscle mass and muscle strength with aging, also called sarcopenia. The aim of this prospective study was to investigate whether low serum 25-hydroxyvitamin D (25-OHD) and high serum PTH concentration were associated with sarcopenia. In men and women aged 65 yr and older, participants of the Longitudinal Aging Study Amsterdam, grip strength (n = 1008) and appendicular skeletal muscle mass (n = 331, using dual-energy x-ray absorptiometry) were measured in 1995–1996 and after a 3-yr follow-up. Sarcopenia was defined as the lowest sex-specific 15th percentile of the cohort, translating into a loss of grip strength greater than 40% or a loss of muscle mass greater than 3%. After adjustment for physical activity level, season of data collection, serum creatinine concentration, chronic disease, smoking, and body mass index, persons with low (<25 nmol/liter) baseline 25-OHD levels were 2.57 (95% confidence interval 1.40–4.70, based on grip strength) and 2.14 (0.73–6.33, based on muscle mass) times more likely to experience sarcopenia, compared with those with high (>50 nmol/liter) levels. High PTH levels (≥4.0 pmol/liter) were associated with an increased risk of sarcopenia, compared with low PTH (<3.0 pmol/liter): odds ratio = 1.71 (1.07–2.73) based on grip strength, odds ratio = 2.35 (1.05–5.28) based on muscle mass. The associations were similar in men and women. The results of this prospective, population-based study show that lower 25-OHD and higher PTH levels increase the risk of sarcopenia in older men and women.
Differential Expression and Regulation of Microsomal Prostaglandin E2 Synthase in Human Fetal Membranes and Placenta with Infection and in Cultured Trophoblast Cells
Authors: Premyslova Marina , Li Wei , Alfaidy Nadia , Bocking Alan D. , Campbell Karen , Gibb William , Challis John R. G.
Published: December 2003

We have evaluated the effect of chorioamnionitis on the protein expression of microsomal and cytosolic prostaglandin E2 synthases (mPGES and cPGES) in preterm human placentae (PL) and fetal membranes (FM), by Western blot and immunohistochemistry, as well as the regulatory effect of IL-1β and TNF-α on mPGES, cPGES, and cyclooxygenase (COX)-2 expression in villous trophoblast (VT) and chorion trophoblast (CT) cell cultures. mPGES localized to the syncytiotrophoblast and vascular endothelium in PL and to the amnion epithelium, CT, and decidual cells in FM. cPGES protein was localized only to the syncytiotrophoblast in PL and had the same profile of expression as mPGES in FM. With infection, there was an increase in mPGES expression in PL and a decrease in the expression in FM. cPGES protein did not change in either PL or FM with infection. In VT cells in culture, IL-1β up-regulated COX-2 protein expression but did not affect mPGES. However, TNF-α increased both mPGES and COX-2 protein expression in these cells. In CT cells in culture, IL-1β and TNF-α increased both mPGES and COX-2 protein levels. Neither IL-1β nor TNF-α affected cPGES in either VT or CT cells. We conclude that protein levels of mPGES, as well as COX-2, can be stimulated by cytokines, potentially contributing to the increased prostaglandin production at the time of infection-driven preterm labor. However, multiple mechanisms, which apparently are inductor- and cell-type-specific, exist for the regulation of these enzymes.
The Lipid Droplet-Associated Protein Adipophilin Is Expressed in Human Trophoblasts and Is Regulated by Peroxisomal Proliferator-Activated Receptor-γ/Retinoid X Receptor
Authors: Bildirici Ibrahim , Roh Cheong-Rae , Schaiff W. Timothy , Lewkowski Beth M. , Nelson D. Michael , Sadovsky Yoel
Published: December 2003

Uptake and transplacental transfer of fatty acids is essential for fetal development. Human adipophilin and its murine ortholog adipocyte differentiation-related protein are lipid droplet-associated proteins that are implicated in cellular fatty acid uptake in adipocytes. The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) stimulates lipid uptake by adipocytes and enhances differentiation of placental trophoblasts. We therefore hypothesized that adipophilin is expressed in human trophoblasts and that its expression is regulated by PPARγ. We initially determined that adipophilin is expressed in human villous trophoblasts and that adipophilin expression is enhanced during differentiation of cultured primary term human trophoblasts. We also found that exposure of cultured human trophoblasts to the PPARγ ligand troglitazone resulted in a concentration-dependent increase in adipophilin expression. We observed a similar increase with LG268, a ligand for retinoid X receptor (RXR), the heterodimeric partner of PPARγ. Lastly, we demonstrated that ligand-activated PPARγ and RXR stimulated the transcriptional activity of adipophilin promoter in CV-1 cells and in the placental JEG3 cell line. We conclude that the expression of adipophilin is enhanced during trophoblast differentiation and is up-regulated by ligand-activated PPARγ/RXR. Enhanced adipophilin expression may contribute to fatty acid uptake by the placenta.
The Trp64Arg Polymorphism of the β3-Adrenergic Receptor Gene Is Not Associated with Body Weight or Body Mass Index in Japanese: A Longitudinal Analysis
Authors: Matsushita Yumi , Yokoyama Tetsuji , Yoshiike Nobuo , Matsumura Yasuhiro , Date Chigusa , Kawahara Kazuo , Tanaka Heizo
Published: December 2003

The β3-adrenergic receptor (ADRB3) is expressed mainly in visceral adipose tissue and is thought to contribute to lipolysis and the delivery of free fatty acids to the portal vein. Although many studies have examined the relationship between the Trp64Arg mutation of ADRB3 and obesity, the results have been inconsistent. We examined the cross-sectional relationship of ADRB3 variants with indexes of obesity, and their longitudinal changes over 10 yr, in men and women, aged 40–69 yr, who were randomly selected from the Japanese rural population. The study considered both dietary energy intake and physical activity levels. Among the 746 participants, the genotype frequencies of the Trp64Trp, Trp64Arg, and Arg64Arg variants were 483, 224, and 39, respectively. The cross-sectional analysis showed no significant differences in height, weight, body mass index, blood pressure, serum total and high density lipoprotein cholesterols, and hemoglobin A1c among the genotype groups even after adjustments for gender, age, smoking, alcohol drinking, physical activity, and energy intake. No significant differences in the weight changes between the genotype groups were evident in the longitudinal analysis. We conclude that the Trp64Arg mutation of ADRB3 has little or no influence on either body weight or body mass index in the general Japanese population.
The −3826 A→G Variant of the Uncoupling Protein-1 Gene Diminishes Postprandial Thermogenesis after a High Fat Meal in Healthy Boys
Authors: Nagai Narumi , Sakane Naoki , Ueno Linda Massako , Hamada Taku , Moritani Toshio
Published: December 2003

This study investigated whether the −3826 A→G nucleotide variant of the uncoupling protein-1 (UCP1) gene is correlated with postprandial thermogenesis after a high fat meal in children. Healthy boys, aged 8–11 yr, were examined for resting energy expenditure and the thermic effect of a meal (TEM), which were measured by indirect calorimetry for 180 min after a high fat (70% fat, 20% carbohydrate, and 10% protein, providing 30% of the daily energy requirement) and a high carbohydrate meal (20% fat, 70% carbohydrate, and 10% protein). The sympatho-vagal activities were assessed by means of spectral analysis of the heart rate variability during the same period. Children were genotyped for UCP1 polymorphism by applying a PCR-restriction fragment length polymorphism using buccal samples. There was no reaction of sympathetic activity to the high carbohydrate meal in eitherthe GG allele or the AA+AG group and no significant difference in TEM. However, after the high fat meal, sympathetic responses were found in both groups; further, the GG allele group showed significantly lower TEM than the AA+AG group. In conclusion, despite fat-induced sympathetic stimulation, GG allele carriers have a lowered capacity of TEM in response to fat intake, suggesting that such impaired UCP1-linked thermogenesis can have adverse effects on the regulation of body weight.
Platelet-Activating Factor Inhibits the Secretion of Platelet-Activating Factor Acetylhydrolase by Human Decidual Macrophages
Authors: Narahara Hisashi , Kawano Yasushi , Nasu Kaei , Yoshimatsu Jun , Johnston John M. , Miyakawa Isao
Published: December 2003

To clarify the role of platelet-activating factor (PAF) in parturition, the effects of PAF on the secretion of PAF-acetylhydrolase (PAF-AH), a PAF-inactivating enzyme, by decidual macrophage populations were examined. The cells were isolated from human decidual tissue by enzymatic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The nonhydrolyzable agonist of PAF, carbamyl-PAF (C-PAF), inhibited the secretion of PAF-AH by either decidual cells or flow cytometrically purified decidual macrophages. A specific PAF receptor antagonist, WEB 2086, blocked the C-PAF-induced inhibition. Lyso-PAF, a metabolite of PAF, had no effect on the enzyme secretion. An intracellular calcium channel blocker, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetra(acetoxymethyl)-ester, partially blocked the inhibition by C-PAF, whereas extracellular calcium channel blockers, nifedipine and verapamil, were without effect. The inhibitory effect of C-PAF was also partially blocked by protein kinase C (PKC) inhibitors, sphingosine and H-7. A PKC activator, 12-O-tetradecanoylphobol 13-acetate, decreased the secretion of PAF-AH. The decrease was abolished by the addition of sphingosine and H-7. It is suggested that PAF inhibits the PAF-AH secretion by decidual macrophages and that the inhibitory action is mediated by a signal transduction mechanism involving intracellular calcium and PKC.
Effects of Chemotherapeutic Agents on the Function of Primary Human Osteoblast-Like Cells Derived from Children
Authors: Davies J. H. , Evans B. A. J. , Jenney M. E. M. , Gregory J. W.
Published: December 2003

Studies in children treated with chemotherapy suggest that chemotherapeutic agents have deleterious effects on bone metabolism. We therefore evaluated the in vitro effects of clinically relevant concentrations of chemotherapeutic agents on the synthesis of type I collagen, alkaline phosphatase (AP) activity, and mineralization by primary human osteoblast-like (HOB) cells derived from children. Because serum 1,25-dihydroxyvitamin D3 concentrations may be reduced during treatment with chemotherapy, the effect of chemotherapeutic agents on HOB cells cultured in the presence or absence of 1,25-dihydroxyvitamin D3 was also evaluated.

Type I collagen synthesis was reduced by all agents (P < 0.01) other than methotrexate, whereas the relative AP activity was increased (P < 0.01) by all agents. The relative number of cells staining intensely for AP after culture with agents increased (P < 0.05), and AP mRNA expression was increased (P < 0.01) with vincristine. 1,25-Dihydroxyvitamin D3 ameliorated (P < 0.01) the depletion of HOB cell numbers by chemotherapeutic agents. Furthermore, vincristine and daunorubicin inhibited 1,25-dihydroxyvitamin D3-mediated AP activity (P < 0.01).

We conclude that chemotherapeutic agents can adversely affect HOB cell function, and we speculate that this observation may account, in part, for the osteopenia observed during and after treatment of children with chemotherapy.
Pegvisomant-Induced Serum Insulin-Like Growth Factor-I Normalization in Patients with Acromegaly Returns Elevated Markers of Bone Turnover to Normal
Authors: Parkinson C. , Kassem M. , Heickendorff L. , Flyvbjerg A. , Trainer P. J.
Published: December 2003

Active acromegaly is associated with increased biochemical markers of bone turnover. Pegvisomant is a GH receptor antagonist that normalizes serum IGF-I in 97% of patients with active acromegaly. We evaluated the effects of pegvisomant-induced serum IGF-I normalization on biochemical markers of bone and soft tissue turnover, as well as levels of PTH and vitamin D metabolites, in 16 patients (nine males; median age, 52 yr; range, 28–78 yr) with active acromegaly (serum IGF-I at least 30% above upper limit of an age-related reference range). Serum procollagen III amino-terminal propeptide (PIIINP) and type I procollagen amino-terminal propeptide, osteocalcin (OC), bone-related alkaline phosphatase, C-terminal cross-linked telopeptide of type I collagen (CTx), albumin-corrected calcium, intact PTH, 25-hydroxy vitamin D, 1,25-dihydroxy vitamin D [1,25-(OH)2 vit D], urinary type 1 collagen cross-linked N-telopeptide/creatinine ratio, and urinary calcium (24 h collection) were measured (single-batch analysis) at study entry and after IGF-I normalization, along with sera from 32 age- and sex-matched controls. Compared with controls, PIIINP, OC, and CTx were significantly elevated in patients at baseline. Pegvisomant-induced serum IGF-I normalization (699 ± 76 to 242 ± 28 μg/liter, P < 0.001) was associated with a significant decrease in PIIINP, markers of bone formation (type I procollagen amino-terminal propeptide, OC, and bone-related alkaline phosphatase), and resorption (CTx and urinary type 1 collagen cross-linked N-telopeptide/creatinine ratio). 1,25-(OH)2 vit D decreased and intact PTH increased significantly, but 25-hydroxy vitamin D was unaffected. A significant decline in calculated calcium clearance was observed. The decrease in serum IGF-I correlated positively with the decrease of serum PIIINP (r = 0.7, P < 0.01). After normalization of serum IGF-I, there was no statistical difference between patients and controls for any parameters for which control data were available. In conclusion, GH excess is associated with increased bone and soft tissue turnover. Pegvisomant-induced normalization of serum IGF-I results in a decrease in markers of bone and soft tissue turnover to levels observed in age-matched controls, and these changes are accompanied by an increase in PTH and a decrease in 1,25-(OH)2 vit D. These data provide further evidence of the effectiveness of pegvisomant in normalizing the altered biological effects of GH hypersecretion.
A Novel Semiquantitative Polymerase Chain Reaction/Enzyme Digestion-Based Method for Detection of Large Scale Deletions/Conversions of the CYP21 Gene and Mutation Screening in Turkish Families with 21-Hydroxylase Deficiency
Authors: Tukel T. , Uyguner O. , Wei J. Q. , Yuksel-Apak M. , Saka N. , Song D. X. , Kayserili H. , Bas F. , Gunoz H. , Wilson R. C. , New M. I. , Wollnik B.
Published: December 2003

21-Hydroxylase deficiency is a recessively inherited disorder resulting from mutations in the CYP21 gene. The CYP21 gene is located along with the CYP21P pseudogene in the human leukocyte antigen major histocompatibility complex region on chromosome 6. Molecular diagnosis is difficult due to the 98% similarity of CYP21 and CYP21P genes and the fact that almost all frequently reported mutations reside on the pseudogene. Allele-specific PCR for the 8 most frequently reported point mutations was performed in 31 Turkish families with at least a single 21-hydroxylase-deficient individual. The allele frequencies of the point mutations were as follows: P30L, 0%; IVS2 (AS,A/C-G,-13), 22.5%; G110Δ8nt, 3.2%; I172N, 11.4%; exon 6 cluster (I236N, V237E, M239K), 3.2%; V281L, 0%; Q318X, 8%; and R356W, 9.6%. Large deletions and gene conversions were detected by Southern blot analysis, and the allele frequencies were 9.6% and 22.5%, respectively. Sequence analysis of the gene, performed on patients with only 1 mutated allele, revealed 2 missense mutations (R339H and P435S). A novel semiquantitative PCR/enzyme digestion-based method for the detection of large scale deletions/conversions of the gene was developed for routine diagnostic purposes, and its accuracy was shown by comparison with the results of Southern blot analysis.
Molecular Changes Underlying Reduced Pineal Melatonin Levels in Alzheimer Disease: Alterations in Preclinical and Clinical Stages
Authors: Wu Ying-Hui , Feenstra Matthijs G. P. , Zhou Jiang-Ning , Liu Rong-Yu , Toranõ Javier Sastre , Van Kan Hendrikus J. M. , Fischer David F. , Ravid Rivka , Swaab Dick F.
Published: December 2003

A disturbed sleep-wake rhythm is common in Alzheimer disease (AD) patients and correlated with decreased melatonin levels and a disrupted circadian melatonin rhythm. Melatonin levels in the cerebrospinal fluid are decreased during the progression of AD neuropathology (as determined by the Braak stages), already in cognitively intact subjects with the earliest AD neuropathology (Braak stages I-II) (preclinical AD). To investigate the molecular mechanisms behind the decreased melatonin levels, we measured monoamines and mRNA levels of enzymes of the melatonin synthesis and its noradrenergic regulation in pineal glands from 18 controls, 33 preclinical AD subjects, and 25 definite AD patients. Pineal melatonin levels were highly correlated with cerebrospinal fluid melatonin levels. The circadian melatonin rhythm disappeared because of decreased nocturnal melatonin levels in both the preclinical AD and AD patients. Also the circadian rhythm of β1-adrenergic receptor mRNA disappeared in both patient groups. The precursor of melatonin, serotonin was stepwise depleted during the course of AD, as indicated by the up-regulated monoamine oxidase A mRNA and activity (5-hydroxyindoleacetic acid:serotonin ratio). We conclude that a dysfunction of noradrenergic regulation and the depletion of serotonin by increased monoamine oxidase A result in the loss of melatonin rhythm already in preclinical AD.
The Role of the Orphan Nuclear Receptor, Liver Receptor Homologue-1, in the Regulation of Human Corpus Luteum 3β-Hydroxysteroid Dehydrogenase Type II
Authors: Peng Noel , Kim Joung W. , Rainey William E. , Carr Bruce R. , Attia George R.
Published: December 2003

After ovulation, ovarian 3β-hydroxysteroid dehydrogenase type II (HSD3B2) expression increases to enhance the shift of steroidogenesis toward progesterone biosynthesis. Steroidogenic factor-1 (SF-1) is a transcription factor for several genes encoding steroidogenic enzymes. However, the level of SF-1 expression decreases in the human corpus luteum (CL) after ovulation. Liver receptor homolog-1 (LRH-1) is another member of the orphan nuclear receptor family. We hypothesize that LRH-1, rather than SF-1, plays an essential role in the regulation of corpus luteum steroidogenesis. Semiquantitative RT-PCR and real-time PCR were performed to quantify the level of LRH-1 expression and correlate with HSD3B2 level. Cell transfection, mutation analysis, and EMSA were performed to examine the role of LRH-1 in the regulation of HSD3B2. LRH-1 expression was higher in CL, compared with mature ovarian follicles. Cotransfection of granulosa cells with HSD3B2 and LRH-1 resulted in a 10-fold increase of transcription. DAX-1 inhibited LRH-1-stimulated HSD3B2, which was maintained in the presence of dibutyryl cAMP. Mutation of the either of the two putative LRH-1 binding sites, which were confirmed by EMSA, in the HSD3B2 promoter decreased LRH-1 stimulation. Our findings suggest that LRH-1 is highly expressed in CL, and it plays an essential role in the regulation of HSD3B2.
Is the Thyrotropin-Releasing Hormone Test Necessary in the Diagnosis of Central Hypothyroidism in Children
Authors: Mehta Ameeta , Hindmarsh Peter C. , Stanhope Richard G. , Brain Caroline E. , Preece Michael A. , Dattani Mehul T.
Published: December 2003

To determine the value of the TRH test, we analyzed the unstimulated serum T4 and TSH concentrations in 54 children with central hypothyroidism. A TRH test was performed in 30 patients. Midline brain defects (septo-optic dysplasia, 28; holoprosencephaly, 2) and combined pituitary hormone deficiencies were present in 30 and 52 patients, respectively. The mean serum free T4, total T4, and basal TSH concentrations were 0.6 ng/dl, 4.0 μg/dl, and 2.8 μU/ml, respectively. Five patients demonstrated elevated basal serum TSH concentrations. A normal TRH test [increase (Δ) in TSH, 4.5–17.8], based on data from 30 controls, was documented in 23.3% of patients. Brisk (ΔTSH, >17.8), absent/blunted (ΔTSH, <4.5), and delayed responses were documented in 16.7%, 30%, and 30% of patients, respectively. The mean age at diagnosis was 2.8 yr, with 8 patients evolving into TSH deficiency. It was not possible to differentiate patients as having pituitary or hypothalamic disease based solely on the TRH test results. Patients with septo-optic dysplasia were diagnosed earlier and had elevated basal serum TSH and PRL concentrations, diabetes insipidus, and evolving disease. Although full pituitary function assessment is mandatory to identify combined pituitary hormone deficiencies, a TRH test is not essential, and the diagnosis should be made by serial T4 measurements.
In Vivo Phosphorylation of the Somatostatin 2A Receptor in Human Tumors
Authors: Liu Qisheng , Reubi Jean-Claude , Wang Yining , Knoll Brian J. , Schonbrunn Agnes
Published: December 2003

Hormone-stimulated receptor internalization and desensitization occur widely in the G protein-coupled receptor (GPCR) family. A critical first step in both these processes is thought to be receptor phosphorylation, a reaction which has been extensively characterized in cell culture. However, little is known about GPCR phosphorylation in vivo. The somatostatin (SS) receptor subtype (sst)2A is widely distributed in human neuroendocrine tumors, and SS analogs are commonly used to target this receptor for both therapy and diagnosis. In cultured pituitary cells sst2A is rapidly phosphorylated and internalized after hormone binding. The aim of the present study was to go one crucial step further and characterize the phosphorylation state of this receptor in human neuroendocrine tumors using a newly developed gel-shift assay. The receptor from a somatostatinoma was completely phosphorylated. In contrast, only unphosphorylated sst2A was present in human tumors that were not exposed to autocrine stimulation. Both in vivo and in cultured cells, the phosphorylation state of the sst2A receptor was correlated with its subcellular localization: phosphorylated receptor was mostly intracellular, whereas unphosphorylated receptor was localized at the cell surface. These results are the first to demonstrate ligand-stimulated GPCR phosphorylation in human tissue in situ, providing a crucial step toward a better understanding of receptor regulation in vivo. Analysis of sst2A phosphorylation promises to provide a sensitive indicator of the effectiveness of SS analogs in diagnostic and therapeutic situations in tumor patients.
P450c17 Deficiency in Brazilian Patients: Biochemical Diagnosis through Progesterone Levels Confirmed by CYP17 Genotyping
Authors: Martin Regina M. , Lin Chin J. , Costa Elaine M. F. , de Oliveira Maria Leocadia , Carrilho Alexandre , Villar Heloisa , Longui Carlos A. , Mendonca Berenice B.
Published: December 2003

P450c17 deficiency is an autosomal recessive disorder and a rare cause of congenital adrenal hyperplasia characterized by hypertension, hypokalemia, and impaired production of sex hormones. We performed a clinical, hormonal, and molecular study of 11 patients from 6 Brazilian families with the combined 17α-hydroxylase/17,20-lyase deficiency phenotype. All patients had elevated basal serum levels of progesterone (1.8–38 ng/ml; 0.57–12 pmol/liter) and suppressed plasma renin activity. CYP17 genotyping identified 5 missense mutations. The compound heterozygous mutation R362C/W406R was found in 1 family, whereas the homozygous mutations R96W, Y329D, and P428L were seen in the other 5 families. The R96W mutation has been described as the cause of P450c17 deficiency in Caucasian patients. The other mutations were not found in 50 normal subjects screened by allele-specific oligonucleotide hybridization (Y329D, R362C, and W406R) or digestion with HphI (P428L) and were recently found in other Brazilian patients. Therefore, we elucidated the genotype of 11 individuals with P450c17 deficiency and concluded that basal progesterone measurement is a useful marker of P450c17 deficiency and that its use should reduce the misdiagnosis of this deficiency in patients presenting with male pseudohermaphroditism, primary or secondary amenorrhea, and mineralocorticoid excess syndrome.
Treatment of Recurrent Thyroid Cysts with Ethanol: A Randomized Double-Blind Controlled Trial
Authors: Bennedbæk Finn Noe , Hegedüs Laszlo
Published: December 2003

Thyroid nodules are prevalent; when evaluated by ultrasonography (US), 15–25% of solitary thyroid nodules are cystic or predominantly cystic, and most are benign. Simple aspiration is the treatment of choice, but the recurrence rate is 10–80% depending on the number of aspirations and the cyst volume. The aim of this study was to evaluate the effect on recurrence rate of benign recurrent thyroid cysts in a double-blind randomized study comparing ethanol instillation with instillation of isotonic saline and subsequent complete emptying. Sixty-six consecutive patients with recurrent and benign (based on US-guided biopsy) thyroid cysts (≥2 ml) were randomly assigned to either subtotal cyst aspiration, flushing with 99% ethanol, and subsequent complete fluid aspiration (n = 33), or to subtotal cyst aspiration, flushing with isotonic saline, and subsequent complete fluid aspiration (n = 33). In case of recurrence (defined as cyst volume >1 ml) at the monthly evaluations, the treatment was repeated but limited to a maximum of three treatments. Procedures were US-guided, and patients were followed for 6 months. Age, sex, number of previous aspirations, pretreatment cyst volume, and serum TSH did not differ in the two groups. Cure (defined as a cyst volume ≤1 ml at the end of follow-up) was obtained in 27 of 33 [82%; confidence interval (CI), 65–93] patients treated with ethanol and in 16 of 33 (48%; CI, 31–66) patients treated with saline (P = 0.006). In the ethanol group, 21 of 33 (64%) patients were cured after one session only, compared with six of 33 (18%) in the saline group (P = 0.002). The number of previous aspirations (P = 0.005) and baseline cyst volume (P = 0.005) had influence on outcome, i.e. the chance of success decreased with the number of previous aspirations and with increasing cyst volume. Seven patients (21%) treated with ethanol had moderate to severe pain (median duration, 5 min; CI, 2–10), and one had transient dysphonia. Indirect laryngoscopy was performed before and after the last session and was normal in all patients.

We concluded that treatment of recurrent thyroid cysts with ethanol is superior to simple aspiration and flushing with saline and devoid of serious side effects. Our study demonstrates that flushing with ethanol is a clinically significant nonsurgical alternative for thyroid cysts that recur despite repeat aspirations.
Intramyocellular Lipid Changes in Men and Women during Aerobic Exercise: A 1H-Magnetic Resonance Spectroscopy Study
Authors: White Lesley J. , Ferguson Michael A. , McCoy Sean C. , Kim HeeWon
Published: December 2003

This study was designed to compare intramyocellular lipid (IMCL) changes during 60 min of submaximal exercise in men and women. Eighteen moderately active (18–38 yr) men (n = 9) and women (n = 9) were recruited. Maximum oxygen consumption (V̇O2max) and body composition were used to match subjects for aerobic fitness and body composition. Subjects performed cycle ergometry for 1 h at 65% of V̇O2max. Expired gases were collected throughout exercise to determine caloric expenditure and substrate use. Blood samples were collected before and after exercise to evaluate markers of lipid metabolism. Pre- and postexercise proton spectra were acquired from the vastus lateralis using a 3-T whole-body imaging system. Spectra were acquired from an 18-mm3 region of interest (echo time = 45 msec; repetition time = 2000 msec) for IMCL evaluation. IMCL decreased significantly with exercise (11.5–28.5% for men and 17.1–21.7% for women) (P < 0.05); however, there were no significant differences between men and women. Although changes were found for many plasma variables [free fatty acids, glycerol, and norepinephrine (P < 0.05)], group differences were only evident for norepinephrine. In conclusion, a significant decrease in IMCL was observed during 60 min of cycling in matched men and women.
Mutational Analysis of the FOXP3 Gene and Evidence for Genetic Heterogeneity in the Immunodysregulation, Polyendocrinopathy, Enteropathy Syndrome
Authors: Owen Catherine J. , Jennings Claire E. , Imrie Helen , Lachaux Alain , Bridges Nicola A. , Cheetham Tim D. , Pearce Simon H. S.
Published: December 2003

The immunodysregulation, polyendocrinopathy, enteropathy syndrome (IPEX), is a rare disorder of immune regulation resulting in multiple autoimmune disorders, which demonstrates X-linked recessive inheritance. The disease gene, FOXP3, was identified in 2001, and several mutations within this gene have since been described in patients with IPEX. We used linkage analysis, mutational screening of the FOXP3 gene, human leukocyte antigen typing, and analysis of X-chromosome inactivation to investigate 2 kindreds (21 subjects in total) with 4 male infants (3 now deceased) and 1 girl affected by IPEX. In 1 family a novel FOXP3 mutation was identified in the proband, with a single base deletion at codon 76 of exon 2, leading to a frameshift, which predicted a truncated protein product (108 residues vs. 431 in wild type). In the second family, the FOXP3 locus was excluded by recombination, and mutational analysis of the gene was negative. The affected girl from this family was shown to have human leukocyte antigen DR2 and DR6 alleles and random X-chromosome inactivation in peripheral blood mononuclear cells. Our analysis has elucidated the molecular basis of IPEX in one family and has, for the first time, provided evidence for an autosomal locus, suggesting genetic heterogeneity in this syndrome.
Pharmacokinetics and Dose Finding of a Potent Aromatase Inhibitor, Aromasin (Exemestane), in Young Males
Authors: Mauras Nelly , Lima John , Patel Deval , Rini Annie , di Salle Enrico , Kwok Ambrose , Lippe Barbara
Published: December 2003

Suppression of estrogen, via estrogen receptor or aromatase blockade, is being investigated in the treatment of different conditions. Exemestane (Aromasin) is a potent and selective irreversible aromatase inhibitor. To characterize its suppression of estrogen and its pharmacokinetic (PK) properties in males, healthy eugonadal subjects (14–26 yr of age) were recruited. In a cross-over study, 12 were randomly assigned to 25 and 50 mg exemestane daily, orally, for 10 d with a 14-d washout period. Blood was withdrawn before and 24 h after the last dose of each treatment period. A PK study was performed (n = 10) using a 25-mg dose. Exemestane suppressed plasma estradiol comparably with either dose [25 mg, 38% (P ≤ 0.002); 50 mg, 32% (P ≤ 0.008)], with a reciprocal increase in testosterone concentrations (60% and 56%; P ≤ 0.003 for both). Plasma lipids and IGF-I concentrations were unaffected by treatment. The PK properties of the 25-mg dose showed the highest exemestane concentrations 1 h after administration, indicating rapid absorption. The terminal half-life was 8.9 h. Maximal estradiol suppression of 62 ± 14% was observed at 12 h. The drug was well tolerated. In conclusion, exemestane is a potent aromatase inhibitor in men and an alternative to the choice of available inhibitors. Long-term efficacy and safety will need further study.
Reinterpreting validation
Authors: Lord S V , McCarthy T J , Aleem H , Zhao Y , Sharratt P N
Published: December 2003

This paper briefly describes the current approach to validation in good manufacturing practice (GMP) and postulates that the main difficulty with the process is determining how much validation is enough. It asserts that the current methodology is focused on documentation rather than verification, and furthermore that the inherent problems of document management are inhibiting resolution of the main difficulty. The paper argues that a data-driven approach instead of a document-driven approach to information management has the capability of removing the document management problems and thus enabling an improvement in verification. It further argues that such an approach is timely because of the increased use of computer applications and databases in design, construction, commissioning and operations, and available standards and implementations. It is also timely because of the recent interest of the US regulatory authority (FDA) in the regulation of electronic records [1]. Furthermore, a migration path exists where documents can be produced as necessary but no longer are the master information source. Opportunities for further research are identified.
An experimental investigation into ink transfer using a roller squeegee in high-speed screen printing
Authors: Fox I J , Claypole T C , Gethin D T
Published: December 2003

The paper describes a series of experiments on precision screen-printing with a roller squeegee. The work showed that this could only be achieved using a screen having a high mesh count, notably using a 150–34 screen, since a lower thread count offers insufficient resistance to flow and leads to image flooding. A detailed investigation into process parameters showed that at up to 60 per cent coverage the performance of a roller squeegee in either sliding and rolling mode is identical. Above this coverage and for smaller snap-off gaps, the roller squeegee constrained to slide led to a higher ink coverage. It is suggested that the main reason for this is the viscosity reduction at the screen surface on account of the ink characteristics and the high local shear rates. This dominates the higher hydrodynamic pressure associated with the rotary motion. However, the rolling action leads to superior tonal gain characteristics. It was observed that, when the squeegee was locked, the screen adhered to the substrate, reducing the snap-off speed and increasing the contact duration. The combined effect led to an increase in ink deposit. When the snap-off gap was increased sufficiently to prevent the screen from adhering to the substrate, the rotating squeegee produced considerably higher ink deposit than the locked squeegee. Presently, this is believed to be due to the increase in hydrodynamic pressure within the squeegee nip junction which now becomes a dominating mechanism.
Film thickness prediction in halftone screen-printing
Authors: Fox I J , Bohan M F J , Claypole T C , Gethin D T
Published: December 2003

The paper presents a numerical model of image transfer in screen-printing that is supported by experimental data. The model focuses on a roller squeegee system. It combines thin film hydrodynamic behaviour with roller squeegee deformation and flow through a porous screen. The work within this investigation has, for the first time, enabled an estimate of deposit thickness at different halftone coverage. For low coverage, ink transfer is governed by hydrodynamic behaviour in the nip contact, and deposited film thickness is represented by an ink spread model. For larger open areas, dependent on squeegee load, the film is removed from the top of the screen and therefore the deposit is likely to be controlled by the screen thickness. The work confirms that a roller squeegee system leads to higher nip pumping capacity in comparison with a sliding squeegee of nominally the same shape. The roller system is therefore appropriate when heavy ink deposits are required. The velocity gradients through the film when ink flows through the screen are reduced as the open area increases. The consequent reduction in shear rate leads to a recovery in viscosity within the nip contact.
Finite Element Simulation of an Industrial hot Rolling Schedule of Aluminium using Integrated Microstructure Models
Authors: Neumann L. , Aretz H. , Kopp R. , Goerdeler M. , Gottstein G.
Published: December 2003

The final properties of aluminum products largely depend on their temperature and forming history. To predict them, it is advantageous to combine process models and microstructural models. Here, the integration of a physically based dislocation density model with a full‐constraints Taylor model into the FEM code Larstran/Shape is shown. Finally, the application of this modeling scheme to an industrial hot rolling schedule is demonstrated.
Continuous Liquid Phase Coating of Carbon Fibers with Kion VL 20® Polysilazane
Authors: Gadow R. , Kern F.
Published: December 2003

Carbon fiber coating with polymer preceramic precursors is an easy, inexpensive, and reproducible method of protecting carbon fibers against chemical and mechanical hazards (see Figure for a roving of HT carbon fibers coated with polysiloxane and calcined). Polysilazane Kion VL 20 was found to be superior to polysiloxanes because it makes coatings insensitive to drying and curing conditions; also, their tensile strength increases with coating thickness.
Dense Reaction Infiltrated Silicon/Silicon Carbide Ceramics Derived from Wood based Composites
Authors: Hofenauer A. , Treusch O. , Tröger F. , Wegener G. , Fromm J. , Gahr M. , Schmidt J. , Krenkel W.
Published: December 2003

Wood‐based SiSiC ceramics with isotropic mechanical behavior were produced from commercial wood‐based composites (e.g., fiber boards). Properties can be tuned by the compression of the starting material, and by the content of added phenolic resin, which influences pore density and the amount of SiC infiltrated. In turn, the abundance of SiC versus residual silicon and carbon in the product determines its mechanical strength.