bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Characterization of a New Member of the Placental Prolactin-Like Protein-C (PLP-C) Subfamily, PLP-Cβ*
Authors: Hwang In-Taek , Lee Yong-Hun , Moon Boung-Cheon , Ahn Kyu-Youn , Lee See-Woo , Chun Jong-Yoon
Published: January 2014

We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55–66% amino acid identity with mouse PLP-Cα and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cβ for consistency. We have also isolated rat PLP-Cβ from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cβ were isolated: one mRNA (rPLP-Cβ) encodes a 241-amino acid product, but another mRNA (rPLP-CβΔ39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-Cα, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cβ form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cβ mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cβ expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cβ mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cβ expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cβ clearly indicate that PLP-Cβ is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cβ during pregnancy.
Transcriptional Regulation of Insulin-Like Growth Factor-I Receptor Gene Expression in Prostate Cancer Cells**This work was supported by V.A. Merit Review Program (to S.R.P.), DAMD 17–98-1–8540 (to J.L.W.), NIH DK-52683 (to S.R.P. and J.L.W.), and NIH DK-50810 (to C.T.R.). The content of this report does not necessarily represent the position or the policy of the United States government, and no official endorsement should be inferred.
Authors: Damon Susan E. , Plymate Stephen R. , Carroll Julie M. , Sprenger Cynthia C. , Dechsukhum Chevaboon , Ware Joy L. , Roberts Charles T.
Published: January 2014

A marked decrease in the type 1 insulin-like growth factor (IGF) receptor (IGF-IR) occurs in prostate epithelial cells during transformation from the benign to the metastatic state. One of the principal regulators of IGF-IR gene expression, the WT1 tumor suppressor, is expressed in prostate cancer and in prostate cancer cell lines. The purpose of this study was to determine whether the decrease in IGF-IR expression was transcriptionally regulated, and whether WT1 action may be involved in the repression of the IGF-IR gene in prostate cancer cells. The P69 cell line was derived by immortalization of human primary prostate epithelial cells with simian virus-40 T antigen and is rarely tumorigenic. The M12 line was derived from the P69 line by selection for tumor formation in nude mice and is tumorigeneic and metastatic. P69 cells express 20,000 IGF-IR/cell, whereas M12 cells express 3,500 IGF-IR/cell. These differences in receptor number are reflected in proportional differences in IGF-IR mRNA levels. To assess IGF-IR promoter activity in these cell lines, each was transiently transfected with luciferase reporter vectors containing the IGF-IR gene transcription start site and 476 bp of 5′-flanking sequence, 640 bp of 5′-untranslated region sequence, or both regions. The promoter activity of the full-length construct was 50% lower (P < 0.01) in M12 cells compared with P69 cells, the activity of the 5′-flanking region construct was 53% lower (P < 0.0001), and that of the 5′-untranslated region construct was 36% lower (P = 0.01). P69 clones stably transfected with a WT1 expression vector exhibited decreased expression of the endogenous IGF-IR gene and decreased promoter activity in transient transfection assays with IGF-IR promoter constructs containing multiple WT1 binding sites. The observed reduction in endogenous IGF-IR expression was sufficient to inhibit IGF-I-stimulated cell proliferation. These data suggest that most of the decreased expression of the IGF-IR seen in malignant prostate epithelium is the result of transcriptional repression of the IGF-IR gene, and that this repression may be due in part to the increased expression of the WT1 tumor suppressor in metastatic prostate cancer.
Phosphatidylinositol-3,4,5-Trisphosphate Is Required for Insulin-Like Growth Factor 1-Mediated Survival of 3T3-L1 Preadipocytes**This work was supported by a grant (to A.S.) from the Medical Research Council of Canada.
Authors: Gagnon AnneMarie , Dods Patti , Roustan-Delatour Nicolas , Chen Ching-Shih , Sorisky Alexander
Published: January 2014

Adipocyte number, a determinant of adipose tissue mass, reflects the balance between the rates of proliferation/differentiation vs. apoptosis of preadipocytes. The percentage of 3T3-L1 preadipocytes undergoing cell death following serum deprivation was reduced by 10 nm insulin-like growth factor (IGF)-1 (from 50.0 ± 0.7% for control starved cells to 27.5 ± 3.1%). TUNEL staining confirmed the apoptotic nature of the cell death. The protective effect of IGF-1 was blocked by phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin, and LY294002, but was unaffected by rapamycin, PD98059, or SB203580, which inhibit mammalian target of rapamycin (mTOR), ERK kinase (MEK1), and p38 MAPK respectively. Exogenous PI(3,4,5)P3 (10 μm), the principal product of IGF-1-stimulated PI3K in 3T3-L1 preadipocytes, had a modest survival effect on its own, reducing cell death from 47.9± 3.4% to 35.6 ± 3.5%. When added to the combination of IGF-1 and LY294002, PI(3,4,5)P3 reversed most of the inhibitory effect of LY294002 on IGF-1-dependent cell survival, protein kinase B/Akt phosphorylation, and caspase-3 activity. Taken together, these results implicate PI(3,4,5)P3 as a necessary signal for the anti-apoptotic action of IGF-1 on 3T3-L1 preadipocytes.
Induction of Type 3 Iodothyronine Deiodinase by Nerve Injury in the Rat Peripheral Nervous System
Authors: Li Wen Wu , Le Goascogne Claude , Ramaugé Martine , Schumacher Michael , Pierre Michel , Courtin Françoise
Published: January 2014

Thyroid hormones are essential for the development and repair of the peripheral nervous system. The type 2 deiodinase, which is responsible for the activation of T4 into T3, is induced in injured sciatic nerve. To obtain information on the type 3 deiodinase (D3) responsible for the degradation of thyroid hormones, we looked for its expression (mRNA and activity) in the sciatic nerve after injury. D3 was undetectable in the intact sciatic nerve of adult rats, but was rapidly and highly increased in the distal and proximal segments after nerve lesion. After cryolesion, D3 up-regulation disappeared after 3 d in the proximal segment, whereas it was sustained for 10 d in the distal segment, then declined to reach basal levels after 28 d, when functional recovery was completed. After a transsection preventing the nerve regeneration, up-regulation of D3 persisted up to 28 d at high levels in the distal segment. D3 was expressed in peripheral connective sheaths and in the internal endoneural compartment. D3 mRNA was inducible by 12-O-tetradecanoylphorbol-13-acetate in cultured fibroblasts or Schwann cells. In conclusion, induction of D3 in the peripheral nervous system after injury may play an important role during the regeneration process by adjusting intracellular T3 levels.
Quantitative Analysis of Estrogen Receptor Proteins in Rat Mammary Gland*
Authors: Saji Shigehira , Sakaguchi Hideki , Andersson Sandra , Warner Margaret , Gustafsson Jan-Åke
Published: January 2014

Estrogen receptor α and β proteins (ERα and ERβ) at various stages of development of the rat mammary gland were quantified by Western blotting. ERα and ERβ recombinant proteins were used as standards, and their molar concentrations were measured by ligand binding assays. In 3-week-old pregnant, lactating, and postlactating rats the ERα content ranged from 0.30–1.55 fmol/μg total protein (mean values). The ERβ content of the same samples ranged between 1.06–7.50 fmol/μg total protein. At every developmental stage, the ERβ content of the mammary gland was higher than that of ERα. When receptor levels were normalized against β-actin, it was evident that ER expression changed during development, with maximum expression of both receptors during the lactation period. With an antibody raised against the 18-amino acid insert of the ERβ variant, originally called ERβ2 but named ERβins in this paper, Western blots revealed that ERβins protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ERβins paralleled those of the protein. Double immunohistochemical staining with anti-ERα and anti-ERβins antibodies revealed that ERβins protein colocalized with ERα in 70–80% of the ERα-expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ERβins is regulated not only quantitatively, but also with regard to its cellular distribution. As ERβins acts as the dominant repressor of ERα, we suggest that its coexpression with ERα quenches ERα function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation.
Inhibin Suppresses and Activin Stimulates Osteoblastogenesis and Osteoclastogenesis in Murine Bone Marrow Cultures
Authors: Gaddy-Kurten D. , Coker J. K. , Abe E. , Jilka R. L. , Manolagas S. C.
Published: January 2014

Using primary murine bone marrow cell cultures, we demonstrate that inhibin suppresses osteoblastogenesis and osteoclastogenesis. In contrast, activin supports osteoblast formation (by alkaline phosphatase-positive and mineralized colony formation); and activin also stimulates osteoclast formation (as measured by staining tartrate-resistant acid phosphatase-positive multinucleated cells). Inhibin, the activin antagonist follistatin, and the bone morphogenetic protein antagonist noggin can all suppress endogenous activin accumulation in bone marrow cultures. Associated with this decrease in activin is the loss of mineralized osteoblastic colony formation (colony forming unit-osteoblast; CFU-OB). However, exogenous activin administration, even in the presence of noggin, permits both alkaline phosphatase-positive and CFU-OB colony formation in vitro. In contrast, the stimulatory effects of locally produced activin on osteoblast and osteoclast development are not likely to be dominant over the suppressive effects of gonadally derived inhibin. The suppressive effect of inhibin is maintained in the presence of either activin or bone morphogenetic protein, suggesting the presence of a distinct inhibin-specific receptor. Taken together, the direct regulation of osteoblastogenesis and osteoclastogenesis by inhibin and activin in vitro suggest that changes in the inhibin/activin ratio detected by bone marrow cells, during the perimenopausal transition, contribute to altered cell differentiation and may be associated with the increased bone resorption observed at this time.
11β-Hydroxysteroid Dehydrogenase 1 Transforms 11-Dehydrocorticosterone into Transcriptionally Active Glucocorticoid in Neonatal Rat Heart
Authors: Sheppard Karen E. , Autelitano Dominic J.
Published: January 2014

The ability of cells to directly respond to glucocorticoids and aldosterone is a function of GR and MR expression, and coexpression of 11β-hydroxysteroid dehydrogenases (11βHSDs), which convert glucocorticoids and their 11-ketometabolites into either receptor inactive or active derivatives. The aim of the present study was to determine the cellular expression of GR, MR, 11βHSD1, and 11βHSD2 in neonatal rat heart and determine the role these enzymes play in modulating glucocorticoid and aldosterone action. Ribonuclease protection analysis and steroid binding assays showed that GR is expressed in both cardiac myocytes and fibroblasts, whereas MR is expressed only in myocytes. 11βHSD2 was not detected in cardiac cells, but 11βHSD1 was expressed at high levels in both cardiac myocytes and fibroblasts. Enzyme activity studies demonstrated that 11βHSD1 acted as a reductase only, converting biologically inactive 11-dehydrocorticosterone to corticosterone, which then stimulated serum and glucocorticoid-induced kinase gene transcription via GR. In both cardiac myocytes and fibroblasts, aldosterone stimulated serum and glucocorticoid-induced kinase gene expression exclusively via GR, but not MR, indicating that aldosterone can have glucocorticoid-like actions in heart. The ability of cardiac cells to use both circulating corticosterone and 11-dehydrocorticosterone as a source of glucocorticoid suggests that the heart is under tonic glucocorticoid control, implying that glucocorticoids play important homeostatic roles in the heart.
Double-Stranded RNA Cooperates with Interferon-γ and IL-1β to Induce Both Chemokine Expression and Nuclear Factor-κB-Dependent Apoptosis in Pancreatic β-Cells: Potential Mechanisms for Viral-Induced Insulitis and β-Cell Death in Type 1 Diabetes Mellitus
Authors: Liu Dongbo , Cardozo Alessandra K. , Darville Martine I. , Eizirik Décio L.
Published: January 2014

Viral infections may trigger the autoimmune assault leading to type 1 diabetes mellitus. Double-stranded RNA (dsRNA) is produced by many viruses during their replicative cycle. The dsRNA, tested as synthetic poly(IC) (PIC), in synergism with the proinflammatory cytokines interferon-γ (IFN-γ) and/or IL-1β, results in nitric oxide production, Fas expression, β-cell dysfunction, and death. Activation of the transcription nuclear factor-κB (NF-κB) is required for PIC-induced inducible nitric oxide synthase expression in β-cells, and we hypothesized that this transcription factor may also participate in PIC-induced Fas expression and β-cell apoptosis. This hypothesis, and the possibility that PIC induces expression of additional chemokines and cytokines (previously reported as NF-κB dependent) in pancreatic β-cells, was investigated in the present study. We observed that the PIC-responsive region in the Fas promoter is located between nucleotides −223 and −54. Site-directed mutations at the NF-κB and CCAAT/enhancer binding protein-binding sites prevented PIC-induced Fas promoter activity. Increased Fas promoter activity was paralleled by enhanced susceptibility of PIC + cytokine-treated β-cells to apoptosis induced by Fas ligand. β-Cell infection with the NF-κB inhibitor AdIκB(SA)2 prevented both necrosis and apoptosis induced by PIC + IL-1β or PIC + IFN-γ. Messenger RNAs for several chemokines and one cytokine were induced by PIC, alone or in combination with IFN-γ, in pancreatic β-cells. These included IP-10, interferon-γ-inducible protein-10, IL-15, macrophage chemoattractant protein-1, fractalkine, and macrophage inflammatory protein-3α. There was not, however, induction of IL-1β expression. We propose that dsRNA, generated during a viral infection, may contribute for β-cell demise by both inducing expression of chemokines and IL-15, putative contributors for the build-up of insulitis, and by synergizing with locally produced cytokines to induce β-cell apoptosis. Activation of the transcription factor NF-κB plays a central role in at least part of the deleterious effects of dsRNA in pancreatic β-cells.
Prenatal LHRH Neurons in Nasal Explant Cultures Express Estrogen Receptor β Transcript
Authors: Sharifi Neda , Reuss Andree E. , Wray Susan
Published: January 2014

Sex steroids influence LHRH neuronal activity, exerting a negative or positive feedback action, depending on the reproductive state of the animal. Recent evidence indicates that LHRH neurons possess the estrogen receptor β (ERβ) subtype postnatally, suggesting that estrogen may act, in part, directly on LHRH neurons. In this study, we identified ERβ transcript in prenatal LHRH neurons as a function of age. Single-cell cDNA pools were made from LHRH neurons maintained for 7, 14, and 28 d in vitro (div). Screening of the cDNA pools by PCR with ERβ-specific primers revealed ERβ-positive LHRH neurons at all three ages. However, the number of LHRH cells coexpressing ERβ transcript decreased dramatically between 14 (6/10) and 28 div (1/10). None of the LHRH cells were positive for ERα transcript. These results indicate that developing LHRH neurons express the transcript for ERβ and suggest that continued expression of ERβ is either a characteristic of LHRH neurons that may require cues from the central nervous system and/or periphery or predetermined to be maintained in a subpopulation of LHRH neurons.
The Somatostatin Analog Octreotide Inhibits GH-Stimulated, But Not IGF-I-Stimulated, Bone Growth in Hypophysectomized Rats
Authors: Zapf Jürgen , Gosteli-Peter Martina , Weckbecker Gisbert , Hunziker Ernst B. , Reinecke Manfred
Published: January 2014

IGF-I mediates growth-promoting actions of GH. In the present study we investigated whether the somatostatin analog octreotide blunts the stimulatory effects of GH and/or IGF-I on bone growth in hypophysectomized rats infused for 6 d with vehicle, GH, or IGF-I. We found that octreotide significantly suppressed the GH-induced rise in liver IGF-I mRNA (−27%) and peptide (−32%) and the serum IGF-I level (−26%) and concomitantly inhibited GH-stimulated, but not IGF-I-stimulated, body weight gain (−31%), tibial epiphyseal width (−14%), and bone growth rate (−24%). Furthermore, octreotide significantly reduced the GH-induced increase in the number of IGF-I immunoreactive chondrocytes in all layers (except in the upper hypertrophic zone) of the tibial growth plate cartilage (P < 0.0001 for stem cell and proliferative zone; P < 0.0005 for lower hypertrophic zone). These findings demonstrate that octreotide does not interfere with IGF-I action, but does interfere with local GH-stimulated IGF-I production in the growth plate. Thus, besides inhibiting pituitary GH secretion, octreotide exerts inhibitory peripheral effects on GH-stimulated longitudinal bone growth.
Coexpression of Dopamine and Somatostatin Receptor Subtypes in Corticotroph Adenomas
Authors: de Bruin Christiaan , Pereira Alberto M. , Feelders Richard A. , Romijn Johannes A. , Roelfsema Ferdinand , Sprij-Mooij Diane M. , van Aken Maarten O. , van der Lelij Aart-Jan , de Herder Wouter W. , Lamberts Steven W. J. , Hofland Leo J.
Published: January 2014

Context: Previous studies have demonstrated the expression of somatostatin receptor subtypes (mainly sst5) and dopamine (DA) receptor subtypes (mainly D2) in smaller series of human corticotroph adenomas. In line with these findings, sst5 and D2-targeting agents have already been used clinically in patients with Cushing’s disease (CD) and have shown promising results in subsets of patients. To what extent these receptor subtypes are coexpressed within individual adenomas, is not known however.

Objective: The aim of the study was to investigate the (co-)expression of both sst and DA receptors in a large series of human corticotroph adenomas.

Design: We conducted in vitro analysis of corticotroph adenoma tissue obtained via transsphenoidal adenomectomy.

Setting: The study was conducted at two university medical centers.

Patients: Thirty patients with CD participated in the study.

Results: Analyzed by quantitative RT-PCR, D2 and sst5 were significantly (co-) expressed in the majority (60%) of adenomas, whereas 23% of adenomas only expressed D2, but not sst5. The remaining 17% of adenomas did not significantly express either sst5 or D2. Overall, expression of sst1–4 and D4 was low to nondetectable. Corticotroph adenomas with invasive growth invariably showed loss of sst5 and D2 expression. Autoradiography revealed clear D2 and/or SS-14 binding in a subset of cases, which correlated well with their respective mRNA data.

Conclusions: sst5 and especially D2 are highly expressed in the majority of human corticotroph adenomas, with coexpression of sst5 and D2 being a common phenomenon. These findings support the current studies with sst5 and D2-targeting agents in patients with CD and highlight the rationale behind sst5-D2 combination therapy.
Increased Hypothalamic-Pituitary-Adrenal Axis Activity in Huntington’s Disease
Authors: Aziz N. Ahmad , Pijl Hanno , Frölich Marijke , van der Graaf A. W. Maurits , Roelfsema Ferdinand , Roos Raymund A. C.
Published: January 2014

Context: Huntington’s disease (HD) is a fatal hereditary neurodegenerative disorder characterized by motor, cognitive, and behavioral disturbances. Hypothalamic-pituitary-adrenal (HPA) axis dysfunction could contribute to a number of HD signs and symptoms; however, no data are available on cortisol diurnal variations and secretory dynamics in HD patients.

Objective: The aim of the study was to perform a detailed analysis of HPA axis function in HD patients in relation to clinical signs and symptoms.

Design, Setting, and Participants: Twenty-four-hour cortisol secretion was studied in eight early-stage, medication-free HD patients and eight age-, sex-, and body mass index-matched controls in a clinical research laboratory. Cortisol levels were measured every 10 min.

Main Outcome Measures: Multiparameter autodeconvolution and cosinor regression were applied to quantify basal, pulsatile, and total cortisol secretion rates as well as diurnal variations in cortisol levels.

Results: Total cortisol secretion rate and the amplitude of the diurnal cortisol profile were both significantly higher in HD patients compared with controls (3490 ± 320 vs. 2500 ± 220 nmol/liter/24 h, P = 0.023; and 111 ± 14 vs. 64 ± 8 nmol/liter, P = 0.012, respectively). Cortisol concentrations in patients were particularly increased in the morning and early afternoon period. In HD patients, mean 24-h cortisol levels significantly correlated with total motor score, total functional capacity, as well as body mass index.

Conclusions: HPA axis hyperactivity is an early feature of HD and is likely to result from a disturbed central glucocorticoid feedback due to hypothalamic pathology. HPA axis dysfunction may contribute to some signs and symptoms in HD patients.
Elevated Corticotropin Releasing Hormone (CRH) during Pregnancy and Risk of Postpartum Depression (PPD)
Authors: Meltzer-Brody Samantha , Stuebe Alison , Dole Nancy , Savitz David , Rubinow David , Thorpba John
Published: January 2014

J Clin Endocrinol Metab, 10.1210/jc.2010-0978
Leptin Replacement Prevents Weight Loss-Induced Metabolic Adaptation in Congenital Leptin-Deficient Patients
Authors: Galgani Jose E. , Greenway Frank L. , Wong Ma-Li , Licinio Julio , Ravussin Eric
Published: January 2014

Context: Leptin regulates energy homeostasis by suppressing food intake; however, its role in energy expenditure and fat oxidation remains uncertain in humans.

Objective: The aim of the study was to assess 24-h energy metabolism before and after weight loss induced by leptin treatment in congenital leptin-deficient subjects or low-calorie diet in controls.

Design and Patients: We measured 24-h energy expenditure, 24-h fat oxidation, and body fat in three null homozygous leptin-deficient obese adults before and after weight loss induced by a 19-wk leptin replacement period (0.02-0.04 mg/kg/d). The same measures were performed in three obese controls pair-matched for sex, age, and weight loss induced by a 10- to 21-wk low-calorie diet. Measurements were preceded for 1 wk of weight stabilization. Energy expenditure was adjusted for fat-free mass, fat mass, sex, and age based on a reference population (n = 842; R2 = 0.85; P < 0.0001). Similarly, fat oxidation was adjusted for fat-free mass, percentage body fat, energy balance, and diet composition during the 24-h respiratory chamber stay (R2 = 0.38; P < 0.0001).

Results: Before weight loss, congenital leptin-deficient and control subjects had similar energy expenditure. However, after weight loss (∼15 kg), controls had energy expenditures lower than expected for their new weight and body composition (−265 ± 76 kcal/d; P = 0.04), whereas leptin-treated subjects had values not different from the reference population (−128 ± 119 kcal/d; P = 0.67). Before weight loss, fat oxidation was similar between groups. However, after weight loss, leptin-treated subjects had higher fat oxidation than controls (P = 0.005) and higher than the reference population (P = 0.0001).

Conclusion: In congenital leptin-deficient subjects, leptin replacement prevented the decrease in energy expenditure and fat oxidation often observed after weight loss.
Materno-Fetal Cardiovascular Complications in Turner Syndrome after Oocyte Donation: Insufficient Prepregnancy Screening and Pregnancy Follow-Up Are Associated with Poor Outcome
Authors: Chevalier Nicolas , Letur Hélène , Lelannou Dominique , Ohl Jeanine , Cornet Dominique , Chalas-Boissonnas Cécile , Frydman René , Catteau-Jonard Sophie , Greck-Chassain Thérèse , Papaxanthos-Roche Aline , Dulucq Marie-Christine , Couet Marie-Laure , Cédrin-Durnerin Isabelle , Pouly Jean-Luc , Fénichel Patrick ,   the French Study Group for Oocyte Donation
Published: January 2014

Context: Recombinant human GH treatment and oocyte donation (OD) have improved the quality of life in women with Turner syndrome (TS). However, life expectancy is reduced, mainly due to cardiovascular complications. Pregnancy may itself increase that risk and be associated with hazardous materno-fetal outcome.

Objective: The objective of this study was to evaluate the materno-fetal outcome of ongoing pregnancies beyond 20 wk of gestation obtained by OD in TS.

Design: This was a multicenter retrospective study including all assisted reproductive technology centers affiliated with the French Study Group for Oocyte Donation.

Results: Among 93 patients, only 37.6% were prescreened with echocardiography or thoracic magnetic resonance imaging. Maternal outcome was dominated by 37.8% of pregnancy-associated hypertensive disorders including preeclampsia in 54.8% and severe eclampsia in four patients. Prematurity occurred in 38.3% and was correlated with PAHD (P = 0.01). The frequency of in utero growth retardation was 27.5%. One fetal demise was linked to eclampsia. Two patients died from aortic rupture after cesarean section in a context of aortic root dilatation. Only 40% of pregnancies were associated with an absolutely normal materno-fetal outcome.

Conclusions: OD pregnancies in TS who have not been managed following recent specific recommendations were at high risk for maternal death by aortic dissection and for preeclampsia and its complications (fetal distress and in utero growth retardation). These recommendations include previous echocardiography, thoracic magnetic resonance imaging, and overnight blood pressure monitoring associated with a tight follow-up during pregnancy. Until future assessment of these recent recommendations, pregnancies obtained in TS after OD must be still considered as very high-risk pregnancies.
Three Novel Mutations and a De Novo Deletion Mutation of the DAX-1 Gene in Patients with X-Linked Adrenal Hypoplasia Congenita
Authors: Nakae Jun , Abe Shuji , Tajima Toshihiro , Shinohara Nozomi , Murashita Mari , Igarashi Yutaka , Kusuda Satoshi , Suzuki Junzou , Fujieda Kenji
Published: January 2014

The DAX-1 [DSS (dosage sensitive sex)-AHC critical region on the X, gene 1] gene is responsible for X-linked adrenal hypoplasia congenita (AHC). However, DAX-1 protein structure-function relationships are not well understood. Identification of missense mutations may help to reveal these relationships. We analyzed the DAX-1 gene from seven patients in six kindreds with X-linked AHC and identified one frameshift mutation, two missense mutations, and three deletion mutations. Case 1 had a 388delAG frameshift mutation, inducing a premature stop codon at position 70. Case 2 had a missense mutation, Lys382Asn, which encodes an asparagine (Asn) for lysine (Lys) at position 382. Sibling cases of 3-1 and 3-2 had a missense mutation of Trp291Cys, which encodes a substitution of cysteine (Cys) for tryptophan (Try) at position 291. The tryptophan (Trp) at position 291 and lysine (Lys) at position 382 in human DAX-1 protein are highly conserved among other related orphan nuclear receptor superfamily members. Cases 4, 5, and 6 showed deletion mutation. In case 6, a de novo deletion mutation was revealed by both southern hybridization and polymerase chain reaction (PCR) of a GGAA tetranucleotide tandem repeat. These findings suggest that: 1) Trp at position 291 and Lys at position 382, located in the C-terminal presumptive ligand binding domain, are important to the functional role of the DAX-1 protein in adrenal embryogenesis and/or in hypothalamic-pituitary activity; and 2) molecular analysis of the DAX-1 gene may help genetic counseling, even in cases with deletion mutation, because a detection of de novo deletion may exclude another affected or carrier child.
Increased Disorderliness of Basal Insulin Release, Attenuated Insulin Secretory Burst Mass, and Reduced Ultradian Rhythmicity of Insulin Secretion in Older Individuals1
Authors: Meneilly Graydon S. , Ryan Alice S. , Veldhuis Johannes D. , Elahi Dariush
Published: January 2014

Insulin is secreted in a pulsatile fashion. Rapid pulses are considered to be important for inhibiting hepatic glucose output, and ultradian pulses for stimulating peripheral glucose disposal. Aging is characterized by a progressive impairment in carbohydrate tolerance. We undertook the current studies to determine whether alterations in pulsatile insulin release accompany the age-related changes in carbohydrate metabolism. Healthy young (n = 8; body mass index, 21 ± 1 kg/m2; age, 24 ± 1 yr) and old (n = 9; body mass index, 24 ± 1 kg/m2; age, 77 ± 2 yr) volunteers underwent two studies. In the first study, insulin was sampled every 1 min for 150 min, and pulse analysis was conducted using a recently validated multiparameter deconvolution technique. In the second study, insulin was sampled every 10 min for 600 min, and insulin release was evaluated by Cluster analysis. In the 150-min studies, insulin secretory burst mass (P < 0.05) and amplitude (P < 0.01) were reduced in the elderly. In addition, approximate entropy, a measure of irregularity or disorderliness of insulin release, was increased in the aged (P < 0.01). In the 600-min studies, interpulse interval was greater in the aged (P < 0.05), and burst number was less (P < 0.05).

We conclude that normal aging is characterized by more disorderly insulin release, a reduction in the amplitude and mass of rapid insulin pulses, and a decreased frequency of ultradian pulses. Whether these alterations in insulin pulsatility contribute directly to the age-related changes in carbohydrate metabolism will require further study.
Measurement of Human Growth Hormone Receptor Messenger Ribonucleic Acid by a Quantitative Polymerase Chain Reaction-Based Assay: Demonstration of Reduced Expression after Elective Surgery*
Authors: Hermansson Majlis , Wickelgren Ruth B. , Hammarqvist Folke , Bjarnason Ragnar , Wennström Ingmar , Wernerman Jan , Carlsson Björn , Carlsson Lena M. S.
Published: January 2014

Studies of GH receptor (GHR) gene expression in human tissues have been hampered by the limited amount of tissue available for analysis and the low sensitivity of conventional methods. We have developed a quantitative reverse transcriptase-PCR assay for measurement of GHR messenger ribonucleic acid levels in small human tissue biopsies. To compensate for sample to sample variation, an internal RNA standard, which differs from the wild-type GHR transcript by only a few nucleotides, was reverse transcribed and amplified together with the GHR transcripts. PCR was carried out using one biotinylated primer to permit the purification of single stranded PCR products on streptavidin-coated microtiter plates. The ratio between the wild-type and mutated transcripts was determined by two separate minisequence reactions in which a primer, annealed immediately 3′ of a variable nucleotide, was extended by a single 3H-labeled nucleotide, complementary to either the wild-type or mutated sequence. The assay range was 0.125–8 × 105 transcripts/sample, the mean intraassay coefficient of variation was 8.7%, and the lower limit of detection was 0.125 × 105 transcripts/sample. GHR messenger ribonucleic acid levels were detectable in small amounts (10–100 ng) of total RNA extracted from adipose tissue, skeletal muscle, and liver. The GHR gene expression in liver was approximately 10-fold higher than that in skeletal muscle, whereas intermediate levels were found in adipose tissue. In nine patients undergoing elective abdominal surgery, GHR gene expression in skeletal muscle was reduced on day 3 after surgery compared to the baseline level. The decrease in GHR gene expression was accompanied by a decrease in skeletal muscle glutamine. This suggests that the postoperative protein catabolism may be caused at least partly by acquired GH insensitivity due to reduced expression of the GHR gene.
Prolactin Receptor Messenger Ribonucleic Acid in Normal and Neoplastic Human Pituitary Tissues1
Authors: Jin Long , Qian Xiang , Kulig Elzbieta , Scheithauer Bernard W. , Calle-Rodrigue Rocio , Abboud Charles , Davis Dudley H. , Kovacs Kalman , Lloyd Ricardo V.
Published: January 2014

We examined the specific cell types in normal human pituitaries that expressed PRL receptor (PRL-R) messenger ribonucleic acid (mRNA) by combined in situ hybridization and immunohistochemistry. The distribution of PRL-R mRNA in 28 pituitary adenomas was examined by in situ hybridization and reverse transcription-PCR in 12 cases of adenomas. In another set of experiments, 34 PRL adenomas from men, women, and bromocriptine-treated patients were analyzed for PRL-R by in situ hybridization.

In the normal pituitary, PRL- and LH-producing cells had significantly more mean grain counts per cell and higher percentages of cells positive for PRL-R than GH and TSH cells. PRL-R mRNA was present in all groups of adenomas by in situ hybridization and reverse transcription-PCR. PRL adenomas had a significantly higher density of labeling compared to other adenoma types. Although there was no difference in the levels of PRL-R mRNA in PRL adenomas from men and premenopausal and postmenopausal women, patients treated with bromocriptine before pituitary surgery had significantly lower levels of PRL-R compared to all other groups. These results indicate that in the normal pituitary, PRL and LH cells have the highest level of PRL-R mRNA, whereas PRL adenomas have significantly higher levels of PRL-R mRNA than other types of adenomas, and bromocriptine treatment decreases the levels of PRL-R mRNA in PRL adenomas.
Cloning and Characterization of the Promoter Regions of the Human Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Gene: Analysis of Deoxyribonucleic Acid from Normal Subjects and Patients with Pseudohypoparathyroidism Type 1b*
Authors: Bettoun J. David , Minagawa Masanori , Kwan Mei Yee , Lee Han S. , Yasuda Toshiyuki , Hendy Geoffrey N. , Goltzman David , White John H.
Published: January 2014

Expression of the PTH/PTH-related peptide (PTHrP) receptor (PTHR) in the mouse is controlled by at least two promoters. The downstream promoter (P2) is ubiquitously expressed, whereas expression of the upstream promoter (P1) is largely restricted to kidney. These observations may provide a genetic basis for a human PTH resistance syndrome, pseudohypoparathyroidism type 1b (PHP1b), in which renal, but not osseous, signaling by PTH is defective. We, therefore, cloned and characterized the 5′-end of the human PTHR gene and found that its organization is very similar to that of the mouse. Transcription initiation sites of human P1 and P2 promoters are in similar, but not identical, positions to those of the mouse gene. The identification of a human P2 promoter is significant because no P2-specific human PTHR complementary DNAs have been isolated to date. Southern analysis of genomic DNA from seven PHP1b patients did not reveal any rearrangements in proximal promoter regions or exons encoding 5′-untranslated region sequences. No significant sequence differences were found in clones of normal and patient DNAs encompassing proximal promoter sequences, and untranslated region and signal sequence exons. Thus, in the seven PHP1b patients analyzed, no defects were identified that would influence initiation site selection, stability, or splicing of renal PTHR transcripts. These data indicate that the genetic defect(s) in PHP1b in these patients lies in distal enhancer elements of the gene, in an essential transcriptional regulator, or in some as yet unidentified cofactor required for renal PTH signaling.
The Use of Tolbutamide-Induced Hypoglycemia to Examine the Intraislet Role of Insulin in Mediating Glucagon Release in Normal Humans*
Authors: Peacey Steven R. , Rostami-Hodjegan Amin , George Emad , Tucker Geoffrey T. , Heller Simon R.
Published: January 2014

Disruption of intraislet mechanisms could account for the impaired glucagon response to hypoglycemia in type 1 diabetes. However, in contrast to animals, there is conflicting evidence that such mechanisms operate in humans. We have used iv tolbutamide (T) (1.7 g bolus + 130 mg/h infusion) to create high portal insulin concentrations and compared this with equivalent hypoglycemia using an iv insulin infusion (I) (30 mU/m2·min). Ten normal subjects underwent two hypoglycemic clamps; mean glucose; I (53 ± 1 mg/dL); and T (53 ± 1 mg/dL) (2.9 ± 0.04 mmol/L vs. 2.9 ± 0.05 mmol/L), held for 30 min. During hypoglycemia, mean peripheral insulin levels were greater with I (59 ± 4 mU/L) than T (18 ± 3 mU/L), P < 0.001. Calculated peak portal insulin concentrations were greater during T (282 ± 28 mU/L) than I (78 ± 4 mU/L), P < 0.00005. The demonstration of a reduced glucagon response during T-induced hypoglycemia (111 ± 8 ng/L vs. 135 ± 12 ng/L, P < 0.05) with higher portal insulin concentrations suggests that intraislet mechanisms may contribute to the release of glucagon during hypoglycemia in man.
Inhibin A and Activin A in the First Trimester of Human Pregnancy
Authors: Birdsall Mary , Ledger William , Groome Nigel , Abdalla Hossam , Muttukrishna Shanthi
Published: January 2014

Recent studies show that high concentrations of inhibin A and activin A are present in the maternal serum throughout human pregnancy. The aim of this study was to determine whether the corpus luteum produces significant quantities of inhibin A and activin A during the first trimester of pregnancy. This prospective study examined two groups of women who had blood samples taken from 5–12 weeks gestation. One group consisted of 14 women with donor egg pregnancies (8 singletons and 6 multiples) who did not have corpora lutea, and the other group consisted 5 women with spontaneous pregnancies who had corpora lutea. Inhibin A and activin A were measured at weekly intervals using specific enzyme immunoassays. All pregnancies progressed to term, with healthy babies being delivered.

Maternal serum concentrations of inhibin A significantly increased throughout the study period in the donor egg pregnancies (P < 0.001) and the control pregnancies (P < 0.001). Circulating concentrations of activin A also increased significantly in both the spontaneous and donor egg pregnancies (P < 0.001) during the study period. However, the concentrations of inhibin A and activin A in the first trimester of human pregnancy were not significantly different in the women with or without corpora lutea, suggesting a fetoplacental origin. Multiple donor egg pregnancies were found to have higher concentrations of inhibin A (P < 0.001) and activin A (P < 0.05) compared with singleton donor egg pregnancies, which also supports a placental source.
Biochemical Markers for Puberty in the Monkey Testis: Desmosterol and Docosahexaenoic Acid1
Authors: Connor William E. , Lin Don S. , Neuringer Martha
Published: January 2014

We previously reported that the sperm of rhesus monkeys and humans uniquely contain large amounts of desmosterol not found in other tissues and have a high concentration of the highly polyunsaturated n-3 fatty acid, docosahexaenoic acid (22:6 n-3). However, the lipid composition of the testis, from which sperm originate, is unknown. During puberty, the testis undergoes remarkable morphological changes as testosterone levels rise and sperm production begins. We hypothesized that testicular maturation might also involve dramatic changes in lipid composition. Accordingly, we characterized the sterol and fatty acid composition of the testis of rhesus monkeys throughout the lifespan, from birth to old age.

Although the cholesterol content in the testis remained relatively unchanged throughout life, the desmosterol content first decreased from 59 μg/g in infants to 6 μg/g in prepubertal monkeys, increased to 83 μg/g during puberty, and reached a plateau of 248 μg/g in the young adult, where it remained into old age. The polyunsaturated fatty acid composition of the testis also changed markedly. Docosahexaenoic acid (22:6 n-3) increased from 5.1% of total fatty acids in infants and juveniles to 18.1% in postpubertal young adults. Although some n-6 fatty acids, arachidonic (20:4 n-6) and linoleic (18:2 n-6), decreased from 16.0% and 10.0% in prepubertal juveniles, respectively, to 7.1% and 3.3% in young adults; dihomogamma-linolenic acid (20:3 n-6), the precursor of 1 series PGs, increased greatly from 1.8% to 10.3%. Similar changes occurred in both membrane and storage lipids (phospholipids and triglycerides), respectively. After puberty, the testicular fatty acid pattern remained stable into old age.

Our data demonstrated that puberty is accompanied by substantial changes in the lipid composition of the primate testis. These changes suggest that desmosterol and both n-3 and n-6 polyunsaturated fatty acids may have important roles in sexual maturation.
An Amphotropic Retroviral Vector Expressing a Mutant gsp Oncogene: Effects on Human Thyroid Cells in Vitro1
Authors: Ivan M. , Ludgate M. , Gire V. , Bond J. A. , Wynford-Thomas D.
Published: January 2014

Point mutations of the gsp protooncogene (encoding theα -subunit of the Gs protein) that constitutively activate the cAMP signaling pathway are a common feature of and a plausible causative mechanism for thyroid hyperfunctioning adenomas (hot nodules). To investigate the extent to which mutant gsp acting alone can induce proliferation of thyroid follicular cells, we generated an amphotropic retroviral vector (based on the pBABE-neo plasmid and psi-CRIP packaging line) to permit stable introduction of a hemagglutinin-tagged Gln227→Leu mutant gsp gene into normal human thyrocytes in vitro. The biological activity of the vector was confirmed by detection of HA-tagged Gsp protein expression and induction of cAMP synthesis in selected target cells. Normal human thyroid follicular cells in primary monolayer culture were infected with the gsp retroviral vector or with corresponding vectors expressing mutant H-ras or neo only as positive and negative controls, respectively. Although, as before, mutant ras generated 10–20 well differentiated epithelial colonies/dish of 105 infected cells, with an average lifespan of 15–20 population doublings, only small groups of no more than 15–50 differentiated thyrocytes were observed with the gsp vector. In addition to standard conditions (10% FCS), infections were performed in reduced serum (1% FCS, TSH, and insulin), in the presence of isobutylylmethylxanthine, or in the presence of agents capable of closing gap junctions, with no significant difference in outcome. Although little or no proliferative response was observed regardless of the conditions, there was clear evidence of morphological response (rearrangement of the actin cytoskeleton and increased cell size). The results suggest that gsp mutation may not be a sufficient proliferogenic stimulus by itself to account for hot nodule formation.
Glucose Homeostasis in Children with Falciparum Malaria: Precursor Supply Limits Gluconeogenesis and Glucose Production1
Authors: Dekker Evelien , Hellerstein Marc K. , Romijn Johannes A. , Neese Richard A. , Peshu Norbert , Endert Erik , Marsh Kevin , Sauerwein Hans P.
Published: January 2014

To evaluate glucose kinetics in children with falciparum malaria, basal glucose production and gluconeogenesis and an estimate of the flux of the gluconeogenic precursors were measured in Kenyan children with uncomplicated falciparum malaria before (n = 11) and during infusion of alanine (1.5 mg/kg·min; n = 6). Glucose production was measured by [6,6-2H2]glucose, gluconeogenesis by mass isotopomer distribution analysis of glucose labeled by [2-13C]glycerol.

Basal plasma glucose concentration ranged from 2.1–5.5 mmol/L, and basal glucose production ranged from 3.3–7.3 mg/kg·min. Glucose production was largely derived from gluconeogenesis (73 ± 4%; range, 52–93%). During alanine infusion, plasma glucose increased by 0.4 mmol/L (P = 0.03), glucose production increased by 0.8 mg/kg·min (P = 0.02), and gluconeogenesis increased by 0.8 mg/kg·min (P = 0.04).

We conclude that glucose production in children with uncomplicated falciparum malaria is largely dependent on gluconeogenesis. However, gluconeogenesis is potentially limited by insufficient precursor supply. These data indicate that in children with falciparum malaria, gluconeogenesis fails to compensate in the presence of decreased glycogen flux to glucose, increasing the risk of hypoglycemia.
Two Years of Growth Hormone (GH) Treatment Increase Isometric and Isokinetic Muscle Strength in GH-Deficient Adults*
Authors: Johannsson Gudmundur , Grimby Gunnar , Sunnerhagen Katharina Stibrant , Bengtsson Bengt-Åke
Published: January 2014

GH deficiency in adults is associated with reduced muscle mass and muscle strength. The objective of this trial was to follow the effect of 2 yr of GH treatment in GH-deficient adults on muscle performance in relation to a reference population. Knee extensor and flexor strengths for isometric and isokinetic concentric muscle strength were measured using a Kin-Com dynamometer. Hand-grip strength was measured in both hands. The fatigue index was calculated as the percent reduction in peak torque at 50 repeated isokinetic knee extensions. Superimposed, single twitch electrical stimulation was performed. The GH-deficient subjects had lower isometric knee extensor, knee flexor, and hand-grip strength than the reference population. Two years of GH treatment increased and normalized the mean isometric knee extensor and flexor strengths. The concentric knee flexor and extensor strength at an angular velocity of π rad/s increased, as did the concentric knee flexor strength at an angular velocity of π/3 rad/s. The increase in muscle strength was more marked in younger patients and in patients with lower initial muscle strength than predicted. Quadriceps endurance decreased, whereas the effect of superimposing single twitches on isometric contraction and hand-grip strength was unaffected by the GH treatment. Two years of GH therapy in GH-deficient adults increased and normalized isokinetic and isometric muscle strength studied in proximal muscle groups. Hand-grip strength and the degree of lack of maximal motor unit activation on voluntary isometric knee extensor force did not change. The dynamic local muscle fatigue index decreased.