bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue
Authors: Scott David W. , Wright George W. , Williams P. Mickey , Lih Chih-Jian , Walsh William , Jaffe Elaine S. , Rosenwald Andreas , Campo Elias , Chan Wing C. , Connors Joseph M. , Smeland Erlend B. , Mottok Anja , Braziel Rita M. , Ott German , Delabie Jan , Tubbs Raymond R. , Cook James R. , Weisenburger Dennis D. , Greiner Timothy C. , Glinsmann-Gibson Betty J. , Fu Kai , Staudt Louis M. , Gascoyne Randy D. , Rimsza Lisa M.
Published: January 2014

Key Points

A 20-gene gene expression-based assay accurately and robustly assigns COO subtypes of DLBCL using formalin-fixed paraffin-embedded tissue.
Dental caries and erosion status of 12-year-old Hong Kong children
Authors: Zhang Shinan , Chau Alex MH , Lo Edward CM , Chu Chun-Hung
Published: January 2014

Background

This study aimed to assess the dental caries and erosion status of 12-year-old Hong Kong children and study the determinants of dental caries and dental erosion of these children.

Methods

The survey was performed from 2011 to 2012 with ethics approval. Stratified random sampling was adopted to select 12-year-old children in 7 primary schools in Hong Kong. The participating parents were asked to complete a self-administered questionnaire concerning their children’s diet and oral health habits. The children were examined for caries status with WHO criteria by 3 calibrated examiners. Detection of dental erosion followed Basic Erosive Wear Examination (BEWE) criteria.

Results

A total of 704 children were recruited and 600 (316 boys, 53%) participated in the survey. There were 124 children (21%) with caries experience (DMFT > 0) and their DMFT was 0.34 ± 0.76. About half of their decay was unfilled (DT = 0.16 ± 0.52) The DMFT of girls and boys were 0.45 ± 0.89 and 0.23 ± 0.61, respectively (p = 0.001). Girls also had a higher DT (0.21 ± 0.62 compared with 0.11 ± 0.41, p = 0.013) and FT than boys (0.23 ± 0.63 compared with 0.12 ± 0.44, p = 0.016). Most children (75%) had at least some sign of erosion (BEWE > 0), but no severe erosion (BEWE = 3). Logistic regression showed girls who consumed soft drinks and took vitamin C supplements had higher caries risk. Dental erosion was more severe among the children who had caries experience and consumed fruit juice.

Conclusions

The 12-year-old Hong Kong children had low caries experience, and almost half of the decay was left untreated. Although severe erosion was not found, many children had early signs of erosion.
Enhanced resistive switching phenomena using low-positive-voltage format and self-compliance IrO
x
/GdO
x
/W cross-point memories
Authors: Jana Debanjan , Maikap Siddheswar , Prakash Amit , Chen Yi-Yan , Chiu Hsien-Chin , Yang Jer-Ren
Published: January 2014

Enhanced resistive switching phenomena of IrO
x
/GdO
x
/W cross-point memory devices have been observed as compared to the via-hole devices. The as-deposited Gd2O3 films with a thickness of approximately 15 nm show polycrystalline that is observed using high-resolution transmission electron microscope. Via-hole memory device shows bipolar resistive switching phenomena with a large formation voltage of -6.4 V and high operation current of >1 mA, while the cross-point memory device shows also bipolar resistive switching with low-voltage format of +2 V and self-compliance operation current of <300 μA. Switching mechanism is based on the formation and rupture of conducting filament at the IrO
x
/GdO
x
interface, owing to oxygen ion migration. The oxygen-rich GdO
x
layer formation at the IrO
x
/GdO
x
interface will also help control the resistive switching characteristics. This cross-point memory device has also Repeatable 100 DC switching cycles, narrow distribution of LRS/HRS, excellent pulse endurance of >10,000 in every cycle, and good data retention of >104 s. This memory device has great potential for future nanoscale high-density non-volatile memory applications.
Neuroendocrinology of the Skin1
Authors: Slominski Andrzej , Wortsman Jacobo
Published: January 2014

The classical observations of the skin as a target for melanotropins have been complemented by the discovery of their actual production at the local level. In fact, all of the elements controlling the activity of the hypothalamus-pituitary-adrenal axis are expressed in the skin including CRH, urocortin, and POMC, with its products ACTH, α-MSH, and β-endorphin. Demonstration of the corresponding receptors in the same cells suggests para- or autocrine mechanisms of action. These findings, together with the demonstration of cutaneous production of numerous other hormones including vitamin D3, PTH-related protein (PTHrP), catecholamines, and acetylcholine that share regulation by environmental stressors such as UV light, underlie a role for these agents in the skin response to stress. The endocrine mediators with their receptors are organized into dermal and epidermal units that allow precise control of their activity in a field-restricted manner. The skin neuroendocrine system communicates with itself and with the systemic level through humoral and neural pathways to induce vascular, immune, or pigmentary changes, to directly buffer noxious agents or neutralize the elicited local reactions. Therefore, we suggest that the skin neuroendocrine system acts by preserving and maintaining the skin structural and functional integrity and, by inference, systemic homeostasis.
Pathophysiology and Pharmacological Treatment of Insulin Resistance*
Authors: Matthaei Stephan , Stumvoll Michael , Kellerer Monika , Häring Hans-Ulrich
Published: January 2014

Diabetes mellitus type 2 is a world-wide growing health problem affecting more than 150 million people at the beginning of the new millennium. It is believed that this number will double in the next 25 yr. The pathophysiological hallmarks of type 2 diabetes mellitus consist of insulin resistance, pancreatic β-cell dysfunction, and increased endogenous glucose production. To reduce the marked increase of cardiovascular mortality of type 2 diabetic subjects, optimal treatment aims at normalization of body weight, glycemia, blood pressure, and lipidemia. This review focuses on the pathophysiology and molecular pathogenesis of insulin resistance and on the capability of antihyperglycemic pharmacological agents to treat insulin resistance, i.e., α-glucosidase inhibitors, biguanides, thiazolidinediones, sulfonylureas, and insulin. Finally, a rational treatment approach is proposed based on the dynamic pathophysiological abnormalities of this highly heterogeneous and progressive disease.
Autoimmune Adrenal Insufficiency and Autoimmune Polyendocrine Syndromes: Autoantibodies, Autoantigens, and Their Applicability in Diagnosis and Disease Prediction
Authors: Betterle Corrado , Dal Pra Chiara , Mantero Franco , Zanchetta Renato
Published: January 2014

Recent progress in the understanding of autoimmune adrenal disease, including a detailed analysis of a group of patients with Addison’s disease (AD), has been reviewed. Criteria for defining an autoimmune disease and the main features of autoimmune AD (history, prevalence, etiology, histopathology, clinical and laboratory findings, cell-mediated andhumoral immunity, autoantigens and their autoepitopes, genetics, animal models, associated autoimmune diseases, pathogenesis, natural history, therapy) have been described. Furthermore, the autoimmune polyglandular syndromes (APS) associated with AD (revised classification, animal models, genetics, natural history) have been discussed.

Of Italian patients with primary AD (n = 317), 83% had autoimmune AD. At the onset, all patients with autoimmune AD (100%) had detectable adrenal cortex and/or steroid 21-hydroxylase autoantibodies. In the course of natural history of autoimmune AD, the presence of adrenal cortex and/or steroid 21-hydroxylase autoantibodies identified patients at risk to develop AD. Different risks of progression to clinical AD were found in children and adults, and three stages of subclinical hypoadrenalism have been defined. Normal or atrophic adrenal glands have been demonstrated by imaging in patients with clinical or subclinical AD.

Autoimmune AD presented in four forms: as APS type 1 (13% of the patients), APS type 2 (41%), APS type 4 (5%), and isolated AD (41%). There were differences in genetics, age at onset, prevalence of adrenal cortex/21-hydroxylase autoantibodies, and associated autoimmune diseases in these groups. “Incomplete” forms of APS have been identified demonstrating that APS are more prevalent than previously reported.

A varied prevalence of hypergonadotropic hypogonadism in patients with AD and value of steroid-producing cells autoantibodies reactive with steroid 17α-hydroxylase or P450 side-chain cleavage enzyme as markers of this disease has been discussed. In addition, the prevalence, characteristic autoantigens, and autoantibodies of minor autoimmune diseases associated with AD have been described.

Imaging of adrenal glands, genetic tests, and biochemical analysis have been shown to contribute to early and correct diagnosis of primary non-autoimmune AD in the cases of hypoadrenalism with undetectable adrenal autoantibodies. An original flow chart for the diagnosis of AD has been proposed.
Are There Any Sensitive and Specific Sex Steroid Markers for Polycystic Ovary Syndrome?
Authors: Stener-Victorin Elisabet , Holm Göran , Labrie Fernand , Nilsson Lars , Janson Per Olof , Ohlsson Claes
Published: January 2014

Context

Despite the high prevalence of hyperandrogenemia, the principal biochemical abnormality in women with polycystic ovary syndrome (PCOS), a definitive endocrine marker for PCOS has so far not been identified.

Objective

To identify a tentative diagnostic marker for PCOS, we compared serum levels of sex steroids, their precursors, and main metabolites in women with PCOS and controls.

Design and Methods

In this cross-sectional study of 74 women with PCOS and 31 controls, we used gas and liquid chromatography/mass spectrometry to analyze serum sex steroid precursors, estrogens, androgens, and glucuronidated androgen metabolites; performed immunoassays of SHBG, LH, and FSH; and calculated the LH/FSH ratio.

Results

Androgens and estrogens, sex steroid precursors, and glucuronidated androgen metabolites were higher in women with PCOS than in controls. In multivariate logistic regression analyses, estrone and free testosterone were independently associated with PCOS. The odds ratios per sd increase were 24.2 for estrone [95% confidence interval (CI), 4.0-144.7] and 12.8 for free testosterone (95% CI, 3.1-53.4). In receiver operating characteristic analyses, the area under curve was 0.93 for estrone (95% CI, 0.88-0.98) and 0.91 for free testosterone (95% CI, 0.86-0.97), indicating high sensitivity and specificity.

Conclusion

Women with PCOS have elevated levels of sex steroid precursors, estrogens, androgens, and glucuronidated androgen metabolites as measured with a specific and sensitive mass spectrometry-based technique. The combination of elevated estrone (>50 pg/ml) and free testosterone (>3.3 pg/ml) appeared to discriminate with high sensitivity and specificity between women with and without PCOS.
Glucagon Deficiency Reduces Hepatic Glucose Production and Improves Glucose Tolerance In Adult Mice
Authors: Hancock Aidan S. , Du Aiping , Liu Jingxuan , Miller Mayumi , May Catherine L.
Published: January 2014

The major role of glucagon is to promote hepatic gluconeogenesis and glycogenolysis to raise blood glucose levels during hypoglycemic conditions. Several animal models have been established to examine the in vivo function of glucagon in the liver through attenuation of glucagon via glucagon receptor knockout animals and pharmacological interventions. To investigate the consequences of glucagon loss to hepatic glucose production and glucose homeostasis, we derived mice with a pancreas specific ablation of the α-cell transcription factor, Arx, resulting in a complete loss of the glucagon-producing pancreatic α-cell. Using this model, we found that glucagon is not required for the general health of mice but is essential for total hepatic glucose production. Our data clarifies the importance of glucagon during the regulation of fasting and postprandial glucose homeostasis.
Adrenomedullin Relaxes Rat Uterine Artery: Mechanisms and Influence of Pregnancy and Estradiol
Authors: Ross Gracious R. , Yallampalli Uma , Gangula Pandu R. R. , Reed Luckey , Sathishkumar K. , Gao Haijun , Chauhan Madhu , Yallampalli Chandra
Published: January 2014

Uterine arteries play a major role in regulating uteroplacental blood flow. Failure to maintain blood flow to the uteroplacental compartment during pregnancy often results in intrauterine growth retardation. Immunohistochemical staining of adrenomedullin (AM), an endogenous vasoactive peptide, in uterine artery was intense in pregnant compared to nonpregnant rats, but it is not known whether AM directly relaxes uterine artery or not. In this study, we elucidated the mechanisms of uterine artery relaxation by AM and its regulation by pregnancy and female sex steroids. AM was able to relax uterine artery, and this relaxation was influenced positively by pregnancy and estradiol as evidenced by the increased pD2 and Emax values of AM. Both pregnancy and estradiol treatment to ovariectomized rats amplified RAMP3 expression in uterine arteries while progesterone had no effect. AM-induced uterine artery relaxation is predominantly endothelium-dependent. The AM receptor antagonist CGRP8-37 is more potent than AM22-52 in inhibiting the AM relaxation, indicating the involvement of AM2 receptor subtype. Moreover, AM uses the classical nitric oxide-cGMP pathway along with KCa channels to mediate the vasodilatory effect in uterine artery. In conclusion, sensitivity of uterine artery to AM-induced relaxation is increased with pregnancy or estradiol treatment by increasing RAMP3 expression, suggesting an important role for AM in regulating the uterine hemodynamics, probably maintaining uterine blood flow during pregnancy and in pre- and postmenopausal cardiovascular adaptation differences.
Large Litter Rearing Enhances Leptin Sensitivity and Protects Selectively Bred Diet-induced Obese Rats from Becoming Obese
Authors: Patterson Christa M. , Bouret Sebastien G. , Park Sunny , Irani Boman G. , Dunn-Meynell Ambrose A. , Levin Barry E.
Published: January 2014

Because rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal d 2 (P) 2 and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and α-MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin sensitivity in LL DIO rats fed chow until 10 wk of age. The enhanced ARC leptin receptor binding and reduced adiposity of LL DIO rats persisted after an additional 5 wk on the HE diet. Female LL DIO rats had similar reductions in weight gain on both chow and HE diet vs. normal litter DIO rats. We postulate that LL rearing enhances DIO leptin sensitivity by lowering plasma leptin levels and thereby increasing leptin receptor availability and that this both enhances the ARC-paraventricular nucleus pathway development and protects them from becoming obese.
Comparable Sensitivity of Postmenopausal and Young Women to the Effects of Intranasal Insulin on Food Intake and Working Memory
Authors: Krug Rosemarie , Benedict Christian , Born Jan , Hallschmid Manfred
Published: January 2014

Context

We have previously shown that enhancing brain insulin signaling by intranasal administration of a single dose of the hormone acutely reduces food intake in young men but not women, whereas its improving effects on spatial and working memory are restricted to young women.

Objective

Against the background of animal studies suggesting that low estrogen concentrations are a prerequisite for the anorexigenic impact of central nervous insulin, we extended our foregoing study by assessing intranasal insulin effects in postmenopausal women with comparatively low estrogen concentrations, expecting them to be more sensitive than young women to the anorexigenic effects of the hormone.

Design, Setting, Participants, and Intervention

In a within-subject, double-blind comparison performed at the University of Lübeck, 14 healthy postmenopausal women (body mass index, 23.71 ± 0.6 kg/m2; age, 57.61 ± 1.14 yr) were intranasally administered 160 IU regular human insulin or vehicle.

Main Outcome Measures

Subjects performed a working memory task (digit span) and a hippocampus-dependent visuospatial memory task. Subsequently, free-choice food intake from an ad libitum breakfast buffet was measured.

Results

Contrary to expectations, results in postmenopausal women mirrored those found in young women (22.44 ± 0.63 yr), i.e. insulin administration did not affect food intake (P > 0.46), but did enhance performance in the prefrontal cortex-dependent working memory task (P < 0.05).

Conclusions

Low estrogen levels as present in postmenopausal women do not modulate the effects of intranasal insulin in females, suggesting that in humans as opposed to rats, estrogen signaling does not critically alter central nervous system sensitivity to the effects of insulin on energy homeostasis and cognition.
Increased Psychopathology and Maladaptive Personality Traits, But Normal Cognitive Functioning, In Patients after Long-Term Cure of Acromegaly
Authors: Tiemensma Jitske , Biermasz Nienke R. , van der Mast Roos C. , Wassenaar Moniek J. E. , Middelkoop Huub A. M. , Pereira Alberto M. , Romijn Johannes A.
Published: January 2014

Objective

Active acromegaly is associated with psychopathology, personality changes, and cognitive dysfunction. It is unknown whether, and to what extent, these effects are present after long-term cure of acromegaly.

Aim

The aim of the study was to assess psychopathology, personality traits, and cognitive function in patients after long-term cure of acromegaly.

Design

This was a cross-sectional study.

Patients and Methods

We studied 68 patients after long-term cure (13 ± 1 yr) of acromegaly and 68 matched controls. We compared these data with 60 patients treated for nonfunctioning pituitary macroadenomas (NFMAs) and 60 matched controls. Psychopathology was assessed using the Apathy Scale, Irritability Scale, Hospital Anxiety and Depression Scale, and Mood and Anxiety Symptoms Questionnaire short-form and personality by the Dimensional Assessment of Personality Pathology short-form (DAPP). Cognitive function was assessed by 11 tests.

Results

Compared with matched controls, patients cured from acromegaly scored significantly worse on virtually all psychopathology questionnaires and on several subscales of the DAPP. Compared with NFMA patients, patients cured from acromegaly scored worse on negative affect (P = 0.050) and somatic arousal (P = 0.009) and seven of 18 subscales of the DAPP (P < 0.05). Cognitive function in patients cured from acromegaly did not differ from matched controls or patients treated for NFMA.

Conclusion

Patients with long-term cure of acromegaly show a higher prevalence of psychopathology and maladaptive personality traits but not cognitive dysfunction, compared with matched controls and patients treated for NFMA. These results suggest irreversible effects of previous GH excess, rather than effects of pituitary adenomas per se and/or their treatment, on the central nervous system.
Molecular Basis for Species and Ligand Specificity of a Monoclonal Antibody Raised Against Human IGF-I
Authors: Van Wyk Judson J. , Bruton Eyvonne T.
Published: January 2014

The anti-hIGF-I monoclonal antibody, α-sm1.2, was found to have substantial crossreactivity with human and rat IGF-II, but recognized rat IGF-I only when this ligand was present at very high concentration. (E50 for hIGF-I ∼3.5 ng/tube vs. ∼12,000 ng/tube for rat IGF-I). In the context of previous studies to define the epitope(s) of α-sm1.2, these findings point to the critical importance of aspartic acid at residue 20 in the B domain in determining the species and ligand specificity of this antibody. Previous studies using this antibody in rodent tissues may require reinterpretation in the light of these findings.
Characterization of a Propylthiouracil-Insensitive Type I Iodothyronine Deiodinase*
Authors: Sanders Jo P. , Van der Geyten Serge , Kaptein Ellen , Darras Veerle M. , Kühn Eduard R. , Leonard Jack L. , Visser Theo J.
Published: January 2014

Mammalian type I iodothyronine deiodinase (D1) activates and inactivates thyroid hormone by outer ring deiodination (ORD) and inner ring deiodination (IRD), respectively, and is potently inhibited by propylthiouracil (PTU). Here we describe the cloning and characterization of a complementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreochromis niloticus, tilapia). This complementary DNA codes for a protein of 248 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 126. The 3′ untranslated region contains two putative Sec insertion sequence (SECIS) elements. Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as native tilapia D1 (tD1), i.e. rT3 ≫ T4 > T3 sulfate > T3. Native and recombinant tD1 show equally low sensitivities to inhibition by PTU, iodoacetate, and gold thioglucose compared with the potent inhibitions observed with mammalian D1s. Because the residue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased ORD and somewhat increased IRD activity, but was still insensitive to PTU. These results provide new information about the structure-activity relationship of D1 concerning two characteristic properties, i.e. catalysis of both ORD and IRD, and inhibition by PTU.
Transforming Growth Factor-β1 Induces a Mesenchyme-Like Cell Shape without Epithelial Polarization in Thyrocytes and Inhibits Thyroid Folliculogenesis in Collagen Gel Culture
Authors: Toda Shuji , Matsumura Sueo , Fujitani Noboru , Nishimura Tomohisa , Yonemitsu Nobuhisa , Sugihara Hajime
Published: January 2014

Transforming growth factor-β1 (TGFβ1) induces a mesenchyme-like cell shape in some epithelial cell types. To clarify the role of TGFβ1 in the morphological regulation of thyrocytes, we performed collagen gel culture of porcine thyrocytes with serum-free medium. TGFβ1-nontreated cells organized follicles. In contrast, the cells treated with 10 ng/ml TGFβ1 became spindle shaped, i.e. they resembled mesenchymal fibroblasts, and did not form follicles. To characterize the spindle-shaped cells, we examined the fine structures and expression of thyroglobulin (Tg) and cytoskeletal proteins using electron microscopy, immunohistochemistry, and immunoblotting. TGFβ1-nontreated cells had microvilli at the apical side facing follicle lumen and had basal lamina at the basal side in contact with collagen gel. TGFβ1-treated cells showed both microvilli and basal lamina at the basal side. TGFβ1-nontreated cells expressed Tg, whereas TGFβ1-treated cells showed no expression. TGFβ1-nontreated cells barely expressed vimentin, but they expressed enough cytokeratin. TGFβ1-treated cells extensively displayed vimentin along with the change in shape to become spindle-like and retained a decreased expression of cytokeratin. TSH (10 mU/ml) did not essentially influence any TGFβ1 effects on the cells. These results indicate that TGFβ1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular change and the loss of both epithelial polarization and a function in thyrocytes, and that it results in inhibiting thyroid folliculogenesis with or without TSH.
The Role of the Active Site Cysteine in Catalysis by Type 1 Iodothyronine Deiodinase*
Authors: Sun Ben C. , Harney John W. , Berry Marla J. , Larsen P. Reed
Published: January 2014

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mm concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in Kipropylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mm reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.
Cloning, Novel Promoter Sequence, and Estrogen Regulation of a Rat Oxytocin Receptor Gene*
Authors: Bale Tracy L. , Dorsa Daniel M.
Published: January 2014

Expression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E2). We have cloned and sequenced 4 kilobases (kb) of 5′-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E2, a palindromic estrogen response element (ERE) that is active in estrogen receptor binding was identified within this new sequence, approximately 4 kb 5′ of the translational start site. The ability of E2 to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E2 only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using 125I-ornithine vasotocin in MCF7 cells revealed that E2 dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E2 treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E2 inducibility of OR gene transcription.
The Effects of Orbital Spaceflight on Bone Histomorphometry and Messenger Ribonucleic Acid Levels for Bone Matrix Proteins and Skeletal Signaling Peptides in Ovariectomized Growing Rats*
Authors: Cavolina Jason M. , Evans Glenda L. , Harris Steven A. , Zhang Minzhi , Westerlind Kim C. , Turner Russell T.
Published: January 2014

A 14-day orbital spaceflight was performed using ovariectomized Fisher 344 rats to determine the combined effects of estrogen deficiency and near weightlessness on tibia radial bone growth and cancellous bone turnover. Twelve ovariectomized rats with established cancellous osteopenia were flown aboard the space shuttle Columbia (STS-62). Thirty ovariectomized rats were housed on earth as ground controls: 12 in animal enclosure modules, 12 in vivarium cages, and 6 killed the day of launch for baseline measurements. An additional 18 ovary-intact rats were housed in vivarium cages as ground controls: 8 rats were killed as baseline controls and the remaining 10 rats were killed 14 days later. Ovariectomy increased periosteal bone formation at the tibia-fibula synostosis; cancellous bone resorption and formation in the secondary spongiosa of the proximal tibial metaphysis; and messenger RNA (mRNA) levels for the prepro-α2(1) subunit of type 1 collagen, osteocalcin, transforming growth factor-β, and insulin-like growth factor I in the contralateral proximal tibial metaphysis and for the collagen subunit in periosteum pooled from tibiae and femora and decreased cancellous bone area. Compared to ovariectomized weight-bearing rats, the flight group experienced decreases in periosteal bone formation, collagen subunit mRNA levels, and cancellous bone area. The flight rats had a small decrease in the cancellous mineral apposition rate, but no change in the calculated bone formation rate. Also, spaceflight had no effect on cancellous osteoblast and osteoclast perimeters or on mRNA levels for bone matrix proteins and signaling peptides. On the other hand, spaceflight resulted in an increase in bone resorption, as ascertained from the diminished retention of a preflight fluorochrome label. This latter finding suggests that osteoclast activity was increased. In a follow-up ground-based experiment, unilateral sciatic neurotomy of ovariectomized rats resulted in cancellous bone loss in the unloaded limb in excess of that induced by gonadal hormone deficiency. This additional bone loss was arrested by estrogen replacement. We conclude from these studies that estrogen alters the expression of signaling peptides believed to mediate skeletal adaptation to changes in mechanical usage and likewise modifies the skeletal response to mechanical unloading.
Characterization of the Cholecystokinin and Gastrin Genes from the Bullfrog, Rana catesbeiana: Evolutionary Conservation of Primary and Secondary Sites of Gene Expression
Authors: Rourke Ian J. , Rehfeld Jens F. , Møller Morten , Johnsen Anders H.
Published: January 2014

The gastrin and cholecystokinin (CCK) genes, and the complementary DNAs they encode, have been isolated and sequenced from the bullfrog, Rana catesbeiana. The CCK gene promoter region possess the same four well characterized transcriptional control elements as the human CCK gene, namely an E-box, AP-1 binding site, Sp1 site, and TATA box. In contrast, no obvious regulatory motifs are conserved in the gastrin gene. Alignment of the bullfrog preprohormone sequences with other members of the CCK/gastrin peptide family showed that preproCCK has been conserved to a greater degree during evolution than preprogastrin. In mammalian species, gastrin gene expression is typically associated with the antrum, and CCK with the small intestine and brain. However numerous secondary sites of CCK/gastrin gene expression have also been found. RT-PCR showed a high degree of conservation of both primary and secondary sites of CCK/gastrin production between mammals and the bullfrog, with gastrin messenger RNA being detected in the antrum, duodenum, colon, pancreas, brain, and testes, whereas CCK mRNA was observed in the brain, lung, testes, and throughout the length of the small intestine. In situ hybridization using radiolabeled gene specific antisense oligonucleotides uncovered CCK and gastrin messenger RNA in distinct areas of the bullfrog central nervous system and pituitary gland. Notably, the gastrin gene was expressed in the pituitary gland and hypothalamus of the bullfrog, as previously seen in mammals. This highly preserved tissue expression pattern suggests that gastrin plays specific roles in the hypothalamus and pituitary gland that are distinct from those of CCK. Our findings show that in spite of the structural resemblance, bullfrog CCK and gastrin constitute independent neuroendocrine peptide systems.
Retinoid X Receptors in the Kidney: Their Protein Expression and Functional Significance
Authors: Sugawara Akira , Sanno Naoko , Takahashi Nobuyuki , Osamura R. Yoshiyuki , Abe Keishi
Published: January 2014

Retinoid X receptors (RXRs) heterodimerize with 1,25-dihydroxyvitamin D3 (VD) receptor (VDR), and play important roles in VD-regulated transactivation. VD acts on many tissues including kidney for the regulation of calcium homeostasis. In the kidney, the expression of VDR in the tubular cells has been well studied. In contrast, little is known about the localization and the functional significance of RXRs there. In order to elucidate these questions, we first performed immunohistochemical analyses of rat kidney using isoform-specific antimouse RXR antibodies we have previously reported. Interestingly, all RXR isoforms, predominantly RXRα, mainly localized to the proximal and the distal tubules, but not to the glomeruli. The serial section staining using anti-VDR antibody showed the colocalization of RXRα and VDR in those tubular cells. In order to elucidate the functional significance of endogenous receptors in the tubular cells, we next performed transient transfection studies using the tubular-cell derived Madin-Darby bovine kidney cells, which express both endogenous VDR and RXR. We transfected a reporter plasmid containing direct repeat 3 (DR3) sequence, to which only RXR/VDR heterodimer can bind, and found that VD and 9-cis retinoic acid, as well as VD and RXR selective agonist LG100153, had an additive effect for the DR3 transactivation. Taken together, we speculate that endogenous RXRs co-localize with VDR, and coregulate VD-dependent genes in the tubular cells of the kidney as RXR/VDR heterodimer.
A Protective Role for Heme Oxygenase Expression in Pancreatic Islets Exposed to Interleukin-1β**This work was supported by NIH Grant DK-25705 (to S.G.L.).
Authors: Ye Jing , Laychock Suzanne G.
Published: January 2014

Heme oxygenase (HO)-1 expression was investigated in rat isolated pancreatic islets. Freshly isolated islets showed no evidence of HO-1 expression. After a 20-h culture, there was a small increase in HO-1 in control islets, and interleukin-1β (IL-1β) induced HO-1 expression above control levels. NG-monomethyl-l-arginine inhibited the IL-1β-induced increase in HO-1. Sodium nitroprusside-generated nitric oxide also increased HO-1 expression. CoCl2 induced a concentration- and time-dependent increase in HO-1, but not heat shock protein 70, expression. Cobalt chloride (CoCl2) protected islets from the inhibitory effects of IL-1β on glucose-stimulated insulin release and glucose oxidation. Nickel chloride did not mimic the effects of CoCl2. An inhibitor of HO-1 activity, zinc-protoporphyrin IX (ZnPP), prevented the protective effect of CoCl2 on insulin release with IL-1β but did not affect HO-1 expression or the inhibitory response to IL-1β alone. ZnPP also inhibited the protective effect of hemin in IL-1β-treated islets. CoCl2 inhibited the marked increase in islet nitrite production in response to IL-1β. Cobalt-protoporphyrin IX (CoPP), which increased HO expression and activity, also protected islets from the inhibitory effects of IL-1β, even though IL-1β largely blocked the CoPP-induced increase in HO-1 expression. In βHC9 cells, CoCl2 increased HO-1 expression and HO activity, whereas CoPP directly activated HO. ZnPP inhibited basal and CoCl2-stimulated HO activity. Thus, increased HO-1 expression and/or HO activity in response to CoCl2, CoPP, and hemin, seems to mediate protective responses of pancreatic islets against IL-1β. HO-1 may be protective of β-cells because of the scavenging of free heme, the antioxidant effects of the end-product bilirubin, or the generation of carbon monoxide, which might have insulin secretion-promoting effects and inhibitory effects on nitric oxide synthase.
Exendin-(9–39) Is an Inverse Agonist of the Murine Glucagon-Like Peptide-1 Receptor: Implications for Basal Intracellular Cyclic Adenosine 3′,5′-Monophosphate Levels and β-Cell Glucose Competence**This work was supported by Grant 31-46958.96 from the Swiss National Science Foundation (to B.T.).
Authors: Serre Véronique , Dolci Wanda , Schaerer Elizabeth , Scrocchi Louise , Drucker Daniel , Efrat Shimon , Thorens Bernard
Published: January 2014

The effect of exendin-(9–39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine βTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nm and can be decreased dose dependently by exendin-(9–39). This latter effect was receptor dependent, as aβ -cell line not expressing the GLP-1 receptor was not affected by exendin-(9–39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of βTC-Tet cells in the presence of exendin-(9–39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, βTC-Tet cells; it was also seen with nontransformed mouse β-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9–39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of βTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of theβ -cells.
Role of Nuclear Factor-κB Activation in Cytokine- and Sphingomyelinase-Stimulated Inducible Nitric Oxide Synthase Gene Expression in Vascular Smooth Muscle Cells**This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan.
Authors: Katsuyama Koichi , Shichiri Masayoshi , Marumo Fumiaki , Hirata Yukio
Published: January 2014

Inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα), are known to activate sphingomyelinase (SMase) and nuclear factor-κB (NF-κB) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-κB activation pathway similar to that of IL-1β and TNFα in cultured rat VSMCs. Neutral SMase, although less potently than IL-1β and TNFα, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1β, and TNFα activated NF-κB, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-κB activation stimulated by TNFα, but not by IL-1β. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-κB activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1β and TNFα, but not neutral SMase, caused a transient decrease in IκB-α protein levels, whereas IκB-β protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated IκB-α degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-κB less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-κB activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated IκB-α degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).
Potential Regulatory Roles for G Protein-Coupled Receptor Kinases and β-Arrestins in Gonadotropin-Releasing Hormone Receptor Signaling*
Authors: Neill Jimmy D. , Duck L. Wayne , Musgrove Lois C. , Sellers Jeffrey C.
Published: January 2014

GnRH stimulates gonadotropin secretion, which desensitizes unless the releasing hormone is secreted or administered in a pulsatile fashion. The mechanism of desensitization is unknown, but as the GnRH receptor is G protein coupled, it might involve G protein-coupled receptor kinases (GRKs). Such kinases phosphorylate the intracellular regions of seven-transmembrane receptors, permitting β-arrestin to bind, which prevents the receptor from activating G proteins. Here, we tested the effect of GRKs and β-arrestins on GnRH-induced inositol trisphosphate (IP3) production in COS cells transfected with the GnRH receptor complementary DNA. GRK2, -3, and -6 overexpression inhibited IP3 production by 50–75% during the 30 sec of GnRH treatment. Coexpression of GRK2 and β-arrestin-2 suppressed GnRH-induced IP3 production more than that of either alone. Immunocytochemical staining of rat anterior pituitary revealed that all cells expressed GRK2, -3, and -6; all cells also expressed theβ -arrestins. Western blots on cytosolic extracts of rat pituitaries revealed the presence of GRK2/3 and β-arrestin-1 and -2. The expression of GRKs and β-arrestins by gonadotropes and their inhibition of GnRH-stimulated IP3 production in COS-1 cells expressing the GnRH receptor suggest a potential regulatory role for the GRK/β arrestin paradigm in GnRH receptor signaling.
Orchidectomy Induces a Wave of Apoptotic Cell Death in the Epididymis*
Authors: Fan Xueping , Robaire Bernard
Published: January 2014

The epididymis is the site where spermatozoa are matured and stored. After orchidectomy, this tissue loses up to 80% of its weight. In the prostate, androgen withdrawal by orchidectomy is associated with apoptotic cell death. The objective of the present study was to investigate whether apoptotic cell death is involved in the androgen-dependent weight loss found in the rat epididymis after orchidectomy. Adult male Sprague-Dawley rats were orchidectomized, and apoptotic cells were identified by in situ TUNEL (TdT-mediated dUTP-digoxigenin nick end-labeling) apoptosis detection. Apoptosis first appeared in the epithelium of the initial segment of the epididymis 18 h after orchidectomy, reached a maximum on day 2, and disappeared by day 5 postorchidectomy. In the caput epididymidis, apoptosis was first found after 24 h, reached a maximum by day 3, and was detectable until day 5. In the corpus epididymidis, apoptosis was first seen on day 4, peaked on day 5, and was undetectable by day 6 postorchidectomy. In the cauda epididymidis, apoptosis was first seen on day 5, peaked on day 6, and was occasionally detected on day 7. Throughout the rat epididymis, apoptotic cell death was localized specifically to principal cells. The presence of apoptosis was confirmed with the observation of a ladder of nucleosomal sized DNA fragmentation by using agarose gel electrophoresis. Androgen replacement therapy after orchidectomy demonstrated that apoptosis in the caput, corpus, and cauda epididymidis was androgen dependent. However, androgens alone could not completely prevent apoptosis in the initial segment of the epididymis. Efferent duct ligation induced a similar pattern of apoptosis in the initial segment of the epididymis as that seen after orchidectomy, but there were fewer apoptotic cells in the caput epididymidis, and no apoptotic cell death in the corpus and cauda epididymidis. We conclude that withdrawal of androgen by orchidectomy induces a wave of apoptotic cell death in the epididymis; we hypothesize that apoptosis in the initial segment is caused primarily by withdrawal of androgen as well as by luminal components coming from the testis.
Regulation of Rat DOC-2 Gene during Castration-Induced Rat Ventral Prostate Degeneration and Its Growth Inhibitory Function in Human Prostatic Carcinoma Cells*
Authors: Tseng Ching-Ping , Ely Brent D. , Li Yingming , Pong Rey-Chen , Hsieh Jer-Tsong
Published: January 2014

Androgen is a mitogen as well as a morphogen for prostatic epithelium. However, the detailed mechanisms of these distinct androgenic actions have not yet been delineated. Therefore, we employed differential display PCR to unveil any potential genes that may be involved in these processes. In this study, we report the isolation and characterization of two alternative splicing forms (p82 and p59) of C9 complementary DNA, the rat homolog of the human deletion of ovarian carcinoma 2 (DOC-2) gene and mouse p96 phosphoprotein, from rat ventral prostate (VP). We found that C9 was up-regulated in rat VP after castration, suggesting that C9 may be regulated by androgen receptor directly or indirectly during prostate degeneration. A similar regulatory pattern was also observed in both the seminal vesicle and dorsolateral prostate, but not in the coagulating gland or other androgen-independent organs. Immunohistochemical analysis of rat VP demonstrated that C9 is detected in the basal epithelia and surrounding stromal cells after prolonged castration. Ribonuclease protection assay and Western blot analysis revealed that p59 is the predominant C9 isoform in rat VP. To unveil the function of C9 in cell growth, we transfected p59 complementary DNA into the C4-2 cells, a derivative of the LNCaP prostatic carcinoma cell line. The p59 stable transfectants exhibited a slower growth rate and an increase in the cell fraction in the G1 phase under our experimental conditions. These data indicate that C9-p59 has growth inhibitory activity for prostatic epithelial cells. Taken together, our results suggest that C9 is up-regulated during prostate degeneration process and may play an active role in the proliferation and differentiation of prostatic epithelium.