bluuebunny/crossref_metadata_2025 huggingface dataset:
AIRCRAFT ICING
Authors: Cebeci Tuncer , Kafyeke Fassi
Published: December 2002

▪ Abstract  Full-scale icing experiments and, therefore, certification time and cost can be significantly reduced by developing calculation methods to evaluate the aircraft and system performance for a wide range of icing conditions. This article summarizes calculation methods for icing that include ice accretion, ice system performance, and icing effects on aircraft.
SMALL-SCALEHYDRODYNAMICS INLAKES
Authors: Wüest Alfred , Lorke Andreas
Published: December 2002

▪ Abstract  Recent small-scale turbulence observations allow the mixing regimes in lakes, reservoirs, and other enclosed basins to be categorized into the turbulent surface and bottom boundary layers as well as the comparably quiet interior. The surface layer consists of an energetic wave-affected thin zone at the very top and a law-of-the-wall layer right below, where the classical logarithmic-layer characteristic applies on average. Short-term current and dissipation profiles, however, deviate strongly from any steady state. In contrast, the quasi-steady bottom boundary layer behaves almost perfectly as a logarithmic layer, although periodic seiching modifies the structure in the details. The interior stratified turbulence is extremely weak, even though much of the mechanical energy is contained in baroclinic basin-scale seiching and Kelvin waves or inertial currents (large lakes). The transformation of large-scale motions to turbulence occurs mainly in the bottom boundary and not in the interior, where the local shear remains weak and the Richardson numbers are generally large.
p53 Is Required for 1,25-Dihydroxyvitamin D3-Induced G0 Arrest But Is Not Required for G1 Accumulation or Apoptosis of LNCaP Prostate Cancer Cells
Authors: Polek Tara C. , Stewart LaMonica V. , Ryu Elizabeth J. , Cohen Michael B. , Allegretto Elizabeth A. , Weigel Nancy L.
Published: December 2002

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is an effective agent for inhibiting the growth of prostate cancer cells including LNCaP and PC-3 cell lines. However, the extent of growth inhibition in these cell lines differs because LNCaP cells are much more responsive than PC-3 cells. Previous studies in LNCaP cells have shown that 1,25-(OH)2D3 treatment results in G0/G1 cell cycle accumulation, loss of Ki67 expression, and induction of apoptosis. One difference between the two cell lines is that PC-3 cells lack functional p53, a protein that plays roles both in cell cycle regulation and induction of apoptosis. In this study, the role of p53 in 1,25-(OH)2D3 action was examined using the p53-negative PC-3 cells and a line of LNCaP cells, called LN-56, in which p53 function was shut off using a dominant negative p53 fragment. We found that treatment with 1,25-(OH)2D3 extensively inhibits growth of LN-56 prostate cancer cells lacking p53, but in contrast to the parental LNCaP cells, the LN-56 cells recover rapidly. Moreover, in prostate cancer cells, the synergism between 1,25-(OH)2D3 and 9-cis retinoic acid appears to be dependent on the presence of functional p53; however, 1,25-(OH)2D3-mediated induction of G1 cell cycle accumulation and induction of apoptosis is not.
Mitochondrial Dysfunction Is Involved in Apoptosis Induced by Serum Withdrawal and Fatty Acids in the β-Cell Line Ins-1
Authors: Maestre Isabel , Jordán Joaquín , Calvo Soledad , Reig Juan Antonio , Ceña Valentín , Soria Bernat , Prentki Marc , Roche Enrique
Published: December 2002

The potential toxic effects of high extracellular concentrations of fatty acids were tested in β(INS-1)-cells cultured in the absence of serum, a condition known to alter cell survival in various systems. This may in part mimic the situation in type 1 or 2 diabetes where β-cells are already insulted by various stressful conditions, such as cytokines and oxidative stress. Serum removal caused, over a 36-h period, oxidative stress and an early impairment of mitochondrial function, as revealed by increased superoxide production and markedly reduced mitochondrial membrane potential, but a lack of cytochrome c and apoptosis-inducing factor release in the cytosol. The fatty acids palmitate and oleate considerably accelerated the apoptosis process in serum-starved cells, as revealed by fluorescence-activated cell sorting analysis, morphological changes, chromatin condensation, DNA laddering, poly(ADP-ribose) polymerase cleavage, cytochrome c and apoptosis-inducing factor release, and increased levels of Bax and cytosolic caspase-2. The fatty acids also increased nitric oxide production, apparently independently of inducible nitric oxide synthase induction. Under the same experimental conditions, elevated glucose alone had only a marginal effect on β-cell apoptosis. Together the results indicate that elevated concentrations of fatty acids are particularly efficient in accelerating the rate of apoptosis of already stressed β(INS-1)-cells displaying altered mitochondrial function, and that the mitochondrial arm of the apoptosis process is involved in β-cell lipotoxicity.
Temporally Sensitive Trophic Responsiveness of the Adrenalectomized Rat Anterior Pituitary to Dexamethasone Challenge: Relationship between Mitotic Activity and Apoptotic Sensitivity
Authors: Nolan L. A. , Levy A.
Published: December 2002

Depending on timing and dose, exogenous glucocorticoids induce a wave of apoptosis in the adult rat anterior pituitary, a response that is enhanced by adrenalectomy. In this study, we show that the size of the glucocorticoid-sensitive apoptotic population progressively increases during the week following surgical adrenalectomy, plateaus for a further week, then spontaneously declines to levels seen in intact animals by 4 wk. Mitotic activity, in contrast, rises rapidly post adrenalectomy but returns to baseline within 2 wk. Increased mitotic activity precedes the increase in the population of cells that undergo glucocorticoid-induced apoptosis and the subsequent decline in mitotic activity precedes the decline in apoptotic sensitivity despite persistent elevation of hypothalamic CRH and pituitary proopiomelanocortin transcripts. If glucocorticoid exposure is delayed until 4 wk post adrenalectomy when the apoptotic response has returned to baseline, glucocorticoid withdrawal, by transiently increasing mitotic activity, again primes the formation of an expanded glucocorticoid-sensitive apoptotic cell population. These data suggest that apoptotic sensitivity is largely confined to cells that have recently entered the cell cycle. This observation is further corroborated by demonstrating an abrupt glucocorticoid-induced step-down in the bromodeoxyuridine-labeling index to basal levels in rats given daily injections of bromodeoxyuridine during the week following adrenalectomy.
pp60c-Src Kinase Mediates Growth Effects of the Full-Length Precursor Progastrin1–80 Peptide on Rat Intestinal Epithelial Cells, in Vitro
Authors: Brown D. , Yallampalli U. , Owlia A. , Singh P.
Published: December 2002

Growth factor effects of precursor forms of gastrins have become evident in recent years. However, intracellular pathways that mediate growth effects of the precursor molecules are not known. In previous studies, we reported an increase in Tyr phosphorylation of pp60c-Src in intestinal epithelial cells (IEC) in response to the fully processed form of gastrin [gastrin1–17 (G17)]. We have now examined whether c-Src kinase is similarly phosphorylated and activated in response to the full-length precursor molecule, progastrin (PG)1–80, (recombinant human PG) in IEC cells. We found a significant increase in pp60c-Src kinase activity in response to both G17 and PG (0.1–1.0 nm), suggesting that growth effects of both the precursor and fully processed gastrin molecules may be mediated via similar pathways. On the other hand, pp62c-Yes was not phosphorylated or activated in response to either G17 or PG. To examine whether c-Src kinase mediates proliferative effects of PG, IEC cells were microinjected with anti-Src-IgG and 3H-thymidine (3H-Tdr) uptake of the cells measured. Control cells received nonimmune IgG. The 3H-Tdr uptake of cells stimulated with 1.0 nm PG was significantly reduced in cells microinjected with anti-c-Src-IgG; control IgG had no effect. In cells stimulated with 1.0% fetal calf serum, microinjection with c-Src-IgG had no effect on 3H-Tdr uptake. The specificity of the effect was further confirmed by blocking the inhibitory effect of anti-c-Src-IgG with antigenic Src peptide. These results suggest that activation of c-Src kinase likely represents a critical step in mediating proliferative effects of both the precursor and fully processed forms of gastrins on IEC.
Estrogen Increases Locomotor Activity in Mice through Estrogen Receptor α: Specificity for the Type of Activity
Authors: Ogawa Sonoko , Chan Johnny , Gustafsson Jan-Åke , Korach Kenneth S. , Pfaff Donald W.
Published: December 2002

Estrogens are known to increase running wheel activity of rodents primarily by acting on the medial preoptic area (mPOA). The mechanisms of this estrogenic regulation of running wheel activity are not completely understood. In particular, little is known about the separate roles of two types of estrogen receptors, ERα and ERβ, both of which are expressed in mPOA neurons. In the present study the effects of continuous estrogen treatment on running wheel activity were examined in male and female mice specifically lacking either the ERα (αERKO) or the ERβ (βERKO) gene. Mice were gonadectomized and 1 wk later implanted with either a low dose (16 ng/d) or a high dose (160 ng/d) of estradiol benzoate (EB) or with a placebo control pellet. Home cage running wheel activity was recorded for 9 d starting 10 d after EB implants. The same mice were also tested for open field activity before and after EB implants. In both female and male αERKO mice, running wheel activity was not different from that in corresponding wild-type (αWT) mice in placebo control groups. In both females and males it was increased by EB only in αWT, not αERKO, mice. In βERKO mice, on the other hand, both doses of EB equally increased running wheel activity in both sexes just as they did in βWT mice. Absolute numbers of daily revolutions of EB-treated groups, however, were significantly lower in βERKO females compared with βWT females. Before EB treatment, gonadectomized αERKO female were significantly less active than αWT mice in open field tests, whereas βERKO females tended to be more active than βWT mice. In male mice there were no effect of ERα or ERβ gene knockout on open field activity. Unlike its effect on running wheel activity, EB treatment induced only a small increase in open field activity in female, but not male, mice. These findings indicate that 1) in both sexes estrogenic regulation of running wheel activity is primarily mediated through the ERα, not the ERβ; and 2) hormone/genotype effects are specific to the type of locomotor activity (i.e. home cage running wheel activity and open field activity) measured.
Anandamide Activity and Degradation Are Regulated by Early Postnatal Aging and Follicle-Stimulating Hormone in Mouse Sertoli Cells
Authors: Maccarrone Mauro , Cecconi Sandra , Rossi Gianna , Battista Natalia , Pauselli Riccardo , Finazzi-Agrò Alessandro
Published: December 2002

Anandamide (AEA), a prominent member of the endogenous ligands of cannabinoid receptors (endocannabinoids), is known to adversely affect female fertility. However, a potential role of AEA in male reproductive functions is unknown. Here we report evidence that immature mouse Sertoli cells have the biochemical tools to bind and inactivate AEA, i.e. a functional type-2 cannabinoid receptor (CB2R), a selective AEA membrane transporter, and an AEA-degrading enzyme fatty acid amide hydrolase. We show that, unlike CB2R, the activity of AEA membrane transporter and the activity and expression of FAAH decrease, whereas the apoptosis-inducing activity of AEA increases with age during the neonatal period. We also show that FSH reduces the apoptotic potential of AEA, but not that of its nonhydrolyzable analog methanandamide. Concomitantly, FSH enhances FAAH activity in a manner dependent on mRNA transcription and protein synthesis and apparently involving cAMP. These data demonstrate that Sertoli cells partake in the peripheral endocannabinoid system, and that FSH reduces the apoptotic potential of AEA by activating FAAH. Taken together, it can be suggested that the endocannabinoid network plays a role in the hormonal regulation of male fertility.
Trafficking of Mutant Carboxypeptidase E to Secretory Granules in a β-Cell Line Derived from Cpefat/Cpefat Mice
Authors: Cawley Niamh X. , Rodriguez Yazmin M. , Maldonado Alex , Loh Y. Peng
Published: December 2002

We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic β-cell line derived from the Cpefat/Cpefat mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.
Stimulation of Combinatorial Expression of Prolactin and Glycoprotein Hormone α-Subunit Genes by Gonadotropin-Releasing Hormone and Estradiol-17β in Single Rat Pituitary Cells during Aggregate Cell Culture
Authors: Hauspie A. , Seuntjens E. , Vankelecom H. , Denef C.
Published: December 2002

Previously we showed the existence of rat and mouse anterior pituitary cells coexpressing mRNA from two or more hormone genes in which production and/or storage of the corresponding hormones were not detectable. To substantiate a putative function for these cells, we investigated whether these phenotypes were retained during long-term reaggregate cell culture and whether protagonist regulatory factors could expand cell populations expressing particular hormone mRNA combinations. After 4-wk culture and treatments, aggregates were trypsinized and single cells collected by means of a fluo-rescence-activated cell sorter. Hormone mRNAs were detected by single-cell RT-PCR. Combinatorial hormone mRNA expression was retained in culture. Both estradiol (E2) and GnRH (1 nm) markedly augmented the proportion of cells expressing prolactin (PRL) mRNA together with other hormone mRNAs and cells expressing glycoprotein subunit (GSU)-α mRNA together with other hormone mRNAs. GnRH strongly increased the proportion of cells containing αGSU mRNA alone, but E2 did not. GnRH and (E2) affected the expansion of a population (∼20% of all cells) coexpressing PRL and αGSU mRNA without βGSUs. Immunostaining of stored hormone on tissue sections revealed colocalization of PRL and αGSU in the E2- but not in the GnRH-treated cells. The present findings suggest that cells coexpressing different pituitary hormone mRNAs form a distinct population that survives without extrapituitary factors. Their occurrence can be markedly modified by regulatory factors. Certain hormone regimens favor unique coexpressions distinctly at mRNA and protein level. These peculiar characteristics support the notion that combinatorial expression of hormone genes in the pituitary serves a biological role.
Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells
Authors: Roh Jae-Sook , Bondestam Jonas , Mazerbourg Sabine , Kaivo-Oja Noora , Groome Nigel , Ritvos Olli , Hsueh Aaron J. W.
Published: December 2002

Ovarian inhibin production is stimulated by FSH and several TGFβ family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFβ/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-α content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using 32P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-α and inhibin-β subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-α gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-α promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-α gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-α promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.
Nongenomic Activity and Subsequent c-fos Induction by Estrogen Receptor Ligands Are Not Sufficient to Promote Deoxyribonucleic Acid Synthesis in Human Endometrial Adenocarcinoma Cells
Authors: Singleton David W. , Feng Yuxin , Burd Craig J. , Khan Sohaib A.
Published: December 2002

Estrogen 17β-estradiol (E2) rapidly modulates several signaling pathways related to cell growth, preservation, and differentiation. The physiological role of these nongenomic effects with regard to downstream outcomes, and the relationship with transcriptional estrogen activity are unclear. Furthermore, the ability of selective estrogen receptor modulators (SERMs) to trigger nongenomic actions is largely unknown. To determine whether estrogen receptor (ER) ligands exert nongenomic activity in endometrial adenocarcinoma cells, and whether this activity affects transcription and DNA synthesis, we challenged human Ishikawa cells with E2 or partial ER agonists 4-hydroxytamoxifen (OHT) and raloxifene (ral). Serum-starved Ishikawa cells exposed for 5 min to 0.1 nm E2 showed induced phosphorylation of MAPK (ERK1/2). Ral and 4-OHT each at 1 nm also stimulated ERK in a rapid transient manner. E2 and 4-OHT induced proto-oncogene c-fos mRNA expression in Ishikawa cells within 30 min, but ral had no effect. In contrast to nongenomic action, only E2 stimulated expression of an estrogen response element (ERE)-driven luciferase (LUC) reporter gene. To examine DNA synthesis, [3H]-thymidine incorporation was measured in serum-starved cultures exposed to E2 or partial agonists for 2 d. E2 at 1 nm stimulated thymidine uptake in an ERK-dependent manner, but 1 nm 4-OHT, 1 nm ral, and 0.1-nm concentrations of E2 had no significant effects. Taken together, these data indicate that both nongenomic and direct transcriptional ER effects are likely required to promote DNA synthesis.
Sex Difference in Hepatic Peroxisome Proliferator-Activated Receptor α Expression: Influence of Pituitary and Gonadal Hormones
Authors: Jalouli Masoumeh , Carlsson Linda , Améen Caroline , Lindén Daniel , Ljungberg Anna , Michalik Liliane , Edén Staffan , Wahli Walter , Oscarsson Jan
Published: December 2002

Peroxisome proliferator-activated receptor (PPAR) α is a nuclear receptor that is mainly expressed in tissues with a high degree of fatty acid oxidation such as liver, heart, and skeletal muscle. Unsaturated fatty acids, their derivatives, and fibrates activate PPARα. Male rats are more responsive to fibrates than female rats. We therefore wanted to investigate if there is a sex difference in PPARα expression. Male rats had higher levels of hepatic PPARα mRNA and protein than female rats. Fasting increased hepatic PPARα mRNA levels to a similar degree in both sexes. Gonadectomy of male rats decreased PPARα mRNA expression to similar levels as in intact and gonadectomized female rats. Hypophysectomy increased hepatic PPARα mRNA and protein levels. The increase in PPARα mRNA after hypophysectomy was more pronounced in females than in males. GH treatment decreased PPARα mRNA and protein levels, but the sex-differentiated secretory pattern of GH does not determine the sex-differentiated expression of PPARα. The expression of PPARα mRNA in heart or soleus muscle was not influenced by gender, gonadectomy, hypophysectomy, or GH treatment. In summary, pituitary-dependent hormones specifically regulate hepatic PPARα expression. Sex hormones regulate the sex difference in hepatic PPARα levels, but not via the sexually dimorphic GH secretory pattern.
New Evidence of Similarity between Human and Plant Steroid Metabolism: 5α-Reductase Activity in Solanum malacoxylon
Authors: Rosati Fabiana , Danza Giovanna , Guarna Antonio , Cini Nicoletta , Racchi Milvia Luisa , Serio Mario
Published: December 2002

The physiological role of steroid hormones in humans is well known, and the metabolic pathway and mechanisms of action are almost completely elucidated. The role of plant steroid hormones, brassinosteroids, is less known, but an increasing amount of data on brassinosteroid biosynthesis is showing unexpected similarities between human and plant steroid metabolic pathways. Here we focus our attention on the enzyme 5α-reductase (5αR) for which a plant ortholog of the mammalian system, DET2, was recently described in Arabidopsis thaliana. We demonstrate that campestenone, the natural substrate of DET2, is reduced to 5α-campestanone by both human 5αR isozymes but with different affinities. Solanum malacoxylon, which is a calcinogenic plant very active in the biosynthesis of vitamin D-like molecules and sterols, was used to study 5αR activity. Leaves and calli were chosen as examples of differentiated and undifferentiated tissues, respectively. Two separate 5αR activities were found in calli and leaves of Solanum using campestenone as substrate. The use of progesterone allowed the detection of both activities in calli. Support for the existence of two 5αR isozymes in S. malacoxylon was provided by the differential actions of inhibitors of the human 5αR in calli and leaves. The evidence for the presence of two isozymes in different plant tissues extends the analogies between plant and mammalian steroid metabolic pathways.
α-Melanocyte Stimulating Hormone Prevents Lipopolysaccharide-Induced Vasculitis by Down-Regulating Endothelial Cell Adhesion Molecule Expression
Authors: Scholzen T. E. , Sunderkötter C. , Kalden D.-H. , Brzoska T. , Fastrich M. , Fisbeck T. , Armstrong C. A. , Ansel J. C. , Luger T. A.
Published: December 2002

The neuroendocrine hormone α-melanocyte stimulating hormone (MSH) has profound antiinflammatory and immunomodulating properties. Here we have examined the possibility that α-MSH may interfere with the expression and function of cell adhesion molecules (CAMs) expressed by human dermal microvascular endothelial cells (HDMECs) in response to lipopolysaccharide (LPS) or TNFα in vitro and in vivo. In HDMEC, α-MSH (10−8/10−12m) profoundly reduced the mRNA and protein expression of E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 induced by LPS or TNFα as determined by semiquantitative RT-PCR, ELISA, and fluorescence-activated cell sorter analysis. In addition, α-MSH significantly impaired the LPS-induced ICAM-1 and VCAM-1-mediated adhesion of lymphocytes to HDMEC monolayer in a functional adhesion assay. Likewise, α-MSH effectively inhibited the transcription factor nuclear factor-κB activation in HDMEC, which is required for CAM gene expression. Importantly in vivo, in murine LPS-induced cutaneous vasculitis (local Shwartzman reaction), a single ip injection of α-MSH significantly suppressed the deleterious vascular damage and hemorrhage by inhibiting the sustained expression of vascular E-selectin and VCAM-1. This persistent expression has been implicated in the dysregulation of diapedesis and activation of leukocytes, which subsequently leads to hemorrhagic vascular damage. Our findings indicate that α-MSH may have an important therapeutical potential for the treatment of vasculitis, sepsis, and inflammatory diseases.
Lysyl Oxidase Gene Expression and Enzyme Activity in the Rat Ovary: Regulation by Follicle-Stimulating Hormone, Androgen, and Transforming Growth Factor-β Superfamily Members in Vitro
Authors: Harlow Christopher R. , Rae Mick , Davidson Lindsay , Trackman Philip C. , Hillier Stephen G.
Published: December 2002

Lysyl oxidase (LOX) catalyzes the final enzymatic reaction required for cross-linking of collagen and elastin fibers and therefore has a crucial role in regulating the formation and maintenance of extracellular matrix in the ovary. LOX mRNA is abundantly expressed in rat granulosa cells. To examine how regulation of LOX in the ovary might influence follicular development, we studied LOX mRNA expression and enzyme activity in rat granulosa cells from late preantral/early antral follicles in vitro. FSH dose dependently inhibited LOX mRNA and enzyme activity (50% reduction at 10 ng/ml) in vitro, and FSH action was mimicked by 8-bromo-cAMP, suggesting FSH action via elevation of cAMP. Dihydrotestosterone alone enhanced LOX mRNA and enzyme activity, but potentiated the effect of FSH, causing a further reduction. TGFβ1 alone dose dependently enhanced LOX mRNA (5-fold increase at 10 ng/ml) and activity (1.5-fold increase). FSH dose dependently inhibited the increase in LOX mRNA and activity caused by TGFβ1 (by up to 84% and 80%, respectively). Growth differentiation factor-9 (GDF-9) and activin A, at the same concentration as TGFβ1 (10 ng/ml), stimulated LOX mRNA and activity within 6 h, although overall expression was higher at 48 h. All three factors when combined with FSH further reduced both mRNA and enzyme activity (by up to 60%) compared with FSH alone. These findings indicate control of LOX at endocrine, paracrine, and autocrine levels within the ovary and suggest coordinated regulation of ovarian extracellular matrix during follicular development, with FSH determining whether local factors act as stimulators or inhibitors of LOX.
Immunolesion of Norepinephrine and Epinephrine Afferents to Medial Hypothalamus Alters Basal and 2-Deoxy-d-Glucose-Induced Neuropeptide Y and Agouti Gene-Related Protein Messenger Ribonucleic Acid Expression in the Arcuate Nucleus
Authors: Fraley G. S. , Ritter S.
Published: December 2002

Neuropeptide Y (NPY) and agouti gene-related protein (AGRP) are orexigenic peptides of special importance for control of food intake. In situ hybridization studies have shown that NPY and AGRP mRNAs are increased in the arcuate nucleus of the hypothalamus (ARC) by glucoprivation. Other work has shown that glucoprivation stimulates food intake by activation of hindbrain glucoreceptor cells and requires the participation of rostrally projecting norepinephrine (NE) or epinephrine (E) neurons. Here we determine the role of hindbrain catecholamine afferents in glucoprivation-induced increase in ARC NPY and AGRP gene expression. The selective NE/E immunotoxin saporin-conjugated antidopamineβ-hydroxylase (anti-dβh) was microinjected into the medial hypothalamus and expression of AGRP and NPY mRNA was analyzed subsequently in the ARC under basal and glucoprivic conditions using 33P-labeled in situ hybridization. Saporin-conjugated anti-dβh virtually eliminated dβh-immunoreactive terminals in the ARC without causing nonspecific damage. These lesions significantly increased basal but eliminated 2-deoxy-d-glucose-induced increases in AGRP and NPY mRNA expression. Results indicate that hindbrain catecholaminergic neurons contribute to basal NPY and AGRP gene expression and mediate the responsiveness of NPY and AGRP neurons to glucose deficit. Our results also suggest that catecholamine neurons couple potent orexigenic neural circuitry within the hypothalamus with hindbrain glucose sensors that monitor brain glucose supply.
Central Administration of Neuropeptide Y Reduces α-Melanocyte-Stimulating Hormone-Induced Cyclic Adenosine 5′-Monophosphate Response Element Binding Protein (CREB) Phosphorylation in Pro-Thyrotropin-Releasing Hormone Neurons and Increases CREB Phosphorylation in Corticotropin-Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus
Authors: Sarkar Sumit , Lechan Ronald M.
Published: December 2002

Neuropeptide Y (NPY) has a potent inhibitory effect on TRH gene expression in the paraventricular nucleus (PVN) and contributes to the fall in circulating thyroid hormone levels during fasting mediated by a reduction in serum leptin levels. Because α-MSH activates the TRH gene by increasing the phosphorylation of CREB in the nucleus of these neurons, we raised the possibility that at least one of the mechanisms by which NPY reduces TRH mRNA in hypophysiotropic neurons is by antagonizing the ability of α-MSH to phosphorylate CREB. As NPY increases CRH mRNA in the hypothalamus, we further determined whether intracerebroventricular (icv) administration of NPY regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. NPY [10 μg in artificial CSF (aCSF)] was administered into the lateral ventricle icv 30 min before the icv administration of aCSF or α-MSH (10 μg in aCSF), the latter in a dose previously demonstrated to increase proTRH mRNA and phosphorylate CREB in TRH neurons. By double-labeling immunocytochemistry, only few TRH neurons in the PVN contained phosphoCREB (PCREB) in animals treated only with aCSF (4 ± 0.2%) or with NPY followed by aCSF (9.7 ± 2.5), whereas α-MSH-infused animals dramatically increased the percentage of TRH neurons containing PCREB (75.3 ± 6.9%). Pretreatment with NPY before α-MSH infusion, however, significantly reduced the percentage of TRH neurons containing PCREB (40.8 ± 3.5%) compared with α-MSH infused animals (P = 0.01). Only 12.2 ± 0.9% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle-treated animals, whereas 30 min following NPY infusion, the number of CRH neurons containing PCREB increased dramatically to 88 ± 2.9%. Whereas α-MSH infusion increased the percentage of CRH neurons that contained PCREB to 56 ± 2.2% compared with control, animals pretreated with NPY further increased the number of CRH neurons colocalizing with PCREB to 87 ± 2.5%. These data demonstrate a functional interaction between NPY and α-MSH in the regulation of proTRH neurons in the PVN, suggesting that NPY can antagonize α-MSH induced activation of the TRH gene by interfering with melanocortin signaling at the postreceptor level, preventing the phosphorylation of CREB. In contrast, NPY infusion increases the phosphorylation of CREB in CRH neurons, indicating that NPY has independent effects on discrete populations of neurons in the PVN, presumably mediated through different signaling mechanisms.
Lysophosphatidic Acid Inhibits C-Type Natriuretic Peptide Activation of Guanylyl Cyclase-B
Authors: Abbey Sarah E. , Potter Lincoln R.
Published: December 2002

C-type natriuretic peptide (CNP), found in endothelial cells, chondrocytes, and neurons, binds its cognate transmembrane receptor, natriuretic peptide receptor-B (NPR-B/GC-B), and stimulates the synthesis of the intracellular signaling molecule, cGMP. The known physiologic consequences of this binding event are vasorelaxation, inhibition of cell proliferation, and the stimulation of long bone growth. Here we report that 10% fetal bovine serum markedly reduced CNP-dependent cGMP elevations in NIH3T3 fibroblast. The purified serum components platelet-derived growth factor and lysophosphatidic acid (LPA) mimicked the effect of serum on CNP-dependent cGMP elevations, but the latter factor resulted in the most dramatic reductions. The LPA-dependent inhibition was rapid and dose dependent, having t1/2 and IC50 values of approximately 5 min and 3.0 μm LPA, respectively. The decreased cGMP concentrations resulted from reduced CNP-dependent NPR-B guanylyl cyclase activity that did not require losses in receptor protein or activation of protein kinase C, indicating a previously undescribed desensitization pathway. These data suggest that NPR-B is repressed by LPA and that one mechanism by which LPA exerts its effects is through the heterologous desensitization of the CNP/NPR-B/cGMP pathway. We hypothesize that cross-talk between the LPA and CNP signaling pathway maximizes the response of fibroblasts in the wound-healing process.
The Morphometry of Astrocytes in the Rostral Preoptic Area Exhibits a Diurnal Rhythm on Proestrus: Relationship to the Luteinizing Hormone Surge and Effects of Age
Authors: Cashion Adrienne B. , Smith Matthew J. , Wise Phyllis M.
Published: December 2002

The morphometry of astrocytes in the arcuate nucleus exhibits cyclic changes during the estrous cycle leading to dynamic changes in the communication between neurotransmitters and neuropeptides that regulate pituitary hormone secretion. Data suggest that remodeling of direct and/or indirect inputs into GnRH neurons may influence the timing and/or amplitude of the preovulatory LH surge in young rats. We have previously found that aging alters the timing and amplitude of the LH surge. Therefore, the purpose of this study was to focus on the rostral preoptic area where GnRH cell bodies reside. We assessed the possibility that the morphometry of astrocytes in the rostral preoptic area displays time-related and age-dependent changes on proestrus.

Our results demonstrate that, in young rats, astrocyte cell surface area decreases between 0800 h and 1200 h, before the initiation of the LH surge. Changes in surface area over the cycle were specific to astrocytes in close apposition to GnRH neurons. In contrast, in middle-aged rats astrocyte surface area was significantly less than in young rats and did not change during the day. These findings suggest that a loss of astrocyte plasticity could lead to the delayed and attenuated LH surge that has been previously observed in middle-aged rats.
Menkes Protein Contributes to the Function of Peptidylglycine α-Amidating Monooxygenase
Authors: Steveson Tami C. , Ciccotosto Giuseppe D. , Ma Xin-Ming , Mueller Gregory P. , Mains Richard E. , Eipper Betty A.
Published: December 2002

Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine α-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.
Anion Selectivity by the Sodium Iodide Symporter
Authors: Van Sande J. , Massart C. , Beauwens R. , Schoutens A. , Costagliola S. , Dumont J. E. , Wolff J.
Published: December 2002

The iodide transporter of the thyroid (NIS) has been cloned by the group of Carrasco. The NIS-mediated transport was studied by electrophysiological methods in NIS-expressing Xenopus oocytes. Using this method, the anion selectivity of NIS was different from that previously reported for thyroid cells, whereas perchlorate and perrhenate were found not transported. In this study we compared the properties of human NIS, stably transfected in COS-7 cells to those of the transport in a thyroid cell line, the FRTL5 cells, by measuring the transport directly. We measured the uptake of 125I−, 186ReO4−, and 99mTcO4− and studied the effect on it of known competing anions, i.e. ClO4−, SCN−, ClO3−, ReO4−, and Br−. We conclude that the properties of the NIS transporter account by themselves for the properties of the thyroid iodide transporter as described previously in thyroid slices. The order of affinity was: ClO4− > ReO4− > I− ≥ SCN− > ClO3− > Br−. NIS is also inhibited by dysidenin (as in dog thyroid).
Ghrelin Protects Against Ethanol-Induced Gastric Ulcers in Rats: Studies on the Mechanisms of Action
Authors: Sibilia V. , Rindi G. , Pagani F. , Rapetti D. , Locatelli V. , Torsello A. , Campanini N. , Deghenghi R. , Netti C.
Published: December 2002

Ghrelin, the endogenous ligand for GH secretagogue receptors, has been reported to influence acid gastric secretion and motility, but its potential gastroprotective effect is unknown. The aims of this study were 1) to examine the effects of central and peripheral administration of ghrelin on ethanol-induced gastric ulcers in conscious rats, and 2) to investigate the possible roles of nitric oxide (NO), vagal nerve, and sensory fibers in the gastric effects of ghrelin.

Ghrelin was administered either intracerebroventricularly or sc 30 min before ethanol, and mucosal lesions were examined macroscopically. Additionally, rats were either treated with the inhibitor of NO synthesis Nω-nitro-l-arginine methyl ester (L-NAME) or underwent bilateral cervical vagotomy or capsaicin-induced sensory denervation. Conventional histology and immunohistochemistry for ghrelin, gastrin, and somatostatin were performed on gastric specimens from representative rats.

Central ghrelin (4–4000 ng/rat) dose-dependently reduced ethanol-induced gastric ulcers by 39–77%. Subcutaneous ghrelin administration (80 μg/kg) reduced ulcer depth only. L-NAME and capsaicin, but not vagotomy, prevented the gastroprotective effect of central ghrelin (4000 ng/rat).

This is the first evidence that ghrelin exerts a potent central gastroprotective activity against ethanol-induced lesions. The gastroprotective effect of ghrelin is mediated by endogenous NO release and requires the integrity of sensory nerve fibers.
Pubertal Acceleration of Pulsatile Gonadotropin-Releasing Hormone Release in Male Rats as Revealed by Microdialysis
Authors: Harris Glenn C. , Levine Jon E.
Published: December 2002

A microdialysis technique was used in male rats to directly assess the postulate that pubertal maturation is associated with accelerated GnRH pulsatility. Juvenile male rats, postnatal d 43 or 45 (n = 4) were stereotaxically fitted with guide cannulas directed toward the lateral median eminence, and repeated microdialysis experiments were conducted over 4–6 d. In each session, samples were collected continuously over 12 h (0900–2100 h) at 5-min intervals Results from individual peripubertal animals were pooled into two time bins for postnatal d 45–47 and 48–50, respectively, and GnRH characteristics were compared between the two epochs. The GnRH pulse frequency and mean GnRH concentration were significantly elevated at 48–50 d compared with 45–47 d. The GnRH pulsatility characteristics for 45–47 d vs. 48–50 d were as follows: pulse frequency, 0.74 ± 0.16 vs. 1.79 ± 0.19 pulses/h (P < 0.05); pulse amplitude, 254.1 ± 22.3 vs. 347.2 ± 15.8 Δpg/ml (difference in value from trough to peak); and mean release, 0.55 ± 0.03 vs. 2.04 ± 0.04 pg/5 min (P < 0.05). An additional two rats were dialyzed only once on postnatal d 50 to assess the effects of repeated sampling; the GnRH pulse characteristics in these animals were similar to those in rats sampled for a third or fourth time on postnatal d 48–50. To further assess the possible effects of repeated sampling on GnRH release profiles, a group of adult male rats (postnatal d 95–105; n = 3) was also dialyzed on four consecutive days. In these rats no significant alteration in GnRH pulse generator activity was observed over the four sessions. Moreover, the increase in GnRH pulse frequency observed in the peripubertal rats was found to be sustained in adult animals. To better understand the temporal relationship of GnRH pulse generator activity to reproductive maturation, groups of male rats were killed from postnatal d 45–56 along with an adult group at 95–105 d (n = 5/group) and examined for physiological signs of reproductive development. Gradual increases in serum levels of LH and testosterone and decreases in FSH and inhibin B were seen from postnatal d 45–56 to adulthood. Mature spermatozoa were found in the vas deferens by postnatal d 53. Our results demonstrate that in the late juvenile stage of male rat development, GnRH pulse generator activity is gradually accelerated over the course of consecutive days. This acceleration occurs over a period during which serum LH and testosterone are rising to adult levels, and it precedes the presence of mature spermatozoa in the vas deferens by 3 d. Our observations provide direct support for the hypothesis that an acceleration of GnRH pulsatility is the critical neural stimulus for the initiation of pubertal maturation in males. The peripheral and central cues that prompt the pubertal activation of the GnRH pulse generator remain to be characterized.
An Evolutionarily Conserved Form of Gonadotropin-Releasing Hormone Coordinates Energy and Reproductive Behavior
Authors: Temple Jennifer L. , Millar Robert P. , Rissman Emilie F.
Published: December 2002

GnRH is the master neuropeptide that coordinates and regulates reproduction in all vertebrates and in some nonvertebrate species. Sixteen forms of GnRH have been isolated in brain. In the vast majority of species, two or more forms occur in anatomically and developmental distinct neuronal populations. In mammalian brain, two GnRH forms, mammalian (GnRH-I) and chicken-II (GnRH-II), exist. The distribution and functions of GnRH-I have been well characterized and intensively studied. However, the function of GnRH-II, which is the most evolutionarily conserved form of GnRH, has been elusive. Here we demonstrate that in a primitive mammal, the musk shrew (Suncus murinus), GnRH-II activates mating behavior in nutritionally challenged females within a few minutes after administration. In addition GnRH-II immunoreactive cell numbers and fibers increase in food-restricted females. Furthermore, GnRH type II receptor immunoreactivity was detected in musk shrew brain in regions associated with mating behavior. Our results lead us to hypothesize that the role of the evolutionarily conserved GnRH-II peptide is to coordinate reproductive behavior as appropriate to the organism’s energetic condition.
Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Deficient Mice Display Reduced Serum Progesterone Levels during Corpus Luteum Development
Authors: Nothnick Warren B.
Published: December 2002

TIMP-1 is a multifunctional protein that is abundantly expressed within the ovary during the period of early corpus luteum formation. TIMP-1 has been suggested to play a role in progesterone production, but reports are conflicting. To more thoroughly examine the role of TIMP-1 in steroidogenesis during early luteal development in vivo, immature wild-type and TIMP-1 null female mice were primed with gonadotropins. Mice were sacrificed at 0, 24, and 48 h post hCG administration and serum was collected for determination of estradiol (E2) or progesterone (P4) concentrations. In the 24 h hCG groups, ovulation was assessed by counting the number of shed ova. Serum P4 concentrations were significantly lower in the TIMP-1 null mice, both at 24 and 48-h post hCG compared to wild-type counterparts. No differences were detected in the number of ovulations between genotypes at the 24 h time point. In both the 24 and 48 h post-hCG groups, null mice had significantly higher ovarian wet weights. Interestingly, ovarian MMP activity was greater in the null mice at 24 h post hCG but higher in the wild-type at 48 h post hCG administration. These observations suggests that expression of TIMP-1 during early luteal development may participate in regulation of progesterone production via its ability to regulate ovarian MMP activity.
Evidence for Genetic Modifiers of Ovarian Follicular Endowment and Development from Studies of Five Inbred Mouse Strains
Authors: Canning Jacqueline , Takai Yasushi , Tilly Jonathan L.
Published: December 2002

The laboratory mouse is the model of choice for genetic studies in mammals due to the availability of many genetically defined inbred strains and inbred congenic strains, as well as the ability to study the effects of over-expression (transgenics) or inactivation (knockouts) of a given gene on cells or tissues. During our studies using these technologies to uncover the importance of various genes to apoptosis in the ovary, we observed that the size of the primordial oocyte reserve was affected by mouse strain in the absence of any other genetic manipulation. To determine if genetic modifiers of oocyte endowment truly exist, herein we examined follicle numbers in one outbred (CD-1) and several inbred (129/Sv, DBA/2, C57BL/6, FVB, AKR/J) strains of mice at day 4 (neonatal) and day 42 (young adult) postpartum. In neonatal life, ovaries of AKR/J females had the lowest total number of follicles, whereas 129/Sv females possessed the highest total number of follicles (P < 0.05 for AKR/J versus 129/Sv). There were more primordial follicles in 129/Sv compared with DBA/2 (P < 0.05), C57BL/6 (P < 0.05), FVB (P < 0.05) or AKR/J (P < 0.05) females, and in CD-1 compared with AKR/J (P < 0.05) females. Although no significant strain-dependent differences in primary follicle numbers were noted, C57BL/6 females had the fewest number of small preantral follicles (P < 0.05 versus all other strains). Evaluation of ovaries at 42 days of age revealed the persistence of strain-dependent differences in early follicle growth patterns, although the total numbers of follicles were comparable. Of interest, marked strain-dependent differences in the rate of primordial follicle growth activation, as well as in the rate of follicle loss (atresia), between days 4 and 42 were observed. These results indicate that genetic modifiers play a major role in follicle endowment, development and atresia in the mouse ovary.