bluuebunny/crossref_metadata_2025 huggingface dataset:
The Booroola mutation in sheep is associated with an alteration of the bone morphogenetic protein receptor-IB functionality
Authors: Fabre S , Pierre A , Pisselet C , Mulsant P , Lecerf F , Pohl J , Monget P , Monniaux D
Published: December 2004

The hyperprolificacy phenotype of Booroola ewes is due to the presence of the FecB(B) allele at the FecB locus, recently identified as a single amino acid substitution (Q249R) in the bone morphogenetic protein (BMP) type-IB receptor (BMPR1B), and is associated with a more precocious differentiation of ovarian granulosa cells (GCs). To evaluate the consequences of the Booroola mutation on BMPR1B functions, the action of ligands of the transforming growth factor-beta (TGFbeta)/BMP family that act through (growth and differentiation factor-5, BMP-4) or independently of (activin A, TGFbeta-1) BMPR1B were studied on primary cultures of GCs from homozygous FecB(+) and FecB(B) ewes. All the tested TGFbeta/BMP family ligands inhibited progesterone secretion by FecB(+) GCs. Those inhibitory effects were lower for GCs from preovulatory (5-7 mm diameter) than from small antral follicles (1-3 mm diameter). The presence of the Booroola mutation was associated with a 3- to 4-fold (P<0.001) decreased responsiveness of GCs from FecB(B) compared with FecB(+) small follicles to the action of BMPR1B ligands. In contrast, TGFbeta-1 and activin A had similar inhibitory effects on progesterone secretion by GCs from FecB(+) and FecB(B) small follicles. No difference between genotypes was observed with GCs from preovulatory follicles. In transfection experiments with HEK-293 cells, co-expression of FecB(+) BMPR1B and BMPR2 resulted in a 2.6-fold (P<0.01) induction of the activity of a BMP-specific luciferase reporter construct by BMP-4. Interestingly, no response to BMP-4 was observed when cells were transfected with the FecB(B) form of the BMPR1B receptor. Overall, these data strongly suggest that the Q249R mutation is associated with a specific alteration of BMPR1B signaling in hyperprolific Booroola ewes.
Distribution and changes in amounts of the androgen receptor in the pig uterus during the estrous cycle, early pregnancy and after treatment with sex steroids
Authors: Cardenas H , Pope WF
Published: December 2004

Two experiments were performed to examine the expression of the androgen receptor (AR) gene in the pig uterus. In experiment 1, immunohistochemistry (IHC) was used to determine the distribution of the AR in uterine tIssue of pigs when collected at the first day of estrus (day 0) and the mid-luteal phase (day 12) of the estrous cycle, or early pregnancy (day 12, n=4 gilts per group). In experiment 2, AR immunostaining and AR mRNA in uterine tIssue were compared among ovariectomized gilts (n=4 per group) following treatment for 4 days with daily injections of: (1) progesterone (2 mg/kg bodyweight (BW)), (2) estradiol-17beta (E(2,) 2 micro g/kg BW), (3) E(2) plus progesterone (same dosages as 1 and 2 combined), (4) 5alpha-dihydrotestosterone (DHT, 7 micro g/kg BW), or (5) vehicle (corn oil). Data were analyzed using ANOVA. In experiment 1, nuclear staining for AR in luminal and glandular epithelia was strong and did not differ in intensity between the two locations. Immunostaining of AR in the myometrium was less (P<0.001) intense than in the luminal and glandular epithelia. Nuclei of stromal cells contained AR immunostaining that varied in intensity from strong (mainly in subepithelial stroma) to weak or no staining. Stages of the estrous cycle or early pregnancy did not influence AR immunostaining in the endometrial epithelia and myometrium. In experiment 2, immunostaining of AR in glandular and luminal epithelia and myometrium of ovariectomized gilts treated with vehicle or DHT was less (P<0.05) than in gilts treated with E(2), progesterone, or E(2) plus progesterone. Immunostaining of AR did not differ between ovariectomized gilts treated with vehicle or DHT, or between gilts treated with E(2), progesterone, or E(2) plus progesterone. In both experiments, intensity of AR immunostaining was greater in glandular epithelium located at the adluminal region compared with glandular epithelium located at the basal region of the endometrium. Competitive reverse-transcription PCR (RT-PCR) indicated a stimulatory effect (P<0.01) of E(2) on amounts of AR mRNA in whole endometrium. This increase in AR mRNA after E(2) treatment was not detected when E(2) was combined with progesterone. Endometrial AR mRNA was not influenced by DHT or progesterone relative to vehicle-treated gilts. In conclusion, immunoreactive AR is mainly present in luminal and glandular epithelia of the pig uterus and to a lesser extent in the myometrium, and does not change significantly during the estrous cycle or early pregnancy. Expression of the AR gene in the pig endometrium and myometrium appears to be regulated by E(2) and progesterone.
Rapid nongenomic effects of glucocorticoids on allergic asthma reaction in the guinea pig
Authors: Zhou J , Kang ZM , Xie QM , Liu C , Lou SJ , Chen YZ , Jiang CL
Published: December 2004

Glucocorticoids (GCs) are routinely believed to work solely through genomic mechanisms. Recent evidence indicates that GCs can act at the membrane to exert rapid nongenomic effects on various tissues and cells. To ascertain whether nongenomic effects of GCs exist on the allergic asthma reaction, Hartley guinea pigs were sensitized with ovalbumin and challenged with the same antigen given by aerosol. Some animals received inhaled budesonide (3 mg/ml suspended in Hydroxypropyl methylcellulose vehicle) for 5 minutes before ovalbumin challenge; Other animals received saline or blank vehicle as control. We measured the changes of lung resistance and dynamic lung compliance, the pulmonary function used to evaluate allergic asthma severity. Inhaled budesonide inhibited allergic reaction within 10 minutes, which would preclude genomic-mediated responses that normally takes several hours to occur. This study infers for the first time that rapid nongenomic effect of GCs exists on allergic asthma reaction, and provides a new way to investigate nongenomic mechanism of GCs. Further study would raise the possibility of new therapeutic strategies for allergic disease including asthma.
MAP kinase phosphatase 1: a novel mediator of biological effects of glucocorticoids?
Authors: Clark AR
Published: December 2004

Synthetic glucocorticoids (GCs) potently inhibit the expression of pro-inflammatory genes and are widely used in the treatment of inflammatory diseases. However, some patients are resistant to the therapeutic effects of GCs, and many suffer deleterious side effects from these drugs. Furthermore, the precise mechanisms by which GCs inhibit pro-inflammatory gene expression remain unclear. A number of recent papers report that GCs induce the sustained expression of MAP kinase (MAPK) phosphatase 1 (MKP-1), a negative regulator of MAPK signal transduction pathways. The potential relevance of MKP-1 to some of the biological effects of GCs is discussed.
Glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implications for the resolution of inflammation
Authors: Heasman SJ , Giles KM , Ward C , Rossi AG , Haslett C , Dransfield I
Published: December 2004

Glucocorticoids represent one of the most effective clinical treatments for a range of inflammatory conditions, including severe acute inflammation. Although glucocorticoids are known to affect processes involved in the initiation of inflammation, the influence of glucocorticoids on the mechanisms by which acute inflammation normally resolves have received less attention. Apoptosis of granulocytes present at inflamed sites leads to their rapid recognition and internalisation by macrophages, a process which may be important for resolution of inflammation. However, if clearance of either eosinophils or neutrophils is impaired, these cells rapidly undergo secondary necrosis leading to release of pro-inflammatory mediators from the phagocyte, potentially prolonging inflammatory responses. Physiologically relevant concentrations of glucocorticoids accelerate eosinophil apoptosis whilst delaying neutrophil apoptosis during in vitro culture. Here we discuss key pathways regulating the granulocyte apoptotic programme and summarise the effects of glucocorticoids on monocyte differentiation and the consequent changes to apoptotic cell clearance capacity. Definition of the mechanisms underlying resolution of inflammatory responses following glucocorticoid treatment may unveil new targets for modulation of inflammatory disease, allowing co-ordinated augmentation of granulocyte apoptosis together with increased macrophage capacity for clearance of apoptotic cells.
Corticosteroids, eosinophils and bronchial epithelial cells: new insights into the resolution of inflammation in asthma
Authors: Walsh GM , Sexton DW , Blaylock MG
Published: December 2004

Anti-inflammatory therapy in asthma is reliant on corticosteroids, particularly in their inhaled form. However, steroids are rather non-specific in their actions and they also raise concerns regarding compliance and side-effect Issues. Furthermore, a small proportion of patients with asthma fail to respond to oral glucocorticoids even at high doses. This Article will review the role that steroids and membrane receptor ligation play in the induction of eosinophil apoptosis together with the mechanisms by which corticosteroids enhance the disposal of apoptotic eosinophils by both professional and non-professional phagocytes. Eosinophils are thought to be the major pro-inflammatory effector cell in asthma and their persistence in the airways is probably enhanced by the presence of several asthma-relevant cytokines that prolong eosinophil survival by inhibition of apoptosis (interleukin (IL)-3, IL-5, granulocyte-macrophage colony-stimulating factor, IL-9, IL-13, IL-15). In contrast, a number of signals have been described that accelerate apoptosis in human eosinophils including corticosteroids or ligation of membrane receptors (CD95, CD45, CD69). Thus, the load of lung eosinophils in asthmatic disease is likely to be related to a balance in the tIssue microenvironment between pro- and anti-apoptotic signals. Furthermore, removal of apoptotic eosinophils by phagocytosis by alveolar macrophages or bronchial epithelial cells in a specific receptor-mediated way is as important as the process of apoptosis induction. Corticosteroids enhance the recognition and engulfment of apoptotic eosinophils by macrophages or bronchial epithelial cells. Caspases are key intracellular molecules in the control of apoptosis and defects in caspase-induced apoptosis in eosinophils from steroid-resistant individuals may contribute to the molecular mechanisms underlying glucocorticoid insensitivity in these cells. These findings point the way to new and more targeted anti-inflammatory therapy for asthma and may provide important clues for the development of alternative therapies for glucocorticoid resistance.
Both stress experience and age determine the impairment or enhancement effect of stress on spatial memory retrieval
Authors: Yang Y , Cao J , Xiong W , Zhang J , Zhou Q , Wei H , Liang C , Deng J , Li T , Yang S , Xu L , Xu L
Published: December 2004

It has been documented that stress or glucocorticoids have conflicting effects on memory under different conditions. However, it is not fully understood why stress can either impair or enhance memory. Here, we have examined the performance of six age groups of Wistar rats in a water maze spatial task to evaluate the effects of stress under different conditions. We found that the impairment or enhancement effect of an 'elevated platform' (EP) stress on memory was dependent on previous stress experience and on age. EP stress impaired memory retrieval in water maze naive animals, but enhanced rather than impaired memory retrieval in young water maze stress-experienced animals. Furthermore, exogenously applied corticosterone or foot shock stress before water maze training prevented the impairment of memory retrieval that should be induced by treatment with corticosterone or foot shock before the 'probe trial'. Again, memory retrieval was enhanced in young animals under these conditions, and this enhancement can be prevented by the glucocorticoid receptor antagonist RU 38486. Thus, glucocorticoid receptor activation not only induced impairment of memory but also increased the capacity of young animals to overcome a later stress. The present findings suggest that the effect of stress on memory can be switched from impairment to enhancement dependent on both stress experience and age.
Hypothalamo-pituitary-adrenal axis dysregulation in patients with rheumatoid arthritis after the dexamethasone/corticotrophin releasing factor test
Authors: Harbuz MS , Korendowych E , Jessop DS , Crown AL , Li pdfan SL ,   Kirwan
Published: December 2004

A defective hypothalamo-pituitary-adrenal axis response to inflammatory cytokines may contribute to the pathophysiology of rheumatoid arthritis (RA). The purpose of this study was to define further the mechanisms responsible for this dysregulation. Six normal individuals and seven patients with active RA were recruited and given an oral dose of dexamethasone at 2300 h the evening before the study. The next day, an i.v. catheter was fitted at 1300 h. Blood samples were collected between 1400 h and 1700 h before and after infusion (at 1500 h) of corticotrophin releasing factor (CRF). Plasma was separated and stored at-20 degrees C before radioimmunoassay for ACTH, cortisol and dihydroepiandrosterone (DHEA). Before the CRF challenge, ACTH and cortisol were significantly increased and DHEA significantly decreased in the patients with RA compared with the controls. Neither ACTH nor DHEA was significantly altered after CRF infusion. Control individuals did not mount a cortisol response to infusion of CRF. Similarly, four of the patients with RA did not respond to CRF. However, in contrast to the controls, three of the patients mounted an immediate and sustained cortisol response after receiving CRF. These data reveal that three of the seven patients with RA were able to escape from dexamethasone suppression and mount a cortisol response to CRF challenge. This suggests that there may be a subpopulation of patients with RA who have impaired glucocorticoid feedback. The implications of this alteration for disease progression remain to be determined.
IGF-I regulates pro-opiomelanocortin and GH gene expression in the mouse pituitary gland
Authors: Honda J , Manabe Y , Matsumura R , Takeuchi S , Takahashi S
Published: December 2004

IGF-I is expressed in somatotrophs, and IGF-I receptors are expressed in most somatotrophs and some corticotrophs in the mouse pituitary gland. Our recent study demonstrated that IGF-I stimulates the proliferation of corticotrophs in the mouse pituitary. These results suggested that somatotrophs regulate corticotrophic functions as well as somatotrophic functions by the mediation of IGF-I molecules. The present study aimed to clarify factors regulating pituitary IGF-I expression and also the roles exerted by IGF-I within the mouse anterior pituitary gland. Mouse anterior pituitary cells were isolated and cultured under serum-free conditions. GH (0.5 or 1 microg/ml), ACTH (10(-8) or 10(-7) M), GH-releasing hormone (GHRH; 10(-8) or 10(-7) M), dexamethasone (DEX; 10(-8) or 10(-7) M) and estradiol-17beta (e2; 10(-11) or 10(-9) M) were given for 24 h. IGF-I mRNA levels were measured using competitive RT-PCR, and GH and pro-opiomelanocortin (POMC) mRNA levels were measured using Northern blotting analysis. GH treatment significantly increased IGF-I mRNA levels (1.5- or 2.1-fold). ACTH treatment did not alter GH and IGF-I mRNA levels. IGF-I treatment decreased GH mRNA levels (0.7- or 0.5-fold), but increased POMC mRNA levels (1.8-fold). GH treatment (4 or 8 microg/ml) for 4 days increased POMC mRNA levels. GHRH treatment increased GH mRNA levels (1.3-fold), but not IGF-I mRNA levels. DEX treatment significantly decreased IGF-I mRNA levels (0.8-fold). e2 treatment did not affect IGF-I mRNA levels. GH receptor mRNA, probably with GH-binding protein mRNA, was detected in somatotrophs, and some mammotrophs and gonadotrophs by in situ hybridization using GH receptor cDNA as a probe. These results suggested that IGF-I expression in somatotrophs is regulated by pituitary GH, and that IGF-I suppresses GH expression and stimulates POMC expression at the transcription level. Pituitary IGF-I produced in somatotrophs is probably involved in the regulation of somatotroph and corticotroph functions.
The CCN family: a new stimulus package
Authors: Brigstock DR
Published: December 2004

The CCN family comprises cysteine-rich 61 (CYR61/CCN1), connective tIssue growth factor (CTGF/CCN2), nephroblastoma overexpressed (NOV/CCN3), and Wnt-induced secreted proteins-1 (WISP-1/CCN4), -2 (WISP-2/CCN5) and -3 (WISP-3/CCN6). These proteins stimulate mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Many of these activities probably occur through the ability of CCN proteins to bind and activate cell surface integrins. Accumulating evidence supports a role for these factors in endocrine pathways and endocrine-related processes. To illustrate the broad role played by the CCN family in basic and clinical endocrinology, this Article highlights the relationship between CCN proteins and hormone action, skeletal growth, placental angiogenesis, IGF-binding proteins and diabetes-induced fibrosis.
Differential distribution and expression of leptin and the functional leptin receptor in major salivary glands of humans
Authors: Bohlender J , Rauh M , Zenk J , Groschl M
Published: December 2004

Leptin plays a central role in the regulation of food intake and energy expenditure in rodents. However, it has become clear that this hormone has more than only a satiety-inducing function, and that there are other sources of leptin, such as the central nervous system, placenta and the gastrointestinal tract in addition to adipose tIssue. Knowing about the important role of the salivary glands in food intake and digestion, it was the objective of the present study to investigate how leptin and its receptor are expressed and distributed in the major salivary glands of humans. We found leptin distributed throughout the major salivary glands with obvious intracellular concentrations in granula. In contrast, immunostaining for the leptin receptor was found exclusively in the membranes of the glandular cells. A high density of the leptin receptor was localised in the epithelia of the duct lumen. PCR analysis proved the autonomous expression of leptin by the salivary glands independently from adipocytes. Accordingly the long receptor isoform was expressed by any examined tIssue. In the light of recent findings of leptin influencing the growth of rodent salivary glands, the presence and distribution of leptin and its receptor suggests an autocrine role of salivary leptin within the glands.
Development of an RIA for salmon 41 kDa IGF-binding protein
Authors: Shimizu M , Hara A , Dickhoff WW
Published: December 2004

Salmon plasma contains at least three IGF-binding proteins (IGFBPs) with molecular masses of 41, 28 and 22 kDa. The 41 kDa IGFBP is similar to mammalian IGFBP-3 in size, type of glycosylation and physiological responses. In this study, we developed an RIA for the 41 kDa IGFBP. The 41 kDa IGFBP purified from serum was used for antibody production and as an assay standard. Binding of three different preparations of tracer were examined: (125)I-41 kDa IGFBP, (125)I-41 kDa IGFBP cross-linked with IGF-I and 41 kDa IGFBP cross-linked with (125)I-IGF-I (41 kDa IGFBP/(125)I-IGF-I). Only binding of 41 kDa IGFBP/(125)I-IGF-I was not affected by added IGFs, and therefore it was chosen for the tracer in the RIA. Plasma 41 kDa IGFBP levels measured by RIA were increased by GH treatment (178.9+/-4.9 ng/ml) and decreased after fasting (95.0+/-7.0 ng/ml). The molarities of plasma 41 kDa IGFBP and total IGF-I were comparable, and they were positively correlated, suggesting that salmon 41 kDa IGFBP is a main carrier of circulating IGF-I in salmon, as is mammalian IGFBP-3 in mammals. During the parr-smolt transformation (smoltification) of coho salmon, plasma 41 kDa IGFBP levels showed a transient peak (182.5+/-10.3 ng/ml) in March and stayed relatively constant thereafter, whereas IGF-I showed peak levels in March and April. Differences in the molar ratio between 41 kDa IGFBP and IGF-I possibly influence availability of IGF-I in the circulation during smoltification.
Stereological evaluation of Sertoli cell ontogeny during fetal and neonatal life in two diverse breeds of swine
Authors: McCoard SA , Wise TH , Lunstra DD , Ford JJ
Published: December 2004

Chinese Meishan (MS) boars have smaller testes due to fewer Sertoli cells compared with White Composite (WC) boars. The objective was to describe Sertoli cell development relative to circulating FSH concentrations in fetal and neonatal MS and WC boars. Testes and blood samples were collected on days 60, 75, 90 and 105 postcoitum (dpc) and 1, 7, 14 and 25 postpartum (dpp). One testis was immunostained for GATA4 or Ki67 antigen to evaluate total and proliferating Sertoli cell numbers respectively. Testicular size was greater (P<0.01) in WC than MS boars at all ages, associated with a greater mass of interstitial tIssue. Tubular mass (P<0.01) was greater in prenatal WC boars, but postnatally increased more rapidly (P<0.001) in MS boars, exceeding WC boars by 25 dpp. Sertoli cell numbers increased with age, was greater (P<0.001) in WC than MS boars during prenatal development but increased rapidly (P<0.01) by 1 dpp in MS and thereafter was similar in both breeds. The proportion of Ki67-positive Sertoli cells was maximal at 90 dpc, declining thereafter, did not differ between breeds through 7 dpp, but was greater (P<0.05) in WC than MS boars at 14 and 25 dpp. Plasma FSH concentrations were greater (P<0.05) in WC than MS boars at 75 dpc. FSH concentrations were elevated at 105 dpc (MS) and 1 dpp (WC) but declined thereafter with advancing postnatal age in both breeds. This study illustrates that late gestation represents the period of maximal Sertoli cell proliferation. Despite asynchronous Sertoli cell population growth between breeds during early postnatal life, differential mature Sertoli cell numbers and testicular size are probably due to differences in duration of the proliferative period after 25 dpp, potentially regulated by Sertoli cell maturation and blood-testis barrier formation. These events were not associated with fetal or early postnatal changes in FSH secretion.
Endocrine and molecular influences on testicular development in Meishan and White Composite boars
Authors: McCoard SA , Wise TH , Ford JJ
Published: December 2004

The aim of this study was to evaluate developmental changes in thyroid hormone and other key endocrine hormones/molecular markers produced by testicular cells, in relation to breed differences in proliferation and maturation of Sertoli cells and general testicular morphological development in Meishan (MS) and White Composite (WC) boars. Blood samples and testes were collected on days 60, 75, 90 and 105 post coitum (dpc) and days 1, 7, 14 and 25 post partum (dpp). Testes were immunostained for thyroid hormone receptor-beta1 (THRbeta1), GATA4, Mullerian-inhibiting substance (MIS), 17-alpha-hydroxylase (P450(c17)) and inhibin subunits (alpha, betaA, betaB). In addition, protein levels were determined by densitometry. Plasma concentrations of free triiodothyronine (T(3)) were greater in MS (hyperthyroid) compared with WC (hypothyroid) boars (P<0.01) during fetal life, but the reverse was evident postnatally. Elevated levels of free T(3) during fetal life were associated with increased levels of THRbeta1, suggesting increased thyroid responsiveness of the testis during this time, contrasting with observations during early postnatal life. Localization patterns of THRbeta1, MIS, GATA4 and the inhibin subunits were consistent with previous studies. MIS protein levels declined more rapidly (P<0.001) in MS compared with WC Sertoli cells postnatally, consistent with earlier maturation of Sertoli cells as indicated by our previous study. In this study, transient neonatal hyperthyroidism in MS boars during late gestation was associated with a decline in proliferation and early maturation of Sertoli cells, followed by early onset of puberty in this breed. These observations indicate a possible role for thyroid hormone in the modification of Sertoli cell development, thereby influencing growth and differentiation of the testis in pigs.
The effects of serine proteinase inhibitors on bone resorption in vitro
Authors: Tumber A , Papaioannou S , Breckon J , Meikle MC , Reynolds JJ , Hill PA
Published: December 2004

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%.Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.
The long-term effects of anti-retroviral protease inhibitors on sugar transport in L6 cells
Authors: Germinario RJ , Colby-Germinario SP , Cammalleri C , Wainberg MA
Published: December 2004

The objective of this study was to investigate the long-term effects of anti-retroviral protease inhibitors (PIs) on 2-deoxy-d -glucose (2-DG) transport in L6 cells in vitro. Exposure of L6 cells to saquinavir, ritonavir, indinavir and amprenavir resulted in significant increases in 2-DG transport using PI concentrations of 1-10 microM with continual exposure to PI. After removal of the PI for up to 48 h, 2-DG transport increases did not change and remained at pre-reversal levels. These changes in 2-DG transport were not related to stress-induced sugar transport or to apoptosis. The examination of glucose transporter (GLUT) 1, 3 or 4 translocation with subcellular fractionation indicated that insulin (i.e. 67 nM) could induce the translocation of all the GLUTs to the plasma membrane. Also, ritonavir (10 microM), which leads to a 2-fold increase in 2-DG transport, demonstrated increased GLUT (i.e. 1, 3 or 4) presence in the plasma membrane fraction, in the presence or absence of insulin. This increased 2-DG transport involved transporter presence in plasma membrane preparations and did not affect the ability of insulin to stimulate 2-DG transport with continual PI exposure. The mechanism(s) involved indicates ready reversibility of PI effects on transporters. The mechanism(s) why reversibility of PI-induced 2-DG transport was similar plus or minus PI was not apparent.
Gene expression of resistin in adipose tissue and mammary gland of lactating and non-lactating cows
Authors: Komatsu T , Itoh F , Mikawa S , Hodate K
Published: December 2004

Resistin has been suggested to induce insulin resistance in obesity and to inhibit adipocyte differentiation. In lactating cows, glucose uptake in the mammary gland is a rate-limiting step in milk synthesis, and to supply glucose to the mammary gland, insulin resistance increases. We examined the expression of the resistin gene by real-time PCR of cDNA in the adipose tIssue and mammary gland of lactating and non-lactating cows. Lactation induced a significant increase of resistin expression in adipose tIssue compared with that in the dry period, and decreased resistin expression in the mammary gland. There were no significant differences in the expression of insulin responsive glucose transporter (GLUT4) mRNA between the adipose tIssue of lactating and non-lactating cows, and GLUT4 mRNA was not detected in the mammary gland. The plasma insulin concentration was lower in lactating cows than in non-lactating cows. These results indicate that the pattern of resistin expression in peripheral tIssues is changed in association with milk production. The increase of resistin expression and maintenance of a lower level of plasma insulin concentration may decrease glucose availability by increasing insulin resistance in adipose tIssue. Additionally, our results suggest that the decrease of resistin expression in the mammary gland may influence on the insulin-dependent glucose uptake in mammary epithelial cells during lactation.
Clock genes in calendar cells as the basis of annual timekeeping in mammals--a unifying hypothesis
Authors: Lincoln GA , Andersson H , Loudon A
Published: December 2004

Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.
Gonadotropin treatment increases homocysteine levels in idiopathic hypogonadotropic hypogonadism: an indirect effect mediated by changes in body composition
Authors: Oktenli C , Yesilova Z , Ozata M , Yaman H , Tuzun A , Dundar S , Sanisoglu SY , Musabak U , Erbil MK , Dagalp K
Published: December 2004

The main objective of the present study was to examine the alterations in plasma total homocysteine (tHcy) concentrations during a testosterone-deficient state and after gonadotropin treatment for 6 Months in patients with idiopathic hypogonadotropic hypogonadism (IHH). Thirty-five newly diagnosed male patients with IHH (mean age 21.34+/-1.53 years) and 29 age- and body mass index-matched healthy males (mean age 21.52+/-1.77 years) were recruited into the study. Pretreatment levels of free testosterone (1.51+/-0.66 pg/ml), estradiol (21.37+/- 4.37 pg/ml), FSH (0.91+/-0.24 IU/l) and LH (1.25+/- 0.53 IU/l) were lower than controls (25.17+/-3.06 pg/ml, 31.00+/-4.96 pg/ml, 3.14+/-1.62 IU/l and 4.83+/-1.65 IU/l respectively) (P<0.001). They increased significantly after treatment (18.18+/-1.59 pg/ml, 27.97+/- 4.25 pg/ml, 2.41+/-0.27 IU/l and 2.79+/-0.19 IU/l respectively) (P<0.001). Patients with IHH had lower tHcy levels than controls (10.14+/-1.34 and 12.58+/- 2.29 micro mol/l respectively) (P<0.001). Plasma tHcy concentrations increased significantly (12.63+/-1.44 micromol/l) after 6 months of treatment (P<0.001). As compared with the controls, pretreatment levels of serum creatinine (63.54+/-13.01 vs 82.84+/-16.69 micromol/l), hemoglobin (12.98+/-0.56 vs 13.83+/-0.71 g/dl) and hematocrit (39.29+/-2.01 vs 41.38+/-1.95%) were significantly lower (P<0.001), and they increased significantly following treatment (80.24+/-11.93 micromol/l, 13.75+/-0.49 g/dl and 41.26+/-1.78% respectively) (P<0.001). The pretreatment folic acid and vitamin B(12) levels were significantly higher in patients when compared with controls (14.87+/-5.68 vs 12.52+/-4.98 nmol/l, P=0.034 and 289.75+/-92.34 vs 237.59+/-108.17 pmol/l, P=0.002 respectively). They decreased significantly after treatment (11.29+/-3.31 nmol/l and 228.51+/-54.33 pmol/l respectively) (P<0.001). The univariate and multivariate regression analysis results showed that only changes in creatinine, creatinine clearance, vitamin B12 and folic acid were independently associated with changes in tHcy levels in patients with IHH. In conclusion, the increase in plasma tHcy concentrations following gonadotropin treatment seems to be largely independent of changes in androgen levels.
Angiotensinogen localization and secretion in the rat pancreas
Authors: Regoli M , Bendayan M , Fonzi L , Sernia C , Bertelli E
Published: December 2004

Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.
Isolation and characterization of Leydig cells from adult bonnet monkeys (Macaca radiata): evidence for low steroidogenic capacity in monkey Leydig cells in contrast to rat Leydig cells
Authors: Anbalagan M , Sriraman V , Jagannadha Rao A
Published: December 2004

Most of the available information on Leydig cells has been obtained using a rodent model system. With an objective to extend the observations made with rat Leydig cells (RLCs) to primates, a method has been developed to isolate Leydig cells from monkey (Macaca radiata) testis. Enzymatic dissociation of monkey testis followed by Percoll-gradient fractionation of the interstitial cells resulted in the recovery of Leydig cells at densities corresponding to 1.064-1.070 g/ml. Purified (90-94%) monkey Leydig cells (MLCs) stained positive for the Leydig cell marker 3beta-hydroxysteroid dehydrogenase. The cells responded to in vitro addition of human chorionic gonadotropin (hCG) and produced testosterone. Comparison of the in vitro testosterone-producing ability of MLCs with RLCs revealed that MLCs have much less steroidogenic capacity compared with the RLCs. Analysis revealed that limitation in substrate availability to mitochondrial P(450) side chain cleavage enzyme and low mitochondrial and smooth endoplasmic reticulum content in MLCs could be the possible reasons for the low steroidogenic capacity of the MLCs.
Gonadal protection from radiation by GnRH antagonist or recombinant human FSH: a controlled trial in a male nonhuman primate (Macaca fascicularis)
Authors: Kamischke A , Kuhlmann M , Weinbauer GF , Luetjens M , Yeung CH , Kronholz HL , Nieschlag E
Published: December 2004

Chemotherapy and radiation often damage spermatogenesis irreversibly in oncological patients and various approaches to gonadal protection have been tested with equivocal results. In rats, hormonal protection of spermatogenesis can be achieved by blocking gonadotropin secretion. However, whether the same mechanisms can effect gonadal protection in primates remains questionable. To clarify this Issue we conducted a placebo-controlled trial in a preclinical animal model using macaques (Macaca fascicularis). Twenty adult male monkeys (five in each group) were randomized to receive either recombinant human FSH, GnRH antagonist or saline injections (two groups) for 36 days. On day 29 all groups except one saline-treated control group were exposed to a single testicular irradiation of 4 Gy. Every 2 weeks before, during and after the treatment, ejaculates, body weight, testicular Volume and hormones were analyzed until day 539. In addition, repeated testicular biopsies were performed. Testicular Volume and inhibin B decreased significantly in all irradiated groups compared with baseline and with the non-irradiated control group, followed by a gradual recovery of these parameters, which was, especially at the earlier time points, significantly better in the FSH-treated group compared with both other irradiated groups. Irradiation caused a drastic decrease of sperm parameters in all groups, followed by a partial recovery of sperm parameters, which was significantly slower in the early phases of recovery in the GnRH antagonist group compared with the vehicle group. Testicular histology showed a significant depletion on study day 261 in all irradiated animals. In conclusion, in clear contrast to rodent studies, GnRH antagonist treatment did not provide gonadal protection in this primate model. FSH treatment resulted in slightly better recovery of spermatogenesis, which appears to be of no or only little clinical relevance.
Migratory trophoblast cells express a newly identified member of the prolactin gene family
Authors: Wiemers DO , Ain R , Ohboshi S , Soares MJ
Published: December 2004

Rodents possess an expanded prolactin (PRL) family of genes. These genes encode for a family of structurally related hormones/cytokines that are expressed most prominently in the anterior pituitary, uterus and placenta. In this study, we have identified a new member of the rat PRL family through a search of the National Center for Biotechnology Information expressed sequence database. The cDNA was sequenced and its corresponding mRNA characterized. On the basis of existing nomenclature, the rat cDNA was termed PRL-like protein-N (PLP-N). PLP-N has structural features indicative of its inclusion in the PRL family and is most closely related to PRL-like protein-F (PLP-F) and proliferin related protein (PLF-RP). A survey of PLP-N mRNA expression by Northern analysis indicated that PLP-N showed extensive expression in the metrial gland and minimal expression in the chorioallantoic placenta or other tIssues. Expression of PLP-N mRNA was restricted to migratory trophoblast cells. Junctional zone trophoblast cells isolated from day 13 of gestation placenta differentiated in vitro and exhibited a capacity for PLP-N expression. In summary, we have discovered a new member of the PRL family that is prominently expressed in migratory trophoblast cells residing in the metrial gland.
Zonal expression of the thyroid hormone receptor alpha isoforms in rodent liver
Authors: Zandieh-Doulabi B , Dop E , Schneiders M , Schiphorst MP , Mansen A , Vennstrom B , Dijkstra CD , Bakker O , Wiersinga WM
Published: December 2004

Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-alpha encoding TRalpha1 and TRalpha2 and their respective Delta isoforms, and c-erbA-beta which encodes TRbeta1, TRbeta2 and TRbeta3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TRbeta1 isoform in rat liver. We were therefore also interested to see whether TRalpha1 and TRalpha2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TRalpha1 and TRalpha2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TRalpha1 and TRalpha2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TRalpha1 is broader than that of TRbeta1. Moreover, the expression of the TRalpha2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TRalpha1 protein.From our data it appears that both the TRalpha1 and TRalpha2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.
Interleukin-1beta (IL-1beta) and IL-6 modulate insulin-like growth factor-binding protein (IGFBP) secretion in colon cancer epithelial (Caco-2) cells
Authors: Street ME , Miraki-Moud F , Sanderson IR , Savage MO , Giovannelli G , Bernasconi S , Camacho-Hubner C
Published: December 2004

Chronic inflammation is characterised by modifications in cytokine concentrations, whereas growth is mainly dependent on the GH-IGF axis. IGF-I bioavailability is modulated by a family of IGF-binding proteins (IGFBPs). The aim of the present study was to evaluate the interactions among interleukin-1beta (IL-1beta), IL-6 and IGFBP secretion by intestinal cells to assess whether cytokines modulate IGFBP secretion, and in turn IGF-I and IGF-II bioavailability. The human colon carcinoma derived cell line Caco-2 was used as an in vitro model for its capacity to differentiate spontaneously. Experiments were carried out on day 4 (undifferentiated state) and day 14 (differentiated state) after plating. Carcinoembryonic antigen (CEA) was used as a marker of differentiation and increased in the conditioned media (CM) from days 4 to 14 (0.2+/-0.01 ng/ml per 10(5) cells vs 3.3+/-0.2 ng/ml per 10(5) cells, P<0.05). IGFBP-2 and IGFBP-4 secretion decreased concomitantly. Cells were stimulated with IL-1beta and IL-6 at 1, 10 and 50 ng/ml, and with IL-1beta and IL-6 in combination at the same dose of 1 and 10 ng/ml. IGF-I at 50 ng/ml was used as a control. Caco-2 cells expressed and secreted mainly IGFBP-2 and IGFBP-4 into the CM. On day 4, IL-1beta (1 ng/ml) and IL-6 (10 and 50 ng/ml) reduced IGFBP-2 by 29+/-8%, and by 32+/-9 and 38+/-8% respectively (P<0.05). IGFBP-4 was also reduced by IL-1beta at 1 and 50 ng/ml (-14+/-4% and -46+/-11% vs serum free medium (SFM) respectively, P<0.05), and IL-6 at 50 ng/ml (-46+/-15%, P<0.05). Both IGFBP-2 and IGFBP-4 were reduced by IL-1beta and IL-6 in combination at 1 and 10 ng/ml (P<0.05). On day 14, IGFBP-2 band intensity was reduced at 10 ng/ml of IL-1beta (-22+/-15% vs SFM, P<0.05) and at 50 ng/ml of both cytokines (-33%+/-8% and -13%+/-13% vs baseline respectively, P<0.05). IGFBP-4 band intensity decreased with 10 and 50 ng/ml of IL-1beta (-35+/-11% and -46+/-15% vs SFM respectively) and IL-6 (-36%+/-10% and -46+/-15% vs SFM respectively). IL-1beta and IL-6 in combination at 1 and 10 ng/ml reduced both IGFBP-2 and IGFBP-4.In conclusion, IGFBP-2 and IGFBP-4 secretion in CM decreased with Caco-2 cell differentiation. IGFBP-2 and IGFBP-4 were significantly decreased by IL-1beta and IL-6 treatment in both the undifferentiated and differentiated state. Furthermore, these cytokines increased cell proliferation whereas total protein content was significantly reduced only at the higher concentrations of IL-6 and IL-1beta. These findings suggest that interleukins modulate the IGF-IGFBP system in Caco-2 cells in vitro.
The inducible isoform of CREM (inducible cAMP early repressor, ICER) is a repressor of CYP19 rat ovarian promoter
Authors: Morales V , Gonzalez-Robayna I , Hernandez I , Quintana J , Santana P , Ruiz de Galarreta CM , Fanjul LF
Published: December 2004

The synthesis of estradiol by the granulosa cells is a prominent event in ovarian physiology and depends on the expression of P450(AROM). FSH induces the expression of P450(AROM) in granulosa cells as a result of the presence in the ovarian promoter of a CRE (cAMP response element)-like sequence (CLS). In rodents, LH downregulates aromatase expression during luteinization by an as yet undescribed mechanism. In granulosa cells, LH increases the expression of the inducible cAMP early repressor (ICER), an isoform of CREM (cAMP-responsive element modulator) that represses cAMP-induced transcription. The possibility that ICER represses the activity of the aromatase ovarian promoter, thus being part of the mechanism underlying the effects of LH was investigated. We have found that: (1) nuclear proteins from forskolin-stimulated granulosa cells were specifically bound to an oligonucleotide containing the CLS sequence of the CYP19 ovarian promoter and one out of the two protein-DNA complexes formed was supershifted by an anti-CREM antibody; (2) in granulosa cells, forskolin-induced increases in P450(AROM) promoter luciferase reporter gene activity were prevented by the transient overexpression of ICER; (3) similar results were obtained in 8-Br-cAMP-stimulated R2C cells, a Leydig tumor cell line routinely used for the study of P450(AROM) promoter activity; (4) both ICER mRNA levels and P450(AROM) promoter-driven luciferase activity were elevated 6 and 12 h after stimulation of R2C cells with 8-Br-cAMP and were decreased 24 and 48 h later; (5) in an R2C polyclonal line overexpressing ICER, the promoter activity at early stages of stimulation was completely attenuated, while 24 and 48 h downregulation was prevented in another R2C line stably transfected with an antisense ICER construct. These results suggest that ICER represses CYP19 ovarian promoter and that LH-induced expression of ICER may serve to downregulate P450(AROM) transcription in granulosa cells during luteinization.