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1.
Peptides ; 91: 20-25, 2017 05.
Article in English | MEDLINE | ID: mdl-28300671

ABSTRACT

Cocaine and Amphetamine-Regulated Transcript (CART) is widely expressed in the central nervous system and in several endocrine organs. CART is an important factor in the regulation of energy homeostasis. The aim of the study was to assess the role of CART in physiological response of pituitary cells in a course of starvation. The pituitary cells harvested from starved and fed ad libitum male rats were cultured for 48h and treated with: 0.1nM, 1nM, 10nM or 100nM doses of CART. The medium was collected after 60min and stored at -70°C until samples were further assayed for: LH, FSH, PRL, GH, TSH and ACTH. We revealed that in cultures of pituitary cells collected from fasted rats the basal levels of the examined hormones were reduced. Incubation of pituitary cells of non-starved rats with any dose of CART reduced the concentration of LH and TSH, while the levels of the other hormones were decreased after administration only specific doses of CART. In cells of fasted rats no change in the concentration of gonadotrophins was observed. The PRL level was increased only in the 1nM dose of CART, while the 10nM and 100nM CART doses markedly enhanced GH and TSH. Moreover, administration of 1nM, 10nM and 100nM of CART to cultured cells of fasted rats resulted in a significant rise of the ACTH. Our results indicate that CART can directly affect the physiological release of PRL, ACTH, TSH and GH in pituitary cells of starved animals. Moreover, CART did not alter the LH and FSH suppression level, which is correlated with food deprivation. This data stays in contrast with the already proposed role of CART as an anorexigenic hypothalamic factor.


Subject(s)
Fasting , Hunger , Nerve Tissue Proteins/physiology , Pituitary Gland/metabolism , Pituitary Hormones/metabolism , Animals , Male , Nerve Tissue Proteins/pharmacology , Pituitary Gland/drug effects , Pituitary Hormones/analysis , Primary Cell Culture , Rats , Rats, Wistar , Statistics, Nonparametric
2.
J Physiol Pharmacol ; 66(5): 673-80, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26579573

ABSTRACT

Adiponectin is a protein secreted primarily by adipose tissue. It has been suggested that adiponectin plays a protective role in the early phase following myocardial infarction. Our primary aim was to investigate the effects of post-myocardial infarction heart failure well-characterized by left ventricular hemodynamic parameters on the total and high molecular weight adiponectin concentrations in plasma, fat and cardiac tissue. Eight weeks after myocardial infarction or sham operation, total and high molecular weight adiponectin concentrations in plasma, fat, and cardiac tissues were assayed in rats. In addition, hemodynamic parameters and expression of the genes encoding atrial natriuretic peptide and brain natriuretic peptide in left ventricle were evaluated. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels in left ventricle tissue were higher in rats with myocardial infarction-induced heart failure compared with the controls. Similarly, total adiponectin concentration was increased in left ventricle (but not in right ventricle) in rats with post-myocardial infarction heart failure. In contrast, adiponectin levels in plasma and cardiac adipose tissue in rats with post-myocardial infarction heart failure were lower than in sham-operated animals. Furthermore, there were no significant differences in levels of high molecular weight adiponectin in plasma, cardiac tissue or adipose tissue between these two groups. We conclude that in the rat model of post-myocardial infarction heart failure, adiponectin level is increased in left ventricle tissue. This is accompanied by decreased adiponectin levels in plasma and cardiac adipose tissue.


Subject(s)
Adiponectin/metabolism , Heart Failure/physiopathology , Myocardial Infarction/complications , Adipose Tissue/metabolism , Animals , Atrial Natriuretic Factor/genetics , Disease Models, Animal , Heart Failure/etiology , Heart Ventricles/metabolism , Hemodynamics , Male , Molecular Weight , Natriuretic Peptide, Brain/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar
3.
Neuropeptides ; 48(4): 199-205, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24931296

ABSTRACT

Orexin A (OxA), also known as hypocretin 1, is a regulatory neuropeptide involved in the control of various autonomic and neuroendocrine functions. It appears to have a significant impact on the regulation of trophic hormones secretion by influencing the hypothalamus and the pituitary. Orexin A acts through two types of receptor found in the pituitary. This suggests the possibility of direct action of OxA at the adenohypophysis level. The aim of this study was to investigate the direct effect of OxA on GnRH (gonadotrophin-releasing hormone)-stimulated LH and FSH secretion from cultured pituitary cells of sexually immature and mature female rats. Anterior pituitary cells obtained from immature and mature female rats (ovariectomized, and ovariectomized and treated with estradiol) were incubated with 10(-10)M or 10(-7)M orexin A for 1 hour and 4h and the effect on GnRH-stimulated (10(-9)M or 10(-6)M) LH and FSH release was examined. The concentrations of secreted gonadotrophins in the culture media were determined by RIA methods. Orexin A significantly inhibited GnRH-stimulated FSH release from pituitary cells isolated from immature female rats, whereas in cells of mature ovariectomized animals, the effect of OxA was dependent on the stimulatory dose of GnRH. When the cells were stimulated with a low dose of GnRH, orexin A inhibited the secretion of gonadotrophins, but when a high dose of GnRH was used, orexin A increased mainly the release of LH. In cultured pituitary cells from ovariectomized, estrogenized mature rats, orexin A inhibited the secretion of LH if the cells were stimulated with a high dose of GnRH. In conclusion, the results of this study revealed that orexin A may modify the sensitivity of gonadotrophic cells to GnRH, and its effect depends on the maturity and estrogen status of the rats from which the cells are isolated.


Subject(s)
Gonadotropin-Releasing Hormone/metabolism , Intracellular Signaling Peptides and Proteins/pharmacology , Neuropeptides/pharmacology , Pituitary Gland, Anterior/metabolism , Animals , Cells, Cultured , Female , Follicle Stimulating Hormone/metabolism , Luteinizing Hormone/metabolism , Male , Orexins , Ovariectomy , Pituitary Gland, Anterior/drug effects , Rats , Rats, Wistar
4.
Acta Neurol Scand ; 121(4): 230-6, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20028343

ABSTRACT

OBJECTIVES: To investigate the role of leptin, ghrelin, GH and IGF-1 in energy balance disturbances in Parkinson's disease (PD). MATERIALS AND METHODS: Thirty-nine patients were included: 11 PD patients with unintentional weight loss, 16 PD patients without weight loss and 12 controls. UPDRS, MMSE, MADRS, appetite scale, BMI, adipose tissue content, plasma leptin and active ghrelin concentrations and serum GH, IGF-1, TSH, T3 and T4, concentrations were evaluated. RESULTS: A lower plasma leptin concentration and a higher serum IGF-1 concentration were found in PD patients with weight loss. BMI and the content of adipose tissue were positively correlated with leptin concentration in all PD patients. Paradoxically, the lower BMI was, the lower plasma active ghrelin concentration was in PD patients with the weight loss. CONCLUSION: These findings confirm that changes of plasma leptin concentration occur in PD patients with loss of weight.


Subject(s)
Energy Metabolism/physiology , Ghrelin/blood , Leptin/blood , Parkinson Disease/metabolism , Weight Loss , Adipose Tissue/metabolism , Aged , Appetite/physiology , Body Mass Index , Female , Growth Hormone/metabolism , Humans , Insulin-Like Growth Factor I/metabolism , Male , Middle Aged , Parkinson Disease/physiopathology , Thyroid Hormones/metabolism , Thyrotropin/metabolism
5.
J Physiol Pharmacol ; 60(1): 151-6, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19439817

ABSTRACT

Cortistatin (CST), a novel neuropeptide, shows high structural homology and functional resemblance with somatostatin. CST binds with high affinity to all somatostatin receptors, and contrary to somatostatin, is also able to bind with MrgX2 and GH secretagogue receptor of ghrelin (GHS-R1) receptors. The aim of the present investigation was to evaluate in vivo the effect of peripheral administration of cortistatin on pituitary hormone release in comparison with somatostatin (SS) treatment. Adult male rats used in the experiment, were given peripheral injection of cortistatin, somatostatin or vehicle. Blood was withdrawn 60 and 120 minutes thereafter. We found short lasting significant decrease of GH concentration as a result of administration of CST and SS when compared with saline injected controls. Prolactin levels were increased 60 min after cortistatin but not to somatostatin injection. There was no effect of CST on both LH and FSH concentration; however, SS administration influenced gonadotropin secretion. We conclude that cortistatin play a regulatory role in pituitary secretion. Moreover, some differences have been found when compared cortistatin to somatostatin. Thus, when analyzing the mechanism of cortistatin activity it is worth to consider the effect of binding with receptors of somatostatin, specific receptor for CST (MrgX2) and GHS-R.


Subject(s)
Growth Hormone/drug effects , Neuropeptides/pharmacology , Prolactin/drug effects , Animals , Follicle Stimulating Hormone/blood , Follicle Stimulating Hormone/metabolism , Growth Hormone/blood , Growth Hormone/metabolism , Luteinizing Hormone/blood , Luteinizing Hormone/drug effects , Luteinizing Hormone/metabolism , Male , Prolactin/blood , Prolactin/metabolism , Radioimmunoassay , Rats , Rats, Inbred WKY , Somatostatin/pharmacology
6.
J Physiol Pharmacol ; 57 Suppl 6: 55-61, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17228087

ABSTRACT

Neuropeptides play a pivotal role in the control of metabolic homeostasis. We aimed to evaluate the release of neuropeptides involved in the control of energy homeostasis in relation to metabolic status in aging humans. The study group consisted of 183 women: 75 centenarians (above 100 yrs old), 26 elderly women (below 70 yrs), 45 younger women (mean 26 yrs) and 37 obese women (mean 41.6 yrs). Fasting plasma concentration of leptin, adiponectin, ghrelin active, neuropeptide Y (NPY) and insulin were measured. Our results showed several differences in the metabolic and neurohormonal status in the centenarian group. The incidence of hypertension, glucose intolerance, insulin resistance and dyslipidemia was lower compared with obese women. Leptin and NPY concentrations were significantly lower than in elderly and obese subjects. Moreover, NPY level was higher than that in the younger group. Plasma adiponectin values were higher than in any of the other group. Insulin levels were significantly lower compared with the young and obese groups. Furthermore, a negative correlation was found between adiponectin and HOMA-IR, and adiponectin and insulin. Ghrelin active concentrations were significantly lower compared with the young subjects. However, ghrelin levels were higher than in obese subjects. We conclude that altered neuropeptide activity in centenarians may play a role in the mechanisms contributing to prolonged survival.


Subject(s)
Aging/blood , Neuropeptides/blood , Neurosecretory Systems/physiology , Peptide Hormones/blood , Adiponectin/blood , Adult , Age Factors , Aged , Aged, 80 and over , Female , Ghrelin/blood , Homeostasis/physiology , Humans , Insulin/blood , Leptin/blood , Longevity/physiology , Middle Aged , Neuropeptide Y/blood , Obesity/blood
7.
Pol J Vet Sci ; 8(4): 275-82, 2005.
Article in English | MEDLINE | ID: mdl-16385851

ABSTRACT

The aim of the study was to determine, using the rat model, whether uterine infections cause an increase in cytokine concentrations in peripheral blood, and whether this increase is accompanied by changes in the pituitary-ovarian axis function. The levels of tumor necrosis factor-alpha, interleukin-1beta, luteinizing hormone, follicle-stimulating hormone, prolactin, progesterone, testosterone and estradiol-17beta in blood plasma as well as the weight of the uterus were determined after intrauterine infusion of lipopolysaccharide (15 microg), peptidoglycan (1 mg) and Escherichia coli (10(6) cfu) suspension on the day of metaestrus. On days 3, 7 and 10 after treatment the rats were sacrificed to collect the blood samples. Inflammation of uterus and vaginal discharge developed in all rats after treatment. The administration of lipopolysaccharide, peptidoglycan and Escherichia coli induced considerable changes in ovarian cyclic activity, mainly diestrus was observed. Application of all these factors resulted in an increase (P<0.05, P<0.01) of plasma levels of tumor necrosis factor-alpha, interleukin-1beta, mainly on day 3 and 7. In the rats receiving pathological factors, the plasma levels of luteinizing hormone, follicle-stimulating hormone, prolactin and estradiol-17beta decreased (P<0.05, P<0.01) whereas progesterone and testosterone increased (P<0.05). These results indicate that in rats, the developing inflammatory process of the uterus following lipopolysaccharide, peptidoglycan and Escherichia coli infusions is connected with an increase of tumor necrosis factor-alpha, interleukin-1beta concentrations in peripheral blood, and is accompanied by changes in the pituitary-ovarian axis function.


Subject(s)
Cytokines/blood , Hormones/blood , Inflammation/blood , Inflammation/chemically induced , Animals , Escherichia coli/physiology , Escherichia coli Infections/blood , Estradiol/blood , Estrous Cycle , Female , Follicle Stimulating Hormone/blood , Lipopolysaccharides/administration & dosage , Luteinizing Hormone/blood , Ovary/physiology , Peptidoglycan/administration & dosage , Pituitary Gland/metabolism , Progesterone/blood , Prolactin/blood , Rats , Rats, Wistar , Uterus/anatomy & histology , Uterus/cytology , Vaginal Discharge
8.
Eur J Ophthalmol ; 13 Suppl 3: S11-8, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12749672

ABSTRACT

Defects in apoptosis (programmed cell death) have recently emerged as being closely involved in the pathogenesis of most ocular diseases and, therefore, apoptosis is now a topic of exponential interest in ophthalmology. This review summarizes recent works on mechanisms of apoptosis, from its initiation and modulation to the switching-on of its execution machinery. Interactions of cell death with cell division programs to orchestrate ontogenesis, aging, and adult life and their alterations in human diseases are pointed out. Two main apoptotic signaling pathways are identified: a death receptor-dependent (extrinsic) pathway and a mitochondrion-dependent (intrinsic) pathway. Mitochondrion harbors both antiapoptotic (Bcl-2, Bcl-XL) and apoptotic factors (Smac/Diablo, Apaf-1, cytochrome c). Its permeability transition pore (mPTP) is the main trigger of cell suicide. The process of mPTP opening, in association with extrusion to cytoplasm of a variety of apoptotic factors, is shown. Cytochrome c is one of these apoptotic factors. When expelled to cytoplasm, this double-faced respiratory chain component assembles with two other modules, Apaf-1 and procaspase 9, to form a protein complex--the apoptosome--that starts apoptosis execution. Another respiratory chain component, the CoQ10, is believed to counteract mPTP opening. What makes apoptosis particularly exciting for medicine is that its dysfunctions play a central role in the pathogenesis of several human diseases. For instance, excesses of apoptosis lead to cell loss that accompanies neurodegenerative diseases, whereas genetically determined defects of apoptosis lead to the deregulated cell proliferation typical of cancer. A variety of ophthalmologic diseases, such as post-keratectomy haze, corneal lesions, cataract, glaucoma, senile maculopathies, and genetic ocular pathologies, that underlie apoptosis dysfunctions are treated in detail in the other reviews of this issue.


Subject(s)
Apoptosis/physiology , Eye Diseases/metabolism , Animals , Biology , Humans , Ion Channels/physiology , Medicine , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Ophthalmology , Signal Transduction/physiology
9.
Biochem Biophys Res Commun ; 287(5): 1063-9, 2001 Oct 12.
Article in English | MEDLINE | ID: mdl-11587529

ABSTRACT

The expression of genes requiring finely tuned control is regulated by a posttranscriptional mechanism involving mRNA A + U-rich elements (AREs) cooperating with ARE-binding proteins (AUBPs) in modulation of mRNA stability. We reported previously that an ARE in the bcl-2 mRNA 3'-untranslated region (3'-UTR) had destabilizing activity and was involved in bcl-2 downregulation during apoptosis in vitro. Here we demonstrate that the bcl-2 ARE complexes with a number of specific AUBPs, whose pattern undergoes changes following application of apoptotic stimuli. The caspase inhibitor Z-VAD-fmk strongly attenuates both bcl-2 mRNA decay and bcl-2 AUBP pattern changes elicited by apoptotic stimuli, indicating the involvement of bcl-2 AUBPs in bcl-2 mRNA stability control.


Subject(s)
Apoptosis , Proto-Oncogene Proteins c-bcl-2/genetics , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , 3' Untranslated Regions/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Caspase Inhibitors , Gene Expression Regulation , Half-Life , Humans , Jurkat Cells , Protein Binding/radiation effects , RNA Stability , Ultraviolet Rays
10.
Neuro Endocrinol Lett ; 22(5): 349-55, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11600877

ABSTRACT

OBJECTIVES: Some hormonal disturbances were demonstrated in starvation. Leptin, NPY and galanin play an important role in the control of appetite and in the mechanism of hormone release. METHODS: In order to evaluate the effect of starvation on the relationship between leptin, neuropeptide Y (NPY) galanin and pituitary and gonadal hormones release, plasma leptin, NPY and galanin as well as serum LH, FSH, prolactin (PRL), estradiol, progesterone levels in non-starved female rats (in diestrus) and after 72 hrs of starvation were measured with RIA methods. Effects of leptin, NPY and galanin administration on pituitary and gonadal hormones were investigated in vivo and in vitro experiments. RESULTS: Plasma leptin, NPY and galanin as well as serum estradiol and progesterone concentrations were significantly lower in starved rats as compared with non-starved rats. However serum prolactin level was significantly higher in starved rats. Opposite effects after leptin and NPY administration on hormone release in vivo and in vitro experiments were observed in non-starved rats. However, in starved rats we did not find changes in pituitary and gonadal hormones release after leptin, NPY and galanin injection or the hormonal response was blunted. CONCLUSIONS: 1) The disturbances in neuropeptides activity and in hormones release were observed in starvation. 2) Leptin, NPY and galanin have direct and indirect effects on pituitary and gonadal hormones release. 3) In starvation the hormonal response to leptin, NPY and galanin is impaired.


Subject(s)
Hormones/blood , Neuropeptides/blood , Starvation/blood , Animals , Appetite Regulation/drug effects , Appetite Regulation/physiology , Energy Metabolism/drug effects , Estradiol/blood , Female , Follicle Stimulating Hormone/blood , Galanin/blood , Galanin/pharmacology , Leptin/analysis , Leptin/pharmacology , Luteinizing Hormone/blood , Neuropeptide Y/blood , Neuropeptide Y/pharmacology , Progesterone/blood , Prolactin/blood , Rats
11.
Neuro Endocrinol Lett ; 22(5): 356-8, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11600878

ABSTRACT

OBJECTIVES: It has been reported that leptin and neuropeptide Y (NPY) play a role in the control of appetite and in the regulation of hormonal secretion. METHODS: Plasma leptin, neuropeptide Y (NPY) and galanin concentrations were estimated in 13 women with bulimia nervosa (BN) 19 women with anorexia nervosa (AN) and in 19 healthy women of the control group (CG). RESULTS: Plasma leptin concentration in BN was significantly higher than that in AN and it was lower as compared with the control group, despite the same BMI (body mass index) in both the groups. Plasma leptin level in AN was significantly lower as compared with the controls. Plasma galanin concentrations in AN and BN did not differ significantly from the control group. Plasma NPY concentration in AN was lower than that in the control group. However, plasma NPY level in BN was significantly higher as compared with AN and with the control group (CG). The observed increase of NPY in BN was independent of BMI because BMI in bulimia nervosa was normal. CONCLUSIONS: The data may suggest that other factors than body weight changes may be involved in the modulation of leptin and NPY release in BN. The pathological behaviour of patients with bulimia nervosa may result from disturbed NPY release which is the strongest orexigenic factor.


Subject(s)
Anorexia Nervosa/blood , Bulimia/blood , Galanin/blood , Leptin/blood , Neuropeptide Y/blood , Adolescent , Adult , Body Mass Index , Female , Humans , Reference Values
12.
Neuro Endocrinol Lett ; 22(3): 208-14, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11449193

ABSTRACT

OBJECTIVES: In many studies it has been reported, that leptin may play an important role not only in the regulation of food intake and body weight but can modify immune response. The aim of our study was to estimate the effects of the administration of exogenous leptin on serum concentration of proinflammatory cytokines (interleukin 6-IL 6 and tumor necrosis factor alpha-TNF alpha) and anti-inflammatory cytokine (interleukin 10 - IL 10) during LPS induced acute inflammation. We also estimated leptin's influence on pituitary, thyroid, adrenal and gonadal hormones in response to lipopolysaccharide (LPS) induced acute inflammation. METHODS: Male rats Wistar-Kyoto were divided into four groups, which received respectively: placebo (0.9% NaCl), LPS, leptin and leptin with LPS. The TNF alpha and IL 6 serum concentrations were measured after 2 hours and IL 10 after 4 hours. The pituitary, thyroid, adrenal and gonadal hormones serum concentrations were measured after 2 and 4 hours. Cytokine concentrations were estimated using ELISA tests and hormones concentrations using RIA tests. RESULTS: Leptin did not have an effect on both cytokine responses (proinflammatory and anti-inflammatory) in the time of LPS-induced acute inflammation. Leptin enhanced LPS-induced increasing of corticosterone secretion after 2 hours and decreased LPS-induced inhibition of testosterone secretion after 4 hours. CONCLUSIONS: Leptin can modulate hormone response during LPS-induced acute inflammation.


Subject(s)
Hormones/blood , Inflammation/blood , Inflammation/immunology , Leptin/pharmacology , Lipopolysaccharides/administration & dosage , Animals , Corticosterone/blood , Cytokines/blood , Inflammation/chemically induced , Interleukin-10/blood , Interleukin-6/blood , Kinetics , Leptin/administration & dosage , Luteinizing Hormone/blood , Male , Prolactin/blood , Rats , Rats, Inbred WKY , Testosterone/blood , Thyroxine/blood , Triiodothyronine/blood , Tumor Necrosis Factor-alpha/analysis
13.
Transplantation ; 66(6): 748-54, 1998 Sep 27.
Article in English | MEDLINE | ID: mdl-9771838

ABSTRACT

BACKGROUND: The key role of anti-galactose alpha1,3-galactose (anti-alphaGal) xenoantibodies in initiating hyperacute xenograft rejection has been clearly demonstrated using a variety of in vitro and in vivo approaches. However, the role of anti-alphaGal antibodies in mediating post-hyperacute rejection mechanisms, such as antibody-dependent cellular cytoxicity, remains to be determined, primarily because of the lack of a small animal model with which to study this phenomena. METHODS: Hearts from wild-type mice were transplanted heterotopically into alpha1,3-galactosyltransferase knockout (Gal KO) mice, which like humans develop antibodies to the disaccharide galactose alpha1,3-galactose (Gal). At the time of rejection, hearts were examined histologically to determine the mechanism of rejection. RESULTS: Hearts from wild-type mice transplanted into high-titer anti-alphaGal recipients were rejected in 8-13 days. Histological examination demonstrated a cellular infiltrate consisting of macrophages (80-90%), natural killer cells (5-10%), and T cells (1-5%). In contrast, wild-type hearts transplanted into low anti-Gal titer recipients demonstrated prolonged (>90 day) survival. However, a significant proportion (30-40%) of these underwent a minor rejection episode between 10 and 13 days, but then recovered ("accommodated"). CONCLUSIONS: The results of this study suggest that the Gal KO mouse is a useful small animal vascularized allograft model, in which the role of anti-alphaGal antibody in graft rejection can be studied in isolation from other rejection mechanisms. The titer of anti-alphaGal antibody was found to be the critical determinant of rejection. The histopathological features of rejection in this model are very similar to other models of delayed xenograft rejection, in both the timing and composition of the cellular infiltrate. The Gal KO mouse therefore provides a new rodent model, which will aid in the identification of the distinct components involved in the pathogenesis of delayed xenograft rejection.


Subject(s)
Antibodies/immunology , Disaccharides/immunology , Galactosyltransferases/genetics , Graft Rejection/immunology , Transplantation, Heterologous/immunology , Animals , Antibodies, Protozoan/immunology , Female , Galactosyltransferases/metabolism , Graft Rejection/genetics , Graft Rejection/pathology , Heart Transplantation/immunology , Leishmania major/immunology , Male , Mice , Mice, Inbred Strains , Mice, Knockout , Myocardium/pathology
14.
J Surg Oncol ; 64(1): 3-11, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9040793

ABSTRACT

BACKGROUND: Sex steroid hormones influence function of the human gastrointestinal tract. Although the specific receptor proteins have been identified in surgical specimens of both intestinal mucosa and colorectal carcinomas, it is still unknown whether they are expressed in intestinal epithelial cells. METHODS: Expression of androgen receptor (AR) protein and estrogen receptor (ER) protein was studied by Scatchard analysis and ELISA (for ER only) in surgical specimens of normal-appearing mucosa, colorectal carcinomas, isolated colonocytes, and human colorectal carcinoma cell lines. Northern analysis was applied to identify the appropriate mRNAs, followed by the sensitive technique of reverse transcription-polymerase-chain-reaction (RT-PCR). RESULTS: AR protein was identified in all surgical specimens analyzed and ER protein in 10 out of 13 normal-appearing mucosa specimens and 4 out of 7 colorectal carcinomas. The receptor proteins were not found in isolated colonocytes or in the transformed cell lines. RT-PCR confirmed that none of the isolated normal colonocytes or transformed colorectal carcinoma-derived cells expressed these mRNAs. Intestinal smooth muscle cells and fibroblasts were found to express sex steroid receptor mRNAs. CONCLUSIONS: Both receptors are present in human large intestine but are expressed in stromal cells and not in intestinal epithelial cells. We hypothesize that sex steroids may influence the function of colonocytes indirectly through stromal-epithelial interactions.


Subject(s)
Colonic Neoplasms/chemistry , Colorectal Neoplasms/chemistry , Receptors, Androgen/metabolism , Receptors, Estrogen/metabolism , Colon/chemistry , Colon/cytology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Polymerase Chain Reaction , Receptors, Androgen/genetics , Receptors, Estrogen/genetics , Stromal Cells/metabolism , Tumor Cells, Cultured
15.
Transplantation ; 62(2): 155-60, 1996 Jul 27.
Article in English | MEDLINE | ID: mdl-8755809

ABSTRACT

Genetic engineering of donor animals in xenotransplantation research has been directed largely toward obtaining expression of various immunoregulatory molecules on vascular endothelium, the initial target of recipient antibody and complement. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. Using the promoter of the gene for intercellular adhesion molecule 2 (ICAM-2), which is constitutively expressed on all vascular endothelium, we have developed a system for endothelial cell gene targeting in vivo. A 334-basepair fragment from the 5' flanking region of the human ICAM-2 gene was used to drive the expression of human CD59 in transgenic mice. Strong and uniform expression of CD59 was observed on the endothelial cells of all blood vessels in the heart, kidney, lung, liver, and pancreas in the three lines of mice examined. Little or no expression was seen in other cell types, with the exception of neutrophils and monocytes. These results suggest that this small promoter region contains most of the signals necessary to endow it with endothelial cell specificity, making it a potentially valuable tool in areas ranging from xenotransplantation to gene therapy.


Subject(s)
Antigens, CD/biosynthesis , Antigens, CD/genetics , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Endothelium, Vascular/metabolism , Promoter Regions, Genetic , Animals , Base Sequence , Endothelium, Vascular/physiology , Gene Expression , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Sensitivity and Specificity
16.
J Biol Chem ; 270(36): 21188-94, 1995 Sep 08.
Article in English | MEDLINE | ID: mdl-7673151

ABSTRACT

The low affinity receptor for IgG, Fc gamma RII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. Fc gamma RII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human Fc gamma RII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by Fc gamma RII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between Fc gamma RII and the structurally related Fc epsilon RI alpha chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by Fc gamma RII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of Fc gamma RII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.


Subject(s)
Immunoglobulin G/metabolism , Receptors, IgG/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , DNA, Complementary , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Receptors, IgG/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
17.
J Biol Chem ; 269(21): 15287-93, 1994 May 27.
Article in English | MEDLINE | ID: mdl-8195166

ABSTRACT

Fc receptor-antibody interactions are key mechanisms through which antibody effector functions are mediated. The low affinity receptor for IgG, Fc gamma RII, is expressed on most hematopoietic cells, and through the binding of immune complexes mediates a large spectrum of biological responses vital for resistance to infection and the regulation of immunity. In this study the key residues of human Fc gamma RII involved in the interaction with IgG1 have been identified. Chimeric receptors composed of extracellular regions of Fc gamma RII and the Fc epsilon RI alpha chain have been used to localize the IgG1 binding site of Fc gamma RII to an 8-residue stretch in the second extracellular domain, Asn154 to Ser161. Site-directed mutagenesis of this region revealed that substitution of Ile155 or Gly156 with alanine ablated the binding of human and mouse IgG1, whereas replacement of Leu159, Phe160, or Ser161 with alanine enhanced binding. Molecular modeling has been used to generate a putative 3-dimensional model structure of the second extracellular domain of Fc gamma RII, suggesting that the binding site lies in an exposed loop region at the interface of domains 1 and 2.


Subject(s)
Immunoglobulin G/metabolism , Mutagenesis, Site-Directed , Receptors, IgG/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/genetics , Cells, Cultured , Humans , Mice , Models, Molecular , Molecular Sequence Data , Oligodeoxyribonucleotides , Protein Conformation , Receptors, IgG/chemistry , Receptors, IgG/genetics , Sequence Alignment
18.
Immunol Cell Biol ; 70 ( Pt 2): 79-87, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1398776

ABSTRACT

Distinct differences in the capacity of monocyte Fc gamma RII of different individuals to bind or not bind mouse IgG1 defines a polymorphism of Fc gamma RIIa and has previously been defined as the high responder (HR) or low responder (LR) polymorphism of Fc gamma RII. The precise definition of the molecular basis of the human HR/LR polymorphism of Fc gamma RIIa from the peripheral blood mononuclear cells of normal individuals has been determined by anti-CD3 induction of T cell proliferation, the polymerase chain reaction (PCR), nucleotide sequencing, transfection and IgG binding. Amplification of first strand cDNA from mRNA isolated from mononuclear cells was performed by PCR using primers specific for the sequences encoding the leader and cytoplasmic sequences of PCR using primers specific for the sequences encoding the leader and cytoplasmic sequences of Fc gamma RIIa, which is normally expressed in monocytes. Sequencing of the PCR products and transfection of these to Fc gamma R- cells indicated that in Fc gamma RIIa of HR or LR individuals: (i) three nucleotide substitutions (CA to TG and G to A) resulted in the change of glutamine to tryptophan at position 27 (first extracellular domain) and arginine to histidine at position 131 (second extracellular domain); (ii) expression of cDNA encoding the various combinations of these indicated that arginine at position 131 was essential for IgG1 binding whereas the amino acid changes at position 27 had no effect; and (iii) IgG1 at high concentration bound to all allomorphic forms of Fc gamma RIIa.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Immunoglobulin G/immunology , Receptors, Fc/immunology , Receptors, IgG/immunology , Transfection , Binding Sites, Antibody/immunology , Cell Line , Gene Expression , Genes, Immunoglobulin/genetics , Humans , Lymphocyte Activation/immunology , Monocytes/immunology , Polymerase Chain Reaction , Polymorphism, Genetic/immunology , RNA, Messenger/genetics , Receptors, Fc/genetics , Receptors, IgG/genetics , T-Lymphocytes/immunology
19.
J Immunol ; 146(1): 369-76, 1991 Jan 01.
Article in English | MEDLINE | ID: mdl-1824594

ABSTRACT

We have isolated and deduced the structure of the beta Fc gamma RII gene from the mouse. This gene spans approximately 18 kb of DNA; the structure and nucleotide sequence has been determined and potential regulatory sites identified. This gene is composed of 10 exons that give rise to the beta 1 and beta 2 Fc gamma RII cDNA. Four exons encode the 5' untranslated region and leader sequence, one exon for each Ig-binding domain and membrane-spanning region and three exons encoding the cytoplasmic tail and 3' untranslated region. Analysis of the gene structure indicated that the beta 1Fc gamma RII and beta 2Fc gamma RII arise by differential mRNA splicing when a single 141-bp exon (exon 8) is alternately spliced to give rise to these isoforms. Sequence analysis of 2 kbp upstream of the transcription start site indicated the presence of regulatory elements, including three binding sites for the transcription factor Sp1, and Ap-4 binding site, a tandem glucocorticoid response element, and an overlapping tandem repeat. Furthermore, as in the Thy-1 gene, no CAT or TATA consensus sequences were observed. In addition, sites of methylation that regulate expression of this gene were also located at the 5' end of the gene and within several introns. A large intron located between exons 4 and 5 also contained a series of purine/pyrimidine-rich regions and a potential enhancer sequence.


Subject(s)
Antigens, Differentiation/genetics , Receptors, Fc/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Genes , Introns , Mice , Molecular Sequence Data , RNA Splicing , RNA, Messenger/genetics , Receptors, IgG , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic Acid , Restriction Mapping
20.
J Mol Endocrinol ; 4(2): 119-25, 1990 Apr.
Article in English | MEDLINE | ID: mdl-2344389

ABSTRACT

The effects of oestradiol and progesterone on LH-subunit mRNA levels were investigated in ovariectomized rats. Four weeks after ovariectomy, rats were implanted with silicone elastomer capsules containing oestradiol and/or injected daily with progesterone in oil (5 mg/rat) for 8 days. The levels of pituitary mRNA encoding alpha and LH-beta were determined using direct hybridization with specific [32P]cDNA probes. After oestradiol implantation in ovariectomized rats, both alpha and LH-beta mRNA decreased with time, with maximum inhibition after 6-8 days of treatment. Progesterone injected alone did not show any effect on alpha and LH-beta mRNA. Cytosolic progesterone receptors, determined using [3H]methyl-17 alpha-progesterone as ligand, were undetectable in control ovariectomized rats. In contrast, 2 days after oestradiol implantation, the number of receptors increased to 287.5 +/- 35.4 (S.E.M.) fmol/pituitary and reached a plateau of 400 +/- 21.8 fmol/pituitary after 4 days. The effects of progesterone were therefore examined by first implanting ovariectomized rats with oestradiol to induce progesterone receptors and then injecting progesterone daily for a further period of 6 days. As a result of this treatment, progesterone induced a decrease in the pituitary gland contents of both alpha and LH-beta mRNAs, and LH release was significantly greater than that observed in the group receiving oestradiol alone. Moreover, the mRNA levels in the animals treated with oestradiol plus progesterone were lower after 8 days of treatment than those observed in ovariectomized rats treated with a tenfold higher dose of oestradiol alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Estradiol/pharmacology , Luteinizing Hormone/genetics , Pituitary Gland/metabolism , Progesterone/pharmacology , RNA, Messenger/metabolism , Animals , Drug Synergism , Female , Luteinizing Hormone/blood , Ovariectomy , RNA, Messenger/genetics , Rats , Rats, Inbred Strains , Receptors, Progesterone/metabolism , Time Factors
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