Glycoprotein Hormone Research Articles

Comprehensive glycan analysis of twelve recombinant human erythropoietin preparations from manufacturers in China and Japan

Recombinant, human, erythropoietin (rhEPO) is a glycoprotein hormone which is prescribed throughout the world to treat anaemia caused by chronic kidney disease or chemotherapy. rhEPO is at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Due to its complex glycosylation profile, which has a crucial influence upon biological activity, therapeutic rhEPO preparations must be closely monitored to ensure consistency, safety and efficacy. Here, we have compared twelve rhEPO preparations from eleven manufacturers in China and one in Japan, measuring in vivo biological activity and exploring its relationship with glycosylation through sialic acid content determination, isoform distribution via capillary electrophoresis (CE), O-glycan profiling, and N-glycan mapping using a novel anion-exchange/hydrophilic interaction chromatography-mass spectrometry (AEX/HILIC-MS) approach. We observed differences between glycosylation profiles, including the varying occurrence of sialic acid O-acetylation, extension of N-glycan antennae with N-acetyllactosamine units, and the distribution of sialic acids across multi-antennary structures. The presence of unusually high levels of suspected penta- and hexa-anionic N-glycans in several samples is consistent with elevated rhEPO isoform acidity, which is reflected by slightly elevated in vivo bioactivities. This aside, the observed differences in glycosylation profile do not appear to have a significant influence upon biological activity in mice. Nonetheless, with the continued emergence of biosimilars, the study highlights the importance of monitoring glycosylation profiles in biological medicines, in order to detect and account for divergence between products, as well as the presence of unusual or unexpected glycans.

Gonadotropins and their receptors: coevolution, genetic variants, receptor imaging, and functional antagonists

Gonadotropins belong to the family of dimeric glycoprotein hormones and regulate gonadal physiology mediated by G protein-coupled, seven-transmembrane receptors. These glycoprotein hormones are widely used in the clinic to promote ovarian follicle development and for treating some cases of male infertility. We traced the coevolution of dimeric gonadotropin hormones and their receptors, together with thyrotropin and its receptor. We updated recent findings on human genetic variants of these genes and their association with dizygotic twining, polycystic ovarian syndrome, primary ovarian insufficiency, male-limited precocious puberty, and infertility. In addition to the known physiological roles of gonadotropin-receptor signaling in gonadal tissues, we also discussed emerging understanding of extragonadal functions of gonadotropins in bones and adipose tissues, together with recent advances in in vivo imaging of gonadotropin receptors in live animals. Recent development of gonadotropin receptor agonists and antagonists were summarized with an emphasis on the development of functional antagonists for FSH receptors to alleviate osteoporosis and obesity associated with menopause.

Immunohistochemical study of the expression of human chorionic gonadotropin-β in salivary gland tumors.

Human chorionic gonadotropin-β (hCGβ) is a hormone glycoprotein usually secreted by trophoblastic cells in early pregnancy and helps in growth and development of the embryo. While many trophoblastic and nontrophoblastic malignancies express elevated levels of hCGβ in serum, increased immunohistochemical reactivity has also been reported in malignant and aggressive tumors, thus serving as a marker. As limited studies exist on tumors of the oral- and para-oral region, it prompted us to observe the immunohistochemical expression of hCGβ in salivary gland tumors. A total of 21 cases of salivary gland tumors - 16 cases of mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and five cases of pleomorphic adenoma - were included in the study. The sections were subjected to immunohistochemical procedures using hCGβ antigen. The degree of intensity and distribution of hCGβ immunostaining was assessed. One case of each (12.5%) MEC and ACC showed positive staining, and no staining was observed in the pleomorphic adenoma. The presence of hCGβ positive tumor cells appears to potentially reflect the aggressive behavior of MEC and ACC.

Expression of two glycoprotein hormone receptors in larval, parasitic phase, and adult sea lampreys

All jawed vertebrates have three canonical glycoprotein hormones (GpHs: luteinizing hormone, LH; follicle stimulating hormone, FSH; and thyroid stimulating hormone, TSH) with three corresponding GpH receptors (GpH-Rs: LH-R, FSH-R, and TSH-R). In contrast, we propose that the jawless vertebrate, the sea lamprey (Petromyzon marinus), only has two pituitary glycoprotein hormones, lamprey (l)GpH and l-thyrostimulin, and two functional glycoprotein receptors, lGpH-R I and II. It is not known at this time whether there is a specific receptor for lGpH and l-thyrostimulin, or if both GpHs can differentially activate the lGpH-Rs. In this report, we determined the RNA expression of lGpH-R I and II in the gonads and thyroids of larval, parasitic phase, and adult lampreys. A highly sensitive dual-label fluorescent in situ hybridization technique (RNAScope™) showed lGpH-R I expression in the ovaries of larval lamprey, and co-localization and co-expression of lGpH-R I and II in the ovaries of parasitic phase and adult lampreys. Both receptors were also highly co-localized and co-expressed in the endostyle of larval lamprey and thyroid follicles of parasitic and adult lampreys. In addition, we performed in vivo studies to determine the actions of lamprey gonadotropin releasing hormones (lGnRHs) on lGpH-R I and II expression by real time PCR, and determined plasma concentrations of estradiol and thyroxine. Administration of lGnRH-III significantly (p ≤ 0.01) increased lGpHR II expression in the thyroid follicles of adult female lampreys but did not cause a significant increase in RNA expression of lGpH-R I and II in ovaries. Concomitantly, there was a significant increase (p ≤ 0.01) of plasma estradiol without any significant changes of plasma thyroxine concentrations in response to treatment to lGnRH-I, -II, or -III. In summary, our results provide supporting evidence that the lamprey pituitary glycoprotein hormones may differentially activate the lamprey GpH-Rs in regulating both thyroid and gonadal activities during each of the three life stages of the sea lamprey.

Effects of erythropoietin on spontaneous and oxytocin induced myometrial contractions in the nonpregnant rat.

Erythropoietin (EPO) is a glycoprotein hormone that regulates erythropoiesis. EPO activity has also been detected in a variety of tissue including the nervous system, and female and male reproductive organs. It has been shown that EPO causes relaxation in vascular smooth muscle. In the present study, we have investigated effects of EPO on spontaneous and oxytocin-induced contractions of non-pregnant rat myometrium. Myometrial stripes were obtained from adult Wistar rats at the oestrous stage. The samples were placed in an isolated organ chamber under physiological conditions and 1 g passive tension. Epoetin beta (rEPO) was added cumulatively at 0.1, 1 and 10 IU/ml concentrations to the myometrial samples showing regular spontaneous contractions for periods of 30 min. Frequency and amplitude of contractions were electrophysiologically recorded and analyzed by using a BIOPAC data acquisition system. rEPO inhibited both area under curve and frequency of spontaneous contractions (ANOVA, n1, 2 = 9, f1 = 20.938, f2 = 20.492, p1,2 = 0.000). The inhibitory effect was insignificant at 0.1 mIU/ml rEPO level (Tukey HSD, p1 = 0.051, p2 = 0.581). In the oxytocin treated myometrial samples, a single dose of 1 IU/ml rEPO was studied. The area under curve and frequency values of these samples were inhibited by rEPO (Student's t-test, n = 9, t1 = 4.776, p1 = 0.000; t2 = 2.835, p2 = 0.012, respectively). rEPO inhibited spontaneous and oxytocin-induced rat myometrial contractions at 1 and 10 IU/ml concentrations. It appeared that the effect was dose-dependent.

Molecular size and charge as dimensions to identify and characterize circulating glycoforms of human FSH, LH and TSH

Background: FSH, LH, and TSH are glycoprotein hormones secreted from the pituitary as fully and low-asparagine-glycosylated hormones. These glycoforms of the hormones exist as a large number of isoforms varying in their glycan contents of terminal anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). Due to the immense heterogeneity and the low concentrations in serum it has been a challenge to develop reliable analytical methods to measure and characterize the circulating glycoforms of these hormones.Methods: The hormones were separated with respect to AMS content per molecule by calibrated 0.1% agarose suspension electrophoreses. Glycoforms in separated fractions were then analyzed with respect to size by 180 calibrated Sephadex G-100 gel filtrations. The hormones were measured with time-resolved sandwich fluoroimmunoassays. All separations and assays were performed in veronal buffer at pH 8.7. Sera and fractions were also analyzed after removal of terminal SA.Results: In addition to the fully glycosylated FSH, LH, and TSH, also tri-glycosylated FSH and di-glycosylated LH and TSH forms could be identified in serum samples. The low- and fully glycosylated hormones differed both with respect to size and to median number of AMS per molecule. Algorithms, based on the distributions by electrophoreses, were developed for each hormone to estimate percent low-glycosylated forms in serum. The median numbers of SA and SU per glycoform molecule were estimated using results obtained after desialylation.Conclusion: The methods can be used for identification and characterization of glycoforms of circulating FSH, LH, and TSH in physiological and clinical studies.

The Exonization and Functionalization of an Alu-J Element in the Protein Coding Region of Glycoprotein Hormone Alpha Gene Represent a Novel Mechanism to the Evolution of Hemochorial Placentation in Primates.

Alu elements contribute considerably to gene regulation and genome evolution in primates. The generation of new exons from Alu elements has been found in various human genes, and the regulatory function of the Alu exon has been investigated in many studies. However, the functionalization of Alu elements in protein coding regions remains unknown. Here, we reported that an Alu-J element exonized in the glycoprotein hormone alpha (GPHA) gene and encoded an additional N-terminal peptide (Alu-J encoding peptide) of the mature GPHA peptide, leading to a splicing variant of Alu-GPHA in anthropoid primates ∼35 Ma. Interestingly, adaptive evolution of the Alu-J exon occurred in the human and ape lineages during anthropoid evolution. The Alu-J encoding peptide is found to be a new biomarker in human early pregnancy and prolongs the serum half-life of human chorionic gonadotropin (HCG) circulation. Moreover, Alu-J encoding peptide enhances the bioactivity of HCG protein, both in vivo and in vitro. Our study reveals the first example of an Alu element functioning as the encoding peptide to increase the whole protein stability and provides insight into the potential multi-functionalization of the Alu exon in the protein coding regions. Furthermore, with the chorionic gonadotropin linking with hemochorial placentation, the exonization and functionalization of the Alu-J exon in GPHA gene represent a novel mechanism to the evolution of hemochorial placentation in primates.

HCG: Biological Functions and Clinical Applications.

Human chorionic gonadotropin (hCG) is produced primarily by differentiated syncytiotrophoblasts, and represents a key embryonic signal that is essential for the maintenance of pregnancy. hCG can activate various signaling cascades including mothers against decapentaplegic homolog 2 (Smad2), protein kinase C (PKC), and/or protein kinase A (PKA) in several cells types by binding to luteinizing hormone/chorionic gonadotropin receptor (LHCGR) or potentially by direct/indirect interaction with transforming growth factor beta receptor (TGFβR). The molecule displays specialized roles in promoting angiogenesis in the uterine endothelium, maintaining myometrial quiescence, as well as fostering immunomodulation at the maternal-fetal interface. It is a member of the glycoprotein hormone family that includes luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and follicle-stimulating hormone (FSH). The α-subunit of hCG displays homologies with TSH, LH, and FSH, whereas the β subunit is 80–85% homologous to LH. The hCG molecule is produced by a variety of organs, exists in various forms, exerts vital biological functions, and has various clinical roles ranging from diagnosis and monitoring of pregnancy and pregnancy-related disorders to cancer surveillance. This review presents a detailed examination of hCG and its various clinical applications.

Extra-pituitary expressed follicle-stimulating hormone: Is it physiologically important?

Pituitary gonadotropes synthesize and secrete follicle-stimulating hormone (FSH). FSH is a heterodimer that consists of an α- and β-subunit. The α-subunit is common to other pituitary and placental glycoprotein hormones, and the β-subunit is the hormone/receptor-specific subunit. Although the pituitary is the main tissue that accounts for circulating hormone, previous and recent reports indicate extra-pituitary sources of FSH production including mouse gonads, human stomach, prostate, umbilical cord vein endothelial cells, uterine myometrium, placenta, and chicken abdominal adipose tissue. Whether extra-pituitary derived FSH exerts any physiologically significant actions is not known. In this review, we have comprehensively analyzed the expression of mRNAs that encode mouse and human FSH subunits and also their corresponding expressed sequence tags in normal tissues, cancer cell lines, and primary tumors by public database mining. We propose criteria to assess the significance of individual FSH subunit or FSH dimer expression as well as genetic approaches to unambiguously define the physiological relevance of extra-pituitary FSH expression.

Neuropeptide discovery in Proasellus cavaticus: Prediction of the first large-scale peptidome for a member of the Isopoda using a publicly accessible transcriptome

In silico transcriptome mining is one of the most effective methods for neuropeptide discovery in crustaceans, particularly for species that are small, rare or from geographically inaccessible habitats that make obtaining the large pools of tissue needed for other peptide discovery platforms impractical. Via this approach, large peptidomes have recently been described for members of many of the higher crustacean taxa, one notable exception being the Isopoda; no peptidome has been predicted for any member of this malacostracan order. Using a publicly accessible transcriptome for the isopod Proasellus cavaticus, a subcentimeter subterranean ground water dweller, the first in silico-predicted peptidome for a member of the Isopoda is presented here. BLAST searches employing known arthropod neuropeptide pre/preprohormone queries identified 49 transcripts as encoding putative homologs within the P. cavaticus transcriptome. The proteins deduced from these transcripts allowed for the prediction of 171 distinct mature neuropeptides. The P. cavaticus peptidome includes members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon α, bursicon β, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone α2, leucokinin, myosuppressin, neuroparsin, neuropeptide F, pigment dispersing hormone, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, sulfakinin, tachykinin-related peptide and trissin families, as well as many linker/precursor-related sequences that may or may not represent additional bioactive molecules. Interestingly, many of the predicted P. cavaticus neuropeptides possess structures identical (or nearly so) to those previously described from members of several other malacostracan orders, i.e., the Decapoda, Amphipoda and Euphausiacea, a finding that suggests broad phylogenetic conservation of bioactive peptide structures, and possibly functions, may exist within the Malacostraca.

Immunoassay for human serum erythroferrone

AbstractErythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to stimulation by erythropoietin (EPO). We previously demonstrated that ERFE messenger RNA expression and serum protein concentration increase in mice subjected to hemorrhage or EPO therapy, that ERFE acts on hepatocytes to suppress hepcidin, and that the resulting decrease in hepcidin augments iron delivery for intensified erythropoiesis. We also showed that ERFE contributes to pathological hepcidin suppression and iron overload in mice with nontransfused β-thalassemia. We now report the development and technical validation of a rabbit monoclonal antibody–based sandwich immunoassay for human ERFE. We use this assay to show that blood loss or EPO administration increases serum ERFE concentrations in humans, and that patients with both nontransfused and transfused β-thalassemia have very high serum ERFE levels, which decrease after blood transfusion. The assay should be useful for human studies of normal and disordered erythropoiesis and its effect on iron homeostasis.

Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix

Equine pituitary gonadotropins (eLH, eFSH, eCG) are heterodimeric glycoprotein hormones with alpha (α) and beta (β) subunits. It is responsible for maintenance of pregnancy in mares during early gestation and fairly valuable for inducing superovulation in animals other than equines. The alpha subunit is common, while beta subunit is species-specific in all glycoprotein hormones. In the present investigation, molecular cloning and in silico characterization including homology modeling and molecular docking analysis of the equine chorionic gonadotropin (eCG) alpha subunit was carried out for gaining structural and functional insights into the eCG alpha subunit and its possible interaction with ganirelix, a gonadotropin-releasing hormone (GnRH) antagonist. The equine chorionic gonadotropin (eCG) alpha subunit expressed in pituitary gland was selected, amplified from total RNA, cloned and sequenced. The in silico analyses were made for homology modelling, structural details, epitope identification and chromosomal localization. Molecular docking studies of eCG alpha were undertaken with a drug ganirelix which is used to control ovulation and has antagonistic activity against GnRH. The protein sequence corresponding to selected open reading frame (ORF) was 99–100% similar with domesticated horse, Przewalski’s horse, and 92–93% with Burchell’s zebra and donkey. Molecular docking studies revealed the possible interaction of eCG alpha with ganirelix. The possible drug-macromolecule interactions were visualized between eCG alpha and ganirelix. The study will provide structural insight into unique sites and an alternate route of gonadotropin suppression applicable to assisted reproductive technologies.

The possible role of AMH in shortening the gubernacular cord in testicular descent: A reappraisal of the evidence

Background/AimAnti-Müllerian hormone (AMH), also called Müllerian inhibiting substance (MIS), is glycoprotein hormone secreted by the fetal Sertoli cells to regulate regression of the Müllerian ducts, the anlagen of the uterus, fallopian tubes, and upper vagina. After its existence was predicted in 1946 and its isolation and purification in the 1970's, a huge amount of information has been gathered on its molecular biology and function in the last 30–40years. Once thought to be a locally acting factor in the male fetus during sexual differentiation, it is now recognized as an endocrine hormone present in both sexes and with functions throughout life. One of the remaining controversies is the possible role of AMH during fetal testicular descent. In the human with aberrant AMH function, the boy has cryptorchidism with persistent Müllerian duct syndrome (PMDS), where the testes are often intraabdominal and on an abnormally long gubernacular cord. By contrast, in rodent models knockout of the AMH gene does not cause cryptorchidism. Methods/ResultsIn this review we examined the evidence in the literature for and against a role for AMH in testicular descent and considered the implications of the different anatomy of the gubernacular cord in rodents versus children. ConclusionWe conclude that AMH may have a role in shortening the gubernacular cord in humans which is concealed in rodent models by differences in anatomy of the gubernacular cord in rodents. The controversy could be resolved by re-examination of the gubernacular cord in boys with PMDS and mice with AMHKO. Type of studyReview. Level of evidenceV

Erythropoietin Rescues Memory Impairment in a Rat Model of Chronic Cerebral Hypoperfusion via the EPO-R/JAK2/STAT5/PI3K/Akt/GSK-3β Pathway.

Vascular dementia is the second most common cause of dementia in older people and is characterized by the sudden onset of impairments in thinking skills and behavior, which generally occur following a stroke. Unfortunately, effective therapy for vascular dementia remains inadequate. Erythropoietin (EPO) is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. Recently, a prominent role for EPO has been defined in the nervous system, and there is growing interest in the potential therapeutic use of EPO for neuroprotection. However, whether it is protective from memory impairments and the underlying mechanisms of vascular dementia (VD) remains unknown. In the current study, we reported that supplements with exogenous erythropoietin (EPO) for 4weeks could restore impaired memory in 2-vessel occlusion (2VO) rats, a well-established vascular dementia animal model. EPO also rescued impairments in dendritic spines and cholinergic dysfunctions in the hippocampus. Moreover, EPO suppressed the overactivation of GSK-3β in the hippocampus by stimulating the JAK2/STAT5/PI3K/Akt signal pathway. Furthermore, we found that genetic knockdown of the EPO receptor (EPO-R) by shRNA blocks the neuroprotection conferred by EPO on memory in VD. We hypothesized that EPO treatment is able to rescue the memory impairments in VD by stimulating the EPO-R/JAK2/STAT5/PI3K/Akt/GSK-3β pathway and suggest the potential usage of EPO in the therapy for VD.

Estrogen Modulates Specific Life and Death Signals Induced by LH and hCG in Human Primary Granulosa Cells In Vitro.

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are glycoprotein hormones used for assisted reproduction acting on the same receptor (LHCGR) and mediating different intracellular signaling. We evaluated the pro- and anti-apoptotic effect of 100 pM LH or hCG, in the presence or in the absence of 200 pg/mL 17β-estradiol, in long-term, serum-starved human primary granulosa cells (hGLC) and a transfected granulosa cell line overexpressing LHCGR (hGL5/LHCGR). To this purpose, phospho-extracellular-regulated kinase 1/2 (pERK1/2), protein kinase B (pAKT), cAMP-responsive element binding protein (pCREB) activation and procaspase 3 cleavage were evaluated over three days by Western blotting, along with the expression of target genes by real-time PCR and cell viability by colorimetric assay. We found that LH induced predominant pERK1/2 and pAKT activation STARD1, CCND2 and anti-apoptotic XIAP gene expression, while hCG mediated more potent CREB phosphorylation, expression of CYP19A1 and procaspase 3 cleavage than LH. Cell treatment by LH is accompanied by increased (serum-starved) cell viability, while hCG decreased the number of viable cells. The hCG-specific, pro-apoptotic effect was blocked by a physiological dose of 17β-estradiol, resulting in pAKT activation, lack of procaspase 3 cleavage and increased cell viability. These results confirm that relatively high levels of steroidogenic pathway activation are linked to pro-apoptotic signals in vitro, which may be counteracted by other factors, i.e., estrogens.

Structural-Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work.

The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to understand the molecular activation mechanisms of this receptor comprehensively. Finally, limitations of current knowledge and lack of information are discussed highlighting the need for intensified efforts toward TSHR structure elucidation.

Immunohistochemical mapping and transcript expression of the GPA2/GPB5 receptor in tissues of the adult mosquito, Aedes aegypti

GPA2/GPB5 is a glycoprotein hormone found in most bilateral metazoans including the mosquito, Aedes aegypti. To elucidate physiological roles and functions of GPA2/GPB5, we aim to identify prospective target tissues by examining the tissue- and sex-specific expression profile of its receptor, the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1) in the adult mosquito. Western analyses using a heterologous system with CHO-K1 cells, transiently expressing A. aegypti LGR1, yielded a 112-kDa monomeric band and high-molecular weight multimers, which associated with membrane-protein fractions. Moreover, immunoblot analyses on protein isolated from HEK 293T cells stably expressing a fusion construct of A. aegypti LGR1-EGFP (LGR1: 105kDa+EGFP: 27kDa) yielded a band with a measured molecular weight of 139kDa that also associated with membrane-protein fractions and upon deglycosylation, migrated as a lower molecular weight band of 132kDa. Immunocytochemical analysis of HEK 293T cells stably expressing this fusion construct confirmed EGFP fluorescence and LGR1-like immunoreactivity colocalized primarily to the plasma membrane. Immunohistochemical mapping in adult mosquitoes revealed LGR1-like immunoreactivity is widespread in the alimentary canal. Importantly, LGR1-like immunoreactivity localizes specifically to basolateral regions of epithelia and, in some regions, appeared as punctate intracellular staining, which together indicates a potential role in feeding and/or hydromineral balance. LGR1 transcript expression was also detected in gut regions that exhibited strong LGR1-like immunoreactivity. Interestingly, LGR1 transcript expression and strong LGR1-like immunoreactivity was also identified in reproductive tissues including the testes and ovaries, which together suggests a potential role linked to spermatogenesis and oogenesis in male and female mosquitoes, respectively.

Differential expression of the Notch1 receptor, and its ligands Dll1, Dll3 and Dll4 in distinct human pituitary adenoma subtypes.

Pituitary adenoma (PA) is a common type of benign tumor of the pituitary gland that is characterized by specific signs and symptoms, primarily associated with hypersecretion of pituitary glycoprotein hormones (thyroid-stimulating, growth and adrenocorticotrophic hormones, and prolactin). Surgery is the first-line treatment, although postoperative residual tissues/cells and subsequent recurrence remain notable complications. Gene therapy is an effective approach for treatment, as previous studies have demonstrated that the Notch signaling pathway participates in the pathogenesis of PA. The focus of the present study was on the associations between the expression of the Notch1 receptor and its ligands δ-like canonical Notch ligand (Dll) 1, Dll3 and Dll4 in patients with PA. Using reverse transcription-quantitative polymerase chain reaction and western blot analyses, to the best of our knowledge, this is the first study to provide a description of the differential expression of the Notch1 receptor and its ligands Dll1, Dll3, and Dll4 in various types of human PA at the mRNA and protein levels. The results of the present study demonstrated that Notch1 protein expression was positively correlated with Dll4 protein expression, but negatively correlated with Dll3 protein expression, indicating synergistic effects between the Notch1 receptor and Dll4 ligand. Furthermore, the Dll3 ligand may be an inhibitor of the Notch1 receptor, indicating an antagonistic association between Notch1 and the Dll3 ligand. These results have identified a potential target for the treatment of patients with PA.

Interpretation of Erythropoietin and Haemoglobin Levels in Patients with Various Stages of Chronic Kidney Disease.

SummaryBackgroundThe production of erythrocytes is regulated by the hormone erythropoietin (EPO), which maintains the blood haemoglobin (Hb) levels constant under normal conditions. Human EPO is a glycoprotein hormone and its synthesis is controlled by the hypoxia-inducible transcription factor. The aim of this study was to establish EPO and Hb levels in patients with chronic kidney disease (CKD), as well as in control subjects, and to investigate the relationship between these parameters.MethodsThis cross-sectional, observational study included 356 subjects with CKD divided into 4 subgroups according to their glomerular filtration rate (GFR). The control group consisted of 206 age and sex matched healthy subjects with GFR rate ≥90 mL/min/1.73 m2. EPO, Hb and serum creatinine levels were determined by using immunochemical and spectrophotometric methods. GFR was determined using the MDRD formula.ResultsThe CKD patients had significantly lower levels of haemoglobin (p<0.0005) and hematocrit (p<0.0005) compared to control group. Our results showed that Hb levels decreased, whereas serum creatinine increased with the increasing renal failure. The CKD patients in all four groups had significantly lower (p<0.0005) Hb levels, and significantly higher (p<0.0005) creatinine levels compared to the control group. The median EPO in group I and II were significantly higher (p=0.002; p=0.018), while median EPO in group III and IV were significantly lower (p=0.03; p=0.011) compared to the control group.ConclusionsIn patients with CKD, GFR positively correlated with Hb and EPO, while the correlation between GFR and serum creatinine was negative.

Mutation of Follicle Ftimulating Hormone Receptor Putative Caveolin Binding Motif Results in Altered Signaling

The human follicle stimulating hormone receptor (hFSHR) is a glycoprotein hormone receptor belonging to the g protein‐coupled receptor family. It is important in both male and female reproductive processes; defects in hFSHR can lead to infertility, delayed puberty, reduced muscle bulk, and osteoporosis. Work in other labs has shown that GPCRs can be localized to microdomains located within the cell membrane called lipid rafts. These regions are highly resistant to detergents because of the high concentration of sphingolipids and cholesterol. Also within these domains, an intracellular protein, caveolin, is present. Our lab has shown that hFSHR also localizes to lipid raft domains. We hypothesize that interaction with caveolin is important in the raft residency of hFSHR through a putative caveolin binding motif (CBM). The interaction between caveolin and hFSHR in these microdomains may regulate intracellular pathways activated by the receptor.To test our hypothesis, we have constructed CBM mutants and are creating stable cell lines expressing the mutant receptors to study the effects of mutating the key phenylalanine residues of the CBM. Interestingly, wild type FSHR presents as a doublet on a western blot while the mutants only have a single band. We theorize that one of these bands may be a glycosylation variant however further investigation is required. The mutated receptors qualitatively show normal p44‐MAPK signaling in response to FSH, indicating that they are reaching the cell surface and are capable of binding hormone and coupling to signaling partners. We have observed a subtle difference in downstream PKA targets between the mutant and wild type hFSHR, suggesting that there might be a difference in the ability to activate downstream targets. Studying the interaction between hFSHR and caveolin has the potential to develop new contraceptive antagonists and alternative agonists to regulate fertility.