FARES F, BAR-SHALOM R, AZZAM N
Department of Molecular Genetics, Carmel Medical Center and the Faculty of Science and Science Education, University of Haifa, Haifa, Israel.
Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, and . The hTSH heterodimer was converted to a biologically active single-peptide chain (hTSHCTP), by fusing the common subunit to the carboxyl terminal end of hTSH subunits in the presence of a ~30 aminoacid peptide from hCG (CTP) as a linker. Ligation of the CTP to the carboxyl-end of hFSH resulted in increasing the biological activity and longivity in vivo. In the present study, the hTSHCTP, was used to investigate the role of the N-linked oligosaccharides of and subunits on secretion and function of hTSH. Two deglycosylated variants were prepared: one lacks both oligosaccharide chains on subunit (hTSHCTP1+2), and the other lacks also the oligosaccharide chain on subunit of the single chain (hTSHCTP(deg)). The single-peptide chain variants were expressed in CHO cells and they are secreted into the medium. Absence of the N-linked oligosaccharides on or subunits and the O-linked oligosaccharides on the CTP, does not affect the secretion of the variants. However, the absence of N-linked oligosaccharide chain on decreased the secretion rate of the single-peptide chain. These results indicate that the signal for the secretion exists in the single peptide chain and is independent of the oligosaccharides. hTSH variants lack of the oligosaccharide chains is less potent than hTSHCTP on cAMP accumulation and T3 secretion in human cultured thyroid follicles. Both deglycosylated variants compete with normal hTSH and hTSI in a dose dependent manner. Maximal concentration of hTSHCTP1+2 (200 U/ml) decreased significantly the hTSH and hTSI -stimulated levels of cAMP and T3 secretion. Moreover, the variants significantly inhibited (50%) TSH activity in vivo, with respect to thyroid hormone secretion in mice. Thus, this variant, behaves as potential antagonist, who may offer a novel therapeutic strategy in the treatment of Grave’s disease, the most common form of hyperthyroidism.
Designing a new agonist of Erythropoietin by Fusing the Carboxyl-Terminal Peptide of Human Chorionic Gonadotropin Subunit to the Coding Sequence of Human Erythropoietin FARES F1,2,, GANEM S2, HAJOUJ T2, AGAI E2 1Department of Molecular genetics, Carmel Medical Center and the Faculty of Science, University of Haifa, Haifa, 31905, and 2ModigeneTech, Weizmann Science Park, Nes-Ziona, 74140, Israel
Human erythropoietin (EPO) is a glycoprotein hormone secreted from the kidney and controls red blood cell production. EPO has a wide clinical use in the treatment of anemia associated with renal disease, certain chronic diseases and anemia related to chemotherapy and radiotherapy. One major issue regarding the clinical use of EPO is its relatively short half-life due to its clearance by glomerular filtration. Thus the therapeutic protocol used in the treatment of patient required frequent injections of EPO. Previous studies indicated that fusing the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin subunit (hCG) to human follitropin (FSH), hCG subunit or to thyrotrpin (TSH), did not affect assembly, secretion, and receptor binding affinity or in vitro bioactivity. However, the in vivo potency and circulatory half-life of the proteins containing CTP were substantially increased. Other report indicated that ligation of CTP to FSH is not immunogenic and this construct is already in clinical trials phase 3.
To address the issue of EPO half-life, we constructed a chimeric gene that contains the sequence of the CTP of human chorionic gonadotropin (hCG) subunit bearing four O-linked oligosaccharide recognition sites and the coding sequence of human EPO cDNA. Fusing the CTP to the carboxyl-terminal of EPO did not affect secretion, receptor binding affinity or in vitro bioactivity. However, both in vivo potency and half-life of EPO-CTP were significantly enhanced. A single injection dose (660 IU/kg) of EPO Wild-type (EPO-WT) administered once a week had no significant effect on haematocrit levels. However, EPO-CTP administered as a 660 IU/kg once a week was effective as well as the same total dose of EPO-WT administered as 220 IU/kg 3 times a week. This may emphasize the importance of sustained blood levels rather than total dose of administration for in vivo bioactivity. These data established the rational for using this chimera as a long-acting EPO analog. The therapeutic efficacy of EPO-CTP analog needs to be established in higher animals and in human clinical trials.
Neuromuscular blocking drugs and magnesium interactions FAWCETT WJ Royal Surrey County Hospital, Guildford GU2 7XX, UK
Background: Parenteral magnesium has become an increasingly popular therapeutic agent over the last fifteen years, with established uses in obstetrics, cardiology (especially management of various arrhythmias), anaesthesia and critical care. It has a number of mechanisms of action, including antagonism of the glutamate N-methyl D-aspartate (NMDA) receptors and calcium antagonism. The latter is involved in inhibition of presynaptic release of acetylcholine (ACh). It is this mechanism that explains its action as a neuromuscular blocking agent and hence its potentiation of neuromuscular blocking (NMB) drugs. Whilst NMBs are a cornerstone of modern balanced anaesthetic practice, one of their principal side effects is persistence into the post operative period – postoperative residual curarisation (PORC). Potentiation of the effects of NMBs from any cause will make PORC more likely.
Methods: The treatment of an elective surgical patient undergoing laparoscopic cholecystectomy who developed rapid atrial fibrillation at the end of the procedure. In order to control the ventricular response rate, iv magnesium was administered.
Results: The patient had recently received NMB drugs and the co-administration of magnesium whilst NMB drugs where still present within the biophase of the neuromuscular junction lead to PORC – with the patient becoming re-paralysed. The patient required sedation and artifical ventilation of the lungs until spontaneous recovery occurred.
Conclusions: This is the first reported recurarisation following the administation of magnesium. Care should be exercised in the use of magnesium if NMBs have recently been administered even if the clinically normal neuromuscular function has returned. The consequences, in addition to severe distress to the patient, include paralysis and loss of airway protection.
Background: Adenosine (Ado) plays an important role in regulating renal vascular tonevia disparate actions of A1 and A2 receptors. While A2b receptor (A2bR) is expressed in preglomerular vessels, there is less functional evidence regarding the role of A2bR in mediating the vasodilator action of afferent arterioles (AA).
Objective: To determine the role of A2bR in buffering the AA constriction caused by Ado by comparing the effects of A2b and A2a receptor blockade on AA.
Methods: We used the isolated blood-perfused juxtamedullary nephron technique combined with videomicroscopy. A single AA from a rat was visualized and superfused with Ado or Ado agonist, or A2b or A2a receptor blockers (1 rat per experiment).
Results: Ado at 10µmol/L constricted AA (-9.6±2.4%, n=9, p<0.05). In the presence of Ado, SCH, an A2aR blocker, at concentrations of 1, 10, 100, 1000 and 10000 nmol/L elicited only slight decreases in AA diameter from 16.1±0.5 to 15.4±0.5, 15.1±0.4, 14.2±0.3 and 14.6±0.4µm, with maximum effect at a concentrations of 1000 nmol/L (-11.3±3.6%, n=5, p<0.05). Superfusion of Ado treated vessels with MAS, an A2bR blocker, at concentrations of 1, 10,100 and 1000 nmol/L caused greater decreases in AA diameter from 15.7±0.5 to 14.8±0.6, 12.9±0.5 and 12.3±0.4µm (-26.0±4.7%, n=6, p<0.01). Adding SCH 1µmol/L did not significantly augment the Ado mediated afferent constriction elicited by MAS 1µmol/L; however, adding MAS 1μmol/L after SCH 10μmol/L caused further vasoconstriction with AA diameter decreasing by 16.8±2.9 %( from 14.6±0.4 to 12.2±0.3µm, n=5, p<0.01). In response to CV101, an Ado agonist, at concentrations of 0.002, 0.02, 0.2, 2μmol/L, AA diameter increased from 17.2±0.4 to 17.1±0.4, 17.7±0.5, 18.5±0.5 and 20.1±0.6 µm (16.8±2.3%, n=5, P<0.01). In the present of CV101 (2 μmol/L), first superfusion with SCH, an A2a receptor blocker, at concentrations of 1μmol/L, AA diameter decreased slightly from 20.1±0.6 to 18.6±0.6µm (-6.1±0.8%, P<0.05). However, superfusion with MAS after SCH, at concentrations of 1μmol/L, AA diameter decreased markedly to 15.1±1.2µm (-24.2±2.2%, n=5, p<0.01). In addition, in the present of CV101 (2μmol/L), first superfusion with MAS, at concentrations of 1μmol/L, AA diameter decreased signficantly from 20.2±0.6 to 15.4±0.7µm (-24.0±2.6%, n=5, P<0.01). However, superfusion with SCH after MAS, at concentrations of 1μmol/L, AA diameter decreased slightly to 14.6±0.9µm (n=5, p>0.05 via MAS group).
Conclusions: Thus, while both A2b and A2a receptors are functionally expressed in juxtamedullary afferent arterioles, the vasodilator effect of adenosine is predominantly via activation A2b receptors.
