Alpha2-adrenoceptors: Structure and ligand binding properties at the molecular level

Alpha₂-Adrenoceptors: structure and ligand binding properties at the molecular level The mouse is the most frequently used animal model in biomedical research, but the use of zebrafish as a model organism to mimic human diseases is on the increase. Therefore it is considered important to understand their pharmacological differences from humans also at the molecular level. The zebrafish Alpha_2-adrenoceptors were expressed in mammalian cells and the binding affinities of 20 diverse ligands were determined and compared to the corresponding human receptors. The pharmacological properties of the human and zebrafish Alpha_2--adrenoceptors were found to be quite well conserved. Receptor models based on the crystal structures of bovine rhodopsin and the human Beta₂-adrenoceptor revealed that most structural differences between the paralogous and orthologous Alpha_2--adrenoceptors were located within the second extracellular loop (XL2). Reciprocal mutations were generated in the mouse and human Alpha_2--adrenoceptors. Ligand binding experiments revealed that substitutions in XL2 reversed the binding profiles of the human and mouse Alpha_2--adrenoceptors for yohimbine, rauwolscine and RS-79948-197, evidence for a role for XL2 in the determination of species-specific ligand binding. Previous mutagenesis studies had not been able to explain the subtype preference of several large Alpha_2--adrenoceptor antagonists. We prepared chimaeric Alpha_2--adrenoceptors where the first transmembrane (TM1) domain was exchanged between the three human Alpha_2--adrenoceptor subtypes. The binding affinities of spiperone, spiroxatrine and chlorpromazine were observed to be significantly improved by TM1 substitutions of the Alpha_2a--adrenoceptor. Docking simulations indicated that indirect effects, such as allosteric modulation, are more likely to be involved in this phenomenon rather than specific side-chain interactions between ligands and receptors.

Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.

Muscarinic toxins : characterization of adrenoceptor binding and applicability of membrane anchoring via glycosylphosphatidylinositol tail

Adrenoceptors (ARs), G-protein coupled receptors (GPCRs) at the plasma membrane, respond to endogenous catecholamines noradrenaline and adrenaline. These receptors mediate several important physiological functions being especially important in the cardiovascular system and in the regulation of smooth muscle contraction. Impairments in the function of these receptors can thus lead to severe diseases and disorders such as to cardiovascular diseases and benign prostatic hyperplasia. The Eastern green mamba (Dendroaspis angusticeps) venom has been shown to contain toxins that can antagonize the functions of GPCRs. The most well-known are muscarinic toxins (MTs) targeting muscarinic acetylcholine receptors (mAChRs) with high affinity and selectivity. However, some reports have indicated that these toxins might also act on the α1- and α2-ARs which can be divided into various subtypes; the α1-ARs to α1A-, α1B- and α1D-ARs and α2-ARs to α2A-, α2B- and α2C-ARs. In this thesis, the interaction of four common MTs (MT1, MT3, MT7 and MTα) with the adrenoceptors was characterized. It was also evaluated whether these toxins could be anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) tail. Results of this thesis reveal that muscarinic toxins are targeting several α-adrenoceptor subtypes in addition to their previously identified target receptors, mAChRs. MTα was found to interact with high affinity and selectivity with the α2B-AR whereas MT7 confirmed its selectivity for the M1 mAChR. Unlike MTα and MT7, MT1 and MT3 have a broad range of target receptors among the α-ARs. All the MTs characterized were found to behave as non-competitive antagonists of receptor action. The interaction between MTα and the α2B-AR was studied more closely and it was observed that the second extracellular loop of the receptor functions as a structural entity enabling toxin binding. The binding of MTα to the α2B-AR appears to be rather complex and probably involves dimerized receptor. Anchoring MTs to the plasma membrane did not interfere with their pharmacological profile; all the GPI-anchored toxins created retained their ability to block their target receptors. This thesis shows that muscarinic toxins are able to target several subtypes of α-ARs and mAChRs. These toxins offer thus a possibility to create new subtype specific ligands for the α-AR subtypes. Membrane anchored MTs on the other hand could be used to block α-AR and mAChR actions in disease conditions such as in hypertension and in gastrointestinal and urinary bladder disorders in a cell-specific manner and to study the physiological functions of ARs and mAChRs in vivo in model organisms.

Effect of sigma factor S (sigmaS) on the stability of penicillin-binding protein 3 (PBP3) of Escherichia colt K12

The stability of penicillin-binding protein 3 (PBP3), a cell septum synthesizing protein, was analyzed at different incubation temperatures in three Escherichia coli K12 strains carrying a PBP3-overproducing plasmid. The stability of PBP3 was significantly reduced in stationary phase cells shifted to 42°C for 4 h, compared to samples incubated at 28 or 37°C. The half-life of PBP3 in the C600 strain was 60 min at 42°C, while samples incubated at 28 or 37°C had PBP3 half-lives greater than 4 h. Analysis of the PBP3 content in mutants deficient in rpoS (coding for the stationary phase sigma factor, sigmaS) and rpoH (coding for the heat shock sigma factor, sigma32) genes after shift to 42°C showed that stability of the protein was controlled by sigmaS but not by sigma32. These results suggest that control of the PBP3 levels in E. coli K12 is through a post-transcriptional mechanism regulated by the stationary phase regulon. We demonstrated that stability of PBP3 in E. coli K12 involves degradation of the protein. Moreover, we observed that incubation of cells at 42°C significantly reduces the stability of PBP3 in early stationary phase cells in a process controlled by sigmaS.

Diclofenac plasma protein binding: PK-PD modelling in cardiac patients submitted to cardiopulmonary bypass

Twenty-four surgical patients of both sexes without cardiac, hepatic, renal or endocrine dysfunctions were divided into two groups: 10 cardiac surgical patients submitted to myocardial revascularization and cardiopulmonary bypass (CPB), 3 females and 7 males aged 65 ± 11 years, 74 ± 16 kg body weight, 166 ± 9 cm height and 1.80 ± 0.21 m2 body surface area (BSA), and control, 14 surgical patients not submitted to CPB, 11 female and 3 males aged 41 ± 14 years, 66 ± 14 kg body weight, 159 ± 9 cm height and 1.65 ± 0.16 m2 BSA (mean ± SD). Sodium diclofenac (1 mg/kg, im Voltaren 75® twice a day) was administered to patients in the Recovery Unit 48 h after surgery. Venous blood samples were collected during a period of 0-12 h and analgesia was measured by the visual analogue scale (VAS) during the same period. Plasma diclofenac levels were measured by high performance liquid chromatography. A two-compartment open model was applied to obtain the plasma decay curve and to estimate kinetic parameters. Plasma diclofenac protein binding decreased whereas free plasma diclofenac levels were increased five-fold in CPB patients. Data obtained for analgesia reported as the maximum effect (EMAX) were: 25% VAS (CPB) vs 10% VAS (control), P<0.05, median measured by the visual analogue scale where 100% is equivalent to the highest level of pain. To correlate the effect versus plasma diclofenac levels, the EMAX sigmoid model was applied. A prolongation of the mean residence time for maximum effect (MRTEMAX) was observed without any change in lag-time in CPB in spite of the reduced analgesia reported for these patients, during the time-dose interval. In conclusion, the extent of plasma diclofenac protein binding was influenced by CPB with clinically relevant kinetic-dynamic consequences

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

Distribution of calcium-binding proteins in the chick visual system

The calcium-binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV) have been extensively studied over the last decade since they appear to be important as buffers of intracellular calcium. In the present study we investigated the distribution of these proteins in the chick visual system by means of conventional immunocytochemistry. The results indicated that CB, CR, and PV are widely distributed in retinorecipient areas of the chick brain. In some regions, all three calcium-binding proteins were present at different intensities and often in different neurons such as in the dorsolateral thalamic complex. In other areas, such as the nucleus geniculatus lateralis ventralis, only CB and CR were detected, whereas PV was absent. These results show that these three calcium-binding proteins are differentially distributed in the visual system of the chick, with varying degrees of co-localization

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Effect of inhibitory avoidance training on [3H]-glutamate binding in the hippocampus and parietal cortex of rats

Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29% increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P<0.01), but not at 0, 15, or 60 min after training, and did not affect glutamate binding in membranes obtained from the parietal cortex. These results are consistent with previous evidence for the involvement of glutamatergic synaptic modification in the hippocampus in the early steps of memory formation.

Selective destruction of nigrostriatal dopaminergic neurons does not alter [3H]-ryanodine binding in rat striatum

Dopamine nigrostriatal neurons are important for motor control and may contain a particularly dense population of ryanodine receptors involved in the control of dopamine release. To test this hypothesis, we used a classical model of unilateral selective lesion of these neurons in rats based on 6-hydroxydopamine (6-OHDA) injection into the substantia nigra. Binding of [3H]-GBR 12935, used as a presynaptic marker since it labels specifically the dopamine uptake complex, was dramatically decreased by 83-100% in striatum homogenates after 6-OHDA lesion. On the contrary, no reduction of [3H]-ryanodine binding was observed. The present data indicate that [3H]-ryanodine binding sites present in rat striatum are not preferentially localized in dopaminergic terminals.

Long-term outcome of 25 children and adolescents with severe aplastic anemia treated with antithymocyte globulin

Severe aplastic anemia (SAA) is probably an immune-mediated disorder, and immunosuppressive therapy is recommended for patients with no available donor for bone marrow transplant. Between October 1984 and November 1987, 25 consecutive children and adolescents with SAA with no HLA-compatible marrow donor received equine antithymocyte globulin (ATG) (15 mg kg-1 day-1) for 10 days. The patients were evaluated 6 weeks, 6 months, and 12 months after starting ATG treatment. Thereafter, patients were evaluated yearly until July 1998. Median age was 10 years (range, 1.5-20 years), granulocyte counts on referral ranged from 0.032 to 1.4 x 10(9)/l (median 0.256 x 10(9)/l), and 12 patients had granulocyte counts <0.2 x 10(9)/l. At a median follow-up of 9.6 years (range, 8.6-11.8 years), 10 patients (40%) remained alive with good marrow function. No morphologic evidence of hematological clonal disorders has been observed, although two patients probably have acquired clonal chromosomal abnormalities (trisomy 8 and del(6)q21, respectively). Responses to ATG were observed between 6 weeks and 6 months from the start of treatment in 60% of evaluable patients. The response rate was not different in patients whose granulocyte count at diagnosis was <0.2 x 10(9)/l, or in those who were <10 years of age. This study supports the view that, when compared with supportive measures, ATG is an effective treatment for children or adolescents with SAA. Although these results are inferior to those reported for marrow transplantation or more intensive immunosuppressive regimens, these patients who responded to ATG are long-term survivors with stable peripheral blood counts and a low rate of relapse.