Hydrodynamic analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. Some detergents (e.g. octylglucoside) maintain the multimeric complex while other detergents (e.g. CHAPS and dodecylmaltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer. Quantitatively deglycosylated GLUT1 displays aberrant electrophoretic mobility, but each protein band contains full-length GLUT1 and the less mobile species, when treated with additional detergent and reductant, converts to the more mobile species. Preliminary structural analysis suggests that denaturing detergent- and thiol chemistry-related changes of α-helical content may mirror mobility shifts. Limited proteolysis of membrane-resident GLUT1 (± ligands) releases membrane-spanning α-helical domains suggesting that (i) some bilayer-resident helices are highly solvent exposed; (ii) membrane-spanning domains 1, 2, & 4 and 7, 8, & 10 are destabilized upon ligand binding; and (iii) helix packing compares well with high-resolution structures of prokaryotic transporters from the same superfamily. Results are consistent with a central, hydrophilic, translocation pathway comprised of amphipathic, membrane-spanning domains that alter associations upon ligand/substrate binding. We have resolved technical difficulties (heterogeneity, lipid/detergent removal, glycosylation, small molecule contamination) associated with GLUT1 analysis by mass spectrometry; and we map global conformational changes between sugar uptake and sugar efflux.

Properties of the nucleotide binding site of the Ca²⁺-ATPase of skeletal muscle sarcoplasmic reticulum have been investigated. The study centred around interaction of the high affinity ATP analog, 2'-3'-0-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, (TNP-ATP), with the Ca²⁺-ATPase. Defined fractions of the sarcoplasmic reticulum (SR), corresponding to the terminal cisternae (TC) and light SR (LSR), were isolated. The TC were shown to have distinctive morphological characteristics that differ from the LSR. The TC vesicles contained electron dense intravesicular material representative of Ca²⁺ binding proteins, and visible membranous "feet" structures, which are reported to interconnect with the transverse tubule. Functional characterisation of the isolated fractions provided evidence for the predominant localisation of Ca²⁺ release channels in TC, and concentration of Ca²⁺-ATPase molecules in LSR. These conclusions were based on the following observations: (a) decreased Ca²⁺ transport of TC versus LSR; ruthenium red, a Ca²⁺ channel blocker, enhanced Ca²⁺ transport and pumping efficiency in TC, (b) higher Ca²⁺-ATPase activity for LSR in the presence and absence of ionophore, (c) rapid Ca²⁺ efflux from TC which is inhibited by ruthenium red. Of special interest was the characterisation of the TC and LSR with respect to turnover-dependent TNP-ATP fluorescence. Fluorescence observed for TC was approximately 65% of that for LSR. This phenomenon may be attributable to either the decreased Ca²⁺ ATPase content of the TC vesicles or open Ca²⁺ release channels. Hence the TNP-ATP fluorescence characteristics appear to reflect the morphological and functional subspecialisation of the defined SR fractions.

No description available.

Yes / Aims/hypothesis Hypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG). Methods To test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios. Results AMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure. Conclusions/interpretation Taken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes. / Diabetes UK (RD Lawrence Fellowship to CB; 13/0004647); the Medical Research Council (MR/N012763/1) to KLJE, ADR and CB; and a Mary Kinross Charitable Trust PhD studentship to CB, ADR and RW to support PGWP. Additional support for this work came from awards from the British Society for Neuroendocrinology (to CB and KLJE), the Society for Endocrinology (CB), Tenovus Scotland (CB) and the University of Exeter Medical School (CB and KLJE). AR was also supported by a Royal Society Industry Fellowship.

This thesis presents a novel hybrid biological solid-state system which makes use of biological components in an in-vitro environment to produce functionality incapable by CMOS circuits alone. A "biocell" comprised of lipids and ion pumps is mated to a CMOS IC in a compact configuration and the IC is powered solely from adenosine triphosphate (ATP), often referred to as the 'life energy currency.' The biocell is a fuel cell that produces a membrane potential in the presence of ATP which is used by the IC as an electrical power supply. The design represents the first of a new class of devices combining both biological and solid-state components, which exploit the unique properties of transmembrane proteins in engineered solid-state systems. This work also suggests that the richness of function of biological ion channels and pumps, functionality that is impossible to achieve in CMOS alone, may be exploited in systems that combine engineered transmembrane proteins as biological components integrated with solid-state devices.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-30Bitstream added on 2014-06-13T20:30:21Z : No. of bitstreams: 1 menezes_mf_me_arafo.pdf: 831526 bytes, checksum: 44cecb40bcf8a2490778eb7d99c815ee (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudos recentes demonstram que os receptores purinérgicos estão presentes no núcleo parabraquial lateral (NPBL), uma estrutura pontina envolvida no controle da ingestão de sódio. No presente estudo, investigamos os efeitos das injeções do , -methyleneadenosine 5 -triphosphate ( , -metileno ATP, agonista dos receptores P2X) sozinho ou combinado com o ácido piridoxalfosfato-6-azofenil-2',4'-disulfônico (PPADS, antagonista dos receptores P2X) ou suramin (antagonista não seletivo dos receptores P2) no NPBL sobre a ingestão de NaCl 1,8% induzida por depleção de sódio. Também investigamos os efeitos da injeção de , -metileno ATP sozinho ou combinado com o PPADS no NPBL sobre a pressão arterial média (PAM) e freqüência cardíaca (FC) em ratos saciados e depletados de sódio. Foram utilizados ratos Holtzman com implante de cânulas implantadas bilateralmente em direção ao NPBL. A depleção de sódio foi induzida pelo tratamento com o diurético furosemida (20 mg/kg do peso corporal) acompanhado de uma dieta deficiente em sódio por 24 horas. As injeções bilaterais de , -metileno ATP (2,0 e 4,0 nmol/0,2 μL) no NPBL aumentaram a ingestão de NaCl 1,8% induzida por depleção de sódio (25,3 ± 0,8 e 26,5 ± 0,9 mL/2 h, respectivamente, vs. salina: 15,2 ± 1,3 mL/2 h). O pré-tratamento com o suramin (2,0 nmol/0,2 μL) ou com o PPADS (4,0 nmol/0,2 μL) no NPBL aboliu os efeitos do , -metileno-ATP na ingestão de NaCl 1,8% (15,2 ± 1,2 e 16,9 ± 0,9 mL/2 h, respectivamente). As injeções de PPADS sozinho no NPBL não alteraram a ingestão de NaCl 1,8% (14,6 ± 0,8 mL/2 h vs. salina: 18,3 ± 1,8 mL/2 h). No entanto, as injeções de suramin sozinho no NPBL quase aboliram a ingestão de NaCl 1,8% (5,7 ± 1,9 mL/120 min, vs. salina: 15,5 ± 1,1 mL/120 min) e aumentaram a ingestão de sacarose 2% somente no tempo de 90 minutos (7,1 ± 1,3 vs. salina: 5,3 ± 0,8 mL/90 min) sem alterar... / Recent studies have shown that purinergic receptors are present in the lateral parabrachial nucleus (LPBN), a pontine structure involved in the control of sodium intake. In the present study, we investigated the effects of , -methyleneadenosine 5 -triphosphate ( , - methylene ATP, selective P2X purinergic agonist) alone or combined with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X purinergic antagonist) or suramin (non-selective P2 purinergic antagonist) injected into the LPBN on sodium depletion-induced by 1.8% NaCl intake. We also investigated the effects of , -methylene ATP alone or combined with PPADS injected into the LPBN on mean arterial pressure (MAP) and heart rate (HR) on replete and sodium depleted rats. Male Holtzman rats with stainless steel cannulas implanted into the LPBN were used. Sodium depletion was induced by treating rats with the diuretic furosemide (20 mg/kg of body weight) followed by 24 h of sodium-deficient diet. Bilateral injections of , -methylene ATP (2.0 and 4.0 nmol/0.2 μL) into the LPBN increased sodium depletion-induced 1.8% NaCl intake (25.3 ± 0.8 and 26.5 ± 0.9 mL/2 h, respectively, vs. saline: 15.2 ± 1.3 mL/2 h). Pre-treatment with suramin (2.0 nmol/0.2 μL) or PPADS (4 nmol/0.2 μL) into the LPBN abolished the effects of , - methylene-ATP on 1.8% NaCl intake (15.2 ± 1.2 and 16.9 ± 0.9 mL/2 h, respectively). Injections of PPADS alone into the LPBN did not change 1.8% NaCl intake (14.6 ± 0.8 ml/2 h vs. saline: 18.3 ± 1.8 mL/2 h). However, injections of suramin alone into the LPBN strongly reduced 1.8% NaCl intake (5.7 ± 1.9 mL/120 min, vs. saline: 15.5 ± 1.1 mL/2 h) and increased the 2% sucrose intake only at 90 min (7.1 ± 1.3 vs. saline: 5.3 ± 0.8 mL/90 min), without changing 24h water deprivation-induced water intake (16.7 ± 1.8 mL/2 h vs. saline: 15.0 ± 2.1 mL/2 h)... (Complete abstract click electronic access below)

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

Human erythrocyte glucose sugar transport displays a complexity that is not explained by available models. Sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions (intracellular [sugar] = extracellular [sugar]). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Two models for biphasic sugar transport are presented in which 3MG must overcome a sugar-specific, physical (diffusional) or chemical (anomerization) barrier to equilibrate with cell water. The anomerization model was rejected through several lines of direct experimental investigation. 1) The sizes of the fast and slow phases of sugar transport do not correlate with the equilibrium anomer distributions of all GLUT1 sugar substrates. 2) Increasing the rate of anomerization by addition of exogenous intracellular mutarotase has no effect on biphasic transport kinetics. 3) Direct measurement of initial rates of sugar uptake or exchange demonstrates that GLUT1 shows no anomer preference. The physical barrier model was further refined by the use of the counterflow condition (intracellular [sugar] >> extracellular [sugar]). The presence of a physical barrier alone was unable to explain the complex counterflow time courses observed. As a result, the model was modified to include the action of a specific sugar export that is compartmentalized from rapidly equilibrating, GLUT1-mediated uptake and exit.

The inherently repressive nature of chromatin presents a sizeable barrier for all nuclear processes in which access to DNA is required. Therefore, eukaryotic organisms ranging from yeast to humans rely on a battery of enzymes that disrupt the chromatin structure as a means of regulating DNA transactions. These enzymes can be divided into two broad classes: those that covalently modify histone proteins, and those that actively disrupt nucleosomal structure using the free energy derived from ATP hydrolysis. The latter group, huge, multisubunit ATP-dependent chromatin remodeling factors, are emerging as a common theme in all nuclear processes in which access to DNA is essential. Although transcription is the process for which a requirement for chromatin remodeling is best documented, it is now becoming clear that other processes like replication, recombination and DNA repair rely on it as well. A growing number of ATP-dependent remodeling machines has been uncovered in the last 10 years. Although they differ in their subunit composition, organism or tissue restriction, substrate specificity, and regulating/recruiting partners, it has become increasingly evident that all ATP-dependent chromatin remodeling factors share a similar underlying mechanism. This mechanism is the subject of the studies presented in this thesis. Chromatin-remodeling factors seem to bind both the histone and DNA components of nucleosomes. From a fixed position on nucleosomes, the remodeling factors appear to translocate on the DNA, generating torsional stress on the double helix. This activity has several consequences, including the distortion of the DNA structure on the surface of the histone octamer, the disruption of histone-DNA interactions, and the mobilization of the nucleosome core with respect to the DNA. The work presented in this thesis, along with data reported by other groups, supports the hypothesis that yeast SWI/SNF chromatin remodeling complex and the recombinational repair factor, Rad54p, both employ similar mechanisms to regulate gene transcription, and facilitate homologous DNA pairing and recombination, respectively.

In this work, we were interested in studying the effect of two main metabolic factors, hypoxia and extracellular ATP metabolism, on the effector function of T helper subsets. The major oxygen sensor is HIF-1α which is continuously degraded in the presence of oxygen but is stabilized in hypoxia, leading to transcription of genes involved in cellular adaptation to low oxygen level. Our data show that the proportion of IFN-& / Doctorat en Sciences / info:eu-repo/semantics/nonPublished