Effects of short- and long-term exposure to c-AMP and c-GMP on the noradrenaline transporter

The effects of short- and long-term exposure of cells to elevated cyclic adenosine monophosphate (c-AMP), using dibutyryl-c-AMP, 8-bromo-c-AMP, cholera toxin or forskolin, or cyclic guanosine monophosphate (c-GMP), using dibutyryl-c-GMP or 8-bromo-c-GMP, on the activity and expression of the noradrenaline transporter (NAT) were examined. Short- or long-term c-GMP elevation had no effects on H-3-noradrenaline uptake by rat PC12 phaeochromocytoma cells or human SK-N-SH-SY5Y neuroblastoma cells. Short-term c-AMP elevation (for 17 min experiment duration) caused a decrease in H-3-noradrenaline uptake by PC12 cells, but had no effects on SK-N-SH-SY5Y cells or COS-7 cells transfected with human or rat NAT cDNA. c-AMP did not affect H-3-nisoxetine binding to PC12 cells. Long-term (24 h) exposure to elevated c-AMP levels caused a decrease in H-3-noradrenaline uptake and NAT mRNA in PC12 cells, but had no effects on SK-N-SH-SY5Y cells and caused a small increase in H-3-noradrenaline uptake in COS-7 cells heterologously expressing rat or human NAT. Hence, c-AMP, but not c-GMP, causes a cell type-dependent reduction in NAT activity after short-term exposure and a reduction in NAT expression after long-term exposure. (C) 2001 Elsevier Science Ltd. All rights reserved.

Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Angstrom resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5-monophosphate (GMP) (2.8 Angstrom resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 Angstrom. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, Usually Mg2+ in vivo. In the free structure, a Mg2+, is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp 163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 Angstrom resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 Angstrom resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 .when the nucleotide product is formed. The 2.6 Angstrom resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by similar to 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex. (C) 1998 Academic Press.

Cancer knowledge and skills of interns in Australia and New Zealand in 2001: comparison with 1990, and between course types

Objective: To compare the cancer knowledge and skills of interns in 2001 who graduated from graduate medical program (GMP) courses with those from non-GMP courses, and to compare the cancer knowledge and skills of interns in 2001 with those who completed a similar survey in 1990. Design: Questionnaire survey of recently graduated interns in a random sample of Australian and New Zealand hospitals. The questionnaire was designed to allow direct comparison with the 1990 survey, and was guided by the Australian Cancer Society's Ideal Oncology Curriculum for Medical Schools. Results: 443 interns completed the survey (response rate, 62%; 42 were excluded, leaving 401 surveys for analysis: 118 from GMP courses and 283 from non-GMP courses). Interns from GMP courses felt more competent than those from non-GMP courses at discussing death (P= 0.02), breaking bad news (P= 0.04) and advising on smoking cessation (P= 0.02), but less competent at preparing a patient for a hazardous procedure (P= 0.02). Mote GMP interns would refer a breast cancer patient to a multidisciplinary clinic (83% versus 70%; P= 0.03). Knowledge about cancer risks and prognosis was significantly less in GMP interns, but GMP interns rated their clinical skills, such as taking a Pap smear, higher than non-GMP interns. The GMP and non-GMP groups did not differ in their exposure to cancer patients, but compared with 1990 interns recent graduates had less exposure to patients with cancer. Conclusions: GMP curricula appear to have successfully introduced new course material and new methods of teaching, but have not always succeeded in producing doctors with better knowledge about cancer. Recent graduates have less exposure to cancer patients than those who trained 10 years ago.

The crystal structure of free human hypoxanthineguanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-U-D-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP. Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg2+.PPi complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9 angstrom resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasina gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T gondii). (c) 2005 Elsevier Ltd. All rights reserved.

Crystal structure of Escherichia coli xanthine phosphoribosyltransferase

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coil is a tetrameric enzyme having 152 residues per subunit. XPRT catalyzes the transfer of the phosphoribosyl group from 5-phospho-alpha-D-ribosyl l-pyrophosphate (PRib-PP) to the 6-oxopurine bases guanine, xanthine, and hypoxanthine to form GMP, XMP, and IMP, respectively. Crystals grown in the absence of substrate or product were used to determine the structure of XPRT at a resolution of 1.8 Angstrom by multiple isomorphous replacement. The core structure of XPRT includes a five-stranded parallel B-sheet surrounded by three or-helices, which is similar to that observed in other known phosphoribosyltransferase (PRTase) structures. The XPRT structure also has several interesting features. A glutamine residue in the purine binding site may be responsible for the altered 6-oxopurine base specificity seen in this enzyme compared to other 6-oxopurine PRTases. Also, we observe both a magnesium ion and a sulfate ion bound at the PRib-PP binding site of XPRT. The sulfate ion interacts with Arg-37 which has a cis-peptide conformation, and the magnesium ion interacts with Asp-89, a highly conserved acidic residue in the PRib-PP binding site motif. The XPRT structure also incorporates a feature which has not been observed in other PRTase structures. The C-terminal 12 residues of XPRT adopt an unusual extended conformation and make interactions with a neighboring subunit. The very last residue, Arg-152, could form part of the active site of a symmetry-related subunit in the XPRT tetramer.

Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa

Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasl::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604,2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket or Vfr. This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-l-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-ajquinoxalin-1-one), and to investigate the possible role of activation of sarco-encloplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 - 10 mug ml(-1)) and adenosine diphosphate (ADP; 2 mum) in platelet rich plasma. It was (i) more effective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mum) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzy] indazole; 1 - 100 mum), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 - 1 mm), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 PM present) were abolished by thapsigargin (200 nm). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Dimethylsulfide dehydrogenase from rhodovulum sulfidophilum: EPR spectroscopy and biochemical analysis reveal its place in the DMSO reductase family of molybdenum enzymes

Dimethylsulfide (DMS) dehydrogenase catalyses the oxidation of DMS to dimethylsulfoxide. The purified enzyme has three subunits of Mr = 94, 38 and 32 kDa and has an optical spectrum dominated by a b-type cytochrome. The metal ion and nucleotide analysis revealed 0.5 g-atom Mo, 9.8 g-atom Fe and 1.96 mol GMP per tool of enzyme. Taken together, these data indicate that DMS dehydrogenase contains a bis(MGD)Mo cofactor. A comparison of the Nterminal amino acid sequence of DMS dehydrogenase revealed that the Mo-containing ct-subunit was most closely related to the c~-subunits of nitrate reductase (NarG) and selenate reductase (SerA). Similarly, the [~-subunit of DMS dehydrogenase was most closely related to the [3-subunits of nitrate reductase (NarH) and selenate reductase (SerB). Variable temperature X-band EPR spectra (120-2K) of 'as isolated' DMS dehydrogenase showed resonances arising from multiple redox centres, Mo(V), [3Fe-4S] +, [4Fe-4S] ÷. A pH dependent EPR study of the Mo(V) centre in lH20 and 2H20 reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). The spin Hamiltonian parameters (2.0158, 1.8870, 1.8620) for the [4Fe-4S] + cluster suggests the presence of histidine (N) coordination to iron in this cluster. It is suggested that this unusual [Fe-S] cluster may be associated with a histidine-cysteine rich sequence at the N-terminus of the ct-subunit of DMS dehydrogenase.

A pore-lining glutamic acid in the olfactory cyclic nucleotide-gated channel controls external spermine block

Spermine is a potent, voltage-dependent blocker of the olfactory cyclic nucleotide-gated channel from both the intracellular and extracellular sides. However, its sites of action are unknown. This study investigated the external spermine binding site in the rat CNC alpha3 subunit. Neutralization of a glutamic acid residue (E342Q) in the P-loop region eliminated voltage-dependence of block by externally applied spermine. The charge-conservative E342D mutation had little effect on spermine block. Thus, E342 forms the binding site for externally applied spermine. However, spermine remained a potent voltage-independent blocker of the E342Q mutant channel, suggesting that the mutation either created a novel binding site outside the membrane electrical field or that it dramatically changed the properties of the existing pore site. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.