The heterotrimer of the membrane-peripheral components of transhydrogenase and the alternating-site mechanism of proton translocation.

Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence for the formation of a dI(2)dIII(2) tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI(2)dIII(1) heterotrimer were investigated. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI(2)dIII(1) complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.

Subchronic cocaine produces training paradigm-dependent learning deficits in laboratory rats.

The effect of cocaine on spatial learning was investigated by exposing male Sprague-Dawley rats to 0, 20, or 40 mg/kg cocaine prior to and during training on a water maze task. Half the animals were pretrained on cued trials prior to hidden platform trials, while the remaining animals completed hidden platform trials immediately. Escape latencies for all animals improved with training, but pretrained animals located the hidden platform faster than untrained animals (P<.001). Pretraining also decreased the effect of cocaine. In pretrained animals, only the high dose of cocaine caused significant increases in escape latency (P<.001), while in the untrained group the lower dose of cocaine also caused a significant increase (P<.001). On working memory measures, cocaine affected both the pretrained (P<.01) and untrained (P<.001) groups. Dwell ratio measurements indicated unaffected reference memory in both pretrained (P<.001) and untrained (P<.001) animals, and no significant differences were detected among the treatment conditions in either group (P>.05). Thus, while cocaine did not abolish learning, the efficiency with which the task was learned was compromised. However, this effect was reduced by pretraining.

A change in ionization of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase regulates both hydride transfer and nucleotide release.

Transhydrogenase couples the transfer of hydride-ion equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The enzyme has three components: dI binds NAD(H), dIII binds NADP(H) and dII spans the membrane. Coupling between transhydrogenation and proton translocation involves changes in the binding of NADP(H). Mixtures of isolated dI and dIII from Rhodospirillum rubrum transhydrogenase catalyse a rapid, single-turnover burst of hydride transfer between bound nucleotides; subsequent turnover is limited by NADP(H) release. Stopped-flow experiments showed that the rate of the hydride transfer step is decreased at low pH. Single Trp residues were introduced into dIII by site-directed mutagenesis. Two mutants with similar catalytic properties to those of the wild-type protein were selected for a study of nucleotide release. The way in which Trp fluorescence was affected by nucleotide occupancy of dIII was different in the two mutants, and hence two different procedures for determining the rate of nucleotide release were developed. The apparent first-order rate constants for NADP(+) release and NADPH release from isolated dIII increased dramatically at low pH. It is concluded that a single ionisable group in dIII controls both the rate of hydride transfer and the rate of nucleotide release. The properties of the protonated and unprotonated forms of dIII are consistent with those expected of intermediates in the NADP(H)-binding-change mechanism. The ionisable group might be a component of the proton-translocation pathway in the complete enzyme.

Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression.

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.

Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum.

Transhydrogenase is a proton pump found in the membranes of bacteria and animal mitochondria. The solution structure of the expressed, 21.5 kDa, NADP(H)-binding component (dIII) of transhydrogenase from Rhodospirillum rubrum has been solved by NMR methods. This is the first description of the structure of dIII from a bacterial source. The protein adopts a Rossmann fold: an open, twisted, parallel beta-sheet, flanked by helices. However, the binding of NADP(+) to dIII is profoundly different to that seen in other Rossmann structures, in that its orientation is reversed: the adenosine moiety interacts with the first betaalphabetaalphabeta motif, and the nicotinamide with the second. Features in the structure that might be responsible for changes in nucleotide-binding affinity during catalysis, and for interaction with other components of the enzyme, are identified. The results are compared with the recently determined, high-resolution crystal structures of human and bovine dIII which also show the reversed nucleotide orientation.

A quality-improvement approach to meeting the needs of critically ill patients and their families.

Family support can have positive effects on critically ill patients, so limiting visitation in critical care units may not be in patients' best interests. This study looked at increasing visitation using a quality-improvement model.

Stopped-flow reaction kinetics of recombinant components of proton-translocating transhydrogenase with physiological nucleotides.

New information on the high resolution structure of the membrane proton pump, transhydrogenase, now provides a framework for understanding kinetic descriptions of the enzyme. Here, we have studied redox reactions catalyzed by mixtures of the recombinant NAD(H)-binding component (dI) of Rhodospirillum rubrum transhydrogenase, and the recombinant NADP(H)-binding component (dIII) of either the R. rubrum enzyme or the human enzyme. By recording changes in the fluorescence emission of native and engineered Trp residues, the rates of the redox reaction with physiological nucleotides have been measured under stopped-flow conditions, for the first time. Rate constants for the binding reaction between NAD(+)/NADH and the R. rubrum dI.dIII complex are much greater than those between nucleotide and isolated dI. For the redox step between the physiological nucleotides on the R. rubrum dI. dIII complex, the rate constant in the forward direction, k(f) approximately 2900 s(-1), and that for the reverse reaction, k(r) approximately 110 s(-1). Comparisons with reactions involving an analogue of NAD(H) indicate that the rate constants at this step are strongly affected by the redox driving force.

Screening for literacy and readability: implications for the advanced practice nurse.

The National Adult Literacy Survey reported that 44 million adult Americans could not read or write well enough to meet the needs of everyday living and working. Low literacy is more prevalent than the average nurse is aware, and often the written materials given to patients are above their reading level and therefore are misunderstood. This article defines literacy and describes literacy screening tools and readability formulas. Recommendations for including assessment of literacy and readability as essential activities of the advanced practice nurse (APN) are presented. The APN can be instrumental in providing leadership and directing education, thus ameliorating the effects of low literacy.

Families first.

Family support can have positive effects on critically ill patients, so limiting visitation in critical care units may not be in their best interest. This study examined the outcomes of increasing visitation using a quality-improvement model.

The mobile loop region of the NAD(H) binding component (dI) of proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum: complete NMR assignment and effects of bound nucleotides.

The dI component of transhydrogenase binds NAD+ and NADH. A mobile loop region of dI plays an important role in the nucleotide binding process, and mutations in this region result in impaired hydride transfer in the complete enzyme. We have previously employed one-dimensional 1H-NMR spectroscopy to study wild-type and mutant dI proteins of Rhodospirillum rubrum and the effects of nucleotide binding. Here, we utilise two- and three-dimensional NMR experiments to assign the signals from virtually all of the backbone and side-chain protons of the loop residues. The mobile loop region encompasses 17 residues: Asp223-Met239. The assignments also provide a much strengthened basis for interpreting the structural changes occurring upon nucleotide binding, when the loop closes down onto the surface of the protein and loses mobility. The role of the mobile loop region in catalysis is discussed with particular reference to a newly-developed model of the dI protein, based on its homology with alanine dehydrogenase.

Structural changes in the recombinant, NADP(H)-binding component of proton translocating transhydrogenase revealed by NMR spectroscopy.

We have analysed 1H, 15N-HSQC spectra of the recombinant, NADP(H)-binding component of transhydrogenase in the context of the emerging three dimensional structure of the protein. Chemical shift perturbations of amino acid residues following replacement of NADP+ with NADPH were observed in both the adenosine and nicotinamide parts of the dinucleotide binding site and in a region which straddles the protein. These observations reflect the structural changes resulting from hydride transfer. The interactions between the recombinant, NADP(H)-binding component and its partner, NAD(H)-binding protein, are complicated. Helix B of the recombinant, NADP(H)-binding component may play an important role in the binding process.

Stopped-flow kinetics of hydride transfer between nucleotides by recombinant domains of proton-translocating transhydrogenase.

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD+/NADH, and domain III for NADP+/NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP+ (k approximately 0.03 s(-1)). Phase A of the burst (k approximately 600 s(-1)) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k approximately 10-50 s(-1)), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcPdAD+ reduction by [4B-2H]NADPH, and by [4B-1H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27535-27538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I:III complex.

Mutation of amino acid residues in the mobile loop region of the NAD(H)-binding domain of proton-translocating transhydrogenase.

The effects of single amino acid substitutions in the mobile loop region of the recombinant NAD(H)-binding domain (dI) of transhydrogenase have been examined. The mutations lead to clear assignments of well-defined resonances in one-dimensional 1H-NMR spectra. As with the wild-type protein, addition of NADH, or higher concentrations of NAD+, led to broadening and some shifting of the well-defined resonances. With many of the mutant dI proteins more nucleotide was required for these effects than with wild-type protein. Binding constants of the mutant proteins for NADH were determined by equilibrium dialysis and, where possible, by NMR. Generally, amino acid changes in the mobile loop region gave rise to a 2-4-fold increase in the dI-nucleotide dissociation constants, but substitution of Ala236 for Gly had a 10-fold effect. The mutant dI proteins were reconstituted with dI-depleted bacterial membranes with apparent docking affinities that were indistinguishable from that of wild-type protein. In the reconstituted system, most of the mutants were more inhibited in their capacity to perform cyclic transhydrogenation (reduction of acetyl pyridine adenine dinucleotide, AcPdAD+, by NADH in the presence of NADP+) than in either the simple reduction of AcPdAD+ by NADPH, or the light-driven reduction of thio-NADP+ by NADH, which suggests that they are impaired at the hydride transfer step. A cross-peak in the 1H-1H nuclear Overhauser enhancement spectrum of a mixture of wild-type dI and NADH was assigned to an interaction between the A8 proton of the nucleotide and the betaCH3 protons of Ala236. It is proposed that, following nucleotide binding, the mobile loop folds down on to the surface of the dI protein, and that contacts, especially from Tyr235 in a Gly-Tyr-Ala motif with the adenosine moiety of the nucleotide, set the position of the nicotinamide ring of NADH close to that of NADP+ in dIII to effect direct hydride transfer.

Growth hormone resistance and somatomedins in children with end-stage liver disease awaiting transplantation.

BACKGROUND: The success of orthotopic liver transplantation as treatment for end-stage liver disease has prompted investigation of strategies to maintain or improve nutrition and growth in children awaiting transplantation, because malnutrition is an adverse prognostic factor. The purpose of this study was to evaluate the effect of recombinant human growth hormone therapy on body composition and indices of liver function in patients awaiting transplant. METHODS: The study was designed as a placebo-controlled, double-blind, crossover trial. Patients received 0.2 U/kg growth hormone, subcutaneously, or placebo daily for 28 days during two treatment periods, separated by a 2-week washout period. Ten patients (mean age, 3.06 +/- 1.15 years; range, 0.51-11.65 years, five men), with extrahepatic biliary atresia (n = 8) or two with Alagille's syndrome (n = 2), with end-stage liver disease, completed the trial while awaiting orthotopic liver transplantation. Height, weight, total body potassium, total body fat, resting energy expenditure, respiratory quotient, hematologic and multiple biochemical profile, number of albumin infusions, insulin-like growth factor-1 and 1, growth hormone binding protein (GHBP), and insulin-like growth factor binding protein-1 (IGFBP-1) and insulin-like growth factor binding protein (IGFBP-3) were measured at the beginning and end of each treatment period. RESULTS: Growth hormone treatment was associated with a significant decline in serum bilirubin (-34.6 +/- 16.5 mumol/l vs. 18.2 +/- 11.59 mumol/l; p < 0.02) but there was no significant effect on any anthropometric or body composition measurements, or on any biochemical or hematologic parameters. CONCLUSIONS: These children with end-stage liver disease displayed growth hormone resistance, particularly in relation to the somatomedin axis. Exogenous growth hormone administration may be of limited value in these patients.

Interdomain hydride transfer in proton-translocating transhydrogenase.

We describe the use of the recombinant, nucleotide-binding domains (domains I and III) of transhydrogenase to study structural, functional and dynamic features of the protein that are important in hydride transfer and proton translocation. Experiments on the transient state kinetics of the reaction show that hydride transfer takes place extremely rapidly in the recombinant domain I:III complex, even in the absence of the membrane-spanning domain II. We develop the view that proton translocation through domain II is coupled to changes in the binding characteristics of NADP+ and NADPH in domain III. A mobile loop region which emanates from the surface of domain I, and which interacts with NAD+ and NADH during nucleotide binding has been studied by NMR spectroscopy and site-directed mutagenesis. An important role for the loop region in the process of hydride transfer is revealed.

Euthanasia in the Commonwealth of Australia.

This article describes the debate over the euthanasia law of the Northern Territory of Australia in its constitutional context. After considering the juridical status of the Northern Territory and related topics, this article outlines the judicial and legislative challenges to the Rights of the Terminally Ill Act (RTI Act) of the Northern Territory and concludes by offering some justifications for the passing of the Euthanasia Laws Act 1997 (Commonwealth), which effectively repeals the RTI Act. In dealing with these issues special emphasis will be given to the problem of Commonwealth law overriding Territory law and the concept of fundamental common law rights. The appropriateness of a Parliamentary solution--as opposed to a resolution through the courts--is defended.

Role of methionine-239, an amino acid residue in the mobile-loop region of the NADH-binding domain (domain I) of proton-translocating transhydrogenase.

Transhydrogenase couples the transfer of hydride equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The one-dimensional proton NMR spectrum of the recombinant NAD(H)-binding domain (domain I) of transhydrogenase from Rhodospirillum rubrum reveals well-defined resonances, several of which arise from a mobile loop at the protein surface. Four have been assigned to Met residues (MetA-MetD). Substitution of Met239 with either Ile (dI.M239I) or Phe (dI.M239F) resulted in loss of MetA from the NMR spectrum. Broadening and shifting of the mobile loop resonances consequent on NAD(H) binding indicate that the loop closes down on the protein surface. More NAD(H) had to be added to mutant domain I than to wild type to give comparable resonance broadening. The Kd of domain I for NADH, measured by equilibrium dialysis, was increased about three-fold by the Met239 mutations. Mutant and wild-type domain I were reconstituted with domain I-depleted membranes from R. rubrum, and with recombinant domain III of transhydrogenase. With membranes, the Km for acetylpyridine adenine dinucleotide during reverse transhydrogenation was 5x and > 6x greater in dI.M239I and dI.M239F, respectively, than in wild-type. Cyclic transhydrogenation (in membranes and the recombinant system) was substantially more inhibited (70% in dI.M239I, and 84% in dI.M239F) than either forward or reverse transhydrogenation. The docking affinities of dI.M239I and dI.M239F to the depleted membranes were similar to those of wild-type. It is concluded that Met239 is MetA in the mobile loop of domain I, and that in proteins with amino acid substitutions at this position, the binding affinity of NAD(H) is decreased, and the hydride transfer step is inhibited.

The ordered phosphorylation of cardiac troponin I by the cAMP-dependent protein kinase--structural consequences and functional implications.

The pattern of phosphorylation of adjacent serine residues in several peptides based on the N-terminal region of human cardiac troponin I has been analysed by PAGE and 1H NMR spectroscopy to identify the products. With cAMP-dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide at Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild-type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N-terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin I is phosphorylated on Ser24. Comparative two-dimensional NOESY data indicate that in the diphosphorylated form at physiological pH values, specific structural constraints are imposed on the N-terminal peptide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N-terminal to the serine pair and stabilisation of the structure in the region of residues 25-29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention.

Evaluation of bioelectrical impedance for prospective nutritional assessment in cystic fibrosis.

We have compared the use of bioelectrical impedance analysis (BIA) with anthropometry for the prediction of changes in total body potassium (TBK) in a group (n = 31) of children with cystic fibrosis. Linear regression analysis showed that TBK was highly correlated (r > 0.93) with height2/impedance, weight, height, and fat-free mass (FFM) estimated from skin-fold measurements. Changes in TBK were also correlated, but less well, with changes in height2/impedance, weight, height, and FFM (r = 0.69, 0.59, 0.44, and 0.40, respectively). The children were divided into two groups: those who had normal accretion of TBK (> 5%/y) and those who had suboptimal accretion of TBK (< 5%/y). Analysis of variance showed that the significant difference in the change in TBK between the groups was detectable by concomitant changes in impedance and weight but not by changes in height, FFM, or weight and height Z scores. The results of this study suggest that serial BIA measures may be useful as a predictor of progressive undernutrition and poor growth in children with cystic fibrosis.