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G D Price

Publications and source records attributed to G D Price.

At least 19 recordsLinked to original sources
PubMed2000

Bicarbonate binding activity of the CmpA protein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in active transport of bicarbonate.

The cmpABCD operon of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an ATP-binding cassette transporter involved in HCO(3)(-) uptake. The three genes, cmpBCD, encode membrane components of an ATP-binding cassette transporter, whereas cmpA encodes a 42-kDa cytoplasmic membrane protein, which is 46.5% identical to the membrane-anchored substrate-binding protein of the nitrate/nitrite transporter. Equilibrium dialysis analysis using H(14)CO(3)(-) showed that a truncated CmpA protein lacking the N-terminal 31 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, specifically binds inorganic carbon (CO(2) or HCO(3)(-)). The addition of the recombinant CmpA protein to a buffer caused a decrease in the concentration of dissolved CO(2) because of the binding of inorganic carbon to the protein. The decrease in CO(2) concentration was accelerated by the addition of carbonic anhydrase, indicating that HCO(3)(-), but not CO(2), binds to the protein. Mass spectrometric measurements of the amounts of unbound and bound HCO(3)(-) in CmpA solutions containing low concentrations of inorganic carbon revealed that CmpA binds HCO(3)(-) with high affinity (K(d) = 5 microm). A similar dissociation constant was obtained by analysis of the competitive inhibition of the CmpA protein on the carboxylation of phosphoenolpyruvate by phosphoenolpyruvate carboxylase at limiting concentrations of HCO(3)(-). These findings showed that the cmpA gene encodes the substrate-binding protein of the HCO(3)(-) transporter.

ATP-Binding Cassette Transporters↗
PubMed2000

The role of chloroplast electron transport and metabolites in modulating Rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase.

Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP(+) ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO(2) assimilation rates decreased with decreasing ribulose 1, 5-bisphosphate concentrations. In anti-b/f plants, CO(2) assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.

Adenosine Monophosphate↗
PubMed2000

Mutation of ndh genes leads to inhibition of CO(2) uptake rather than HCO(3)(-) uptake in Synechocystis sp. strain PCC 6803.

Six mutants (B1 to B6) that grew poorly in air on BG11 agar plates buffered at pH 8.0 were rescued after mutations were introduced into ndhB of wild-type (WT) Synechocystis sp. strain PCC 6803. In these mutants and a mutant (M55) lacking ndhB, CO(2) uptake was much more strongly inhibited than HCO(3)(-) uptake, i.e., the activities of CO(2) and HCO(3)(-) uptake in B1 were 9 and 85% of those in the WT, respectively. Most of the mutants grew very slowly or did not grow at all at pH 6.5 or 7.0 in air, and their ability to grow under these conditions was correlated with CO(2) uptake capacity. Detailed studies of B1 and M55 indicated that the mutants grew as fast as the WT in liquid at pH 8.0 under air, although they grew poorly on agar plates. The contribution of CO(2) uptake appears to be larger on solid medium. Five mutants were constructed by inactivating each of the five ndhD genes in Synechocystis sp. strain PCC 6803. The mutant lacking ndhD3 grew much more slowly than the WT at pH 6.5 under 50 ppm CO(2), although other ndhD mutants grew like the WT under these conditions and showed low affinity for CO(2) uptake. These results indicated the presence of multiple NAD(P)H dehydrogenase type I complexes with specific roles.

Bicarbonates↗
PubMed1999

Identification of an ATP-binding cassette transporter involved in bicarbonate uptake in the cyanobacterium Synechococcus sp. strain PCC 7942.

Exposure of cells of cyanobacteria (blue-green algae) grown under high-CO(2) conditions to inorganic C-limitation induces transcription of particular genes and expression of high-affinity CO(2) and HCO(3)(-) transport systems. Among the low-CO(2)-inducible transcription units of Synechococcus sp. strain PCC 7942 is the cmpABCD operon, encoding an ATP-binding cassette transporter similar to the nitrate/nitrite transporter of the same cyanobacterium. A nitrogen-regulated promoter was used to selectively induce expression of the cmpABCD genes by growth of transgenic cells on nitrate under high CO(2) conditions. Measurements of the initial rate of HCO(3)(-) uptake after onset of light, and of the steady-state rate of HCO(3)(-) uptake in the light, showed that the controlled induction of the cmp genes resulted in selective expression of high-affinity HCO(3)(-) transport activity. The forced expression of cmpABCD did not significantly increase the CO(2) uptake capabilities of the cells. These findings demonstrated that the cmpABCD genes encode a high-affinity HCO(3)(-) transporter. A deletion mutant of cmpAB (M42) retained low CO(2)-inducible activity of HCO(3)(-) transport, indicating the occurrence of HCO(3)(-) transporter(s) distinct from the one encoded by cmpABCD. HCO(3)(-) uptake by low-CO(2)-induced M42 cells showed lower affinity for external HCO(3)(-) than for wild-type cells under the same conditions, showing that the HCO(3)(-) transporter encoded by cmpABCD has the highest affinity for HCO(3)(-) among the HCO(3)(-) transporters present in the cyanobacterium. This appears to be the first unambiguous identification and description of a primary active HCO(3)(-) transporter.

ATP-Binding Cassette Transporters↗
PubMed1999

Cloning and expression of a prokaryotic sucrose-phosphate synthase gene from the cyanobacterium Synechocystis sp. PCC 6803.

Sucrose is one of several low-molecular-weight compounds that cyanobacteria accumulate in response to osmotic stress and which are believed to act as osmoprotectants. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains a 2163 bp open reading frame (ORF) that shows similarity to genes from higher plants encoding sucrose-phosphate synthase (SPS), the enzyme responsible for sucrose synthesis. The deduced amino acid sequence shows 35-39% identity with known higher-plant SPS sequences. The putative Synechocystis sps gene was cloned from genomic DNA by PCR amplification and expressed as a His6-tagged amino-terminal fusion protein in Escherichia coli. The expressed protein was purified and shown to be a functional SPS enzyme, confirming the identity of the ORF, which is the first sps gene to be cloned from a prokaryotic organism. The Synechocystis SPS has a molecular mass of 81.5 kDa, which is smaller than the typical higher-plant SPS subunit (117-119 kDa), and lacks the phosphorylation site motifs associated with light- and osmotic stress-induced regulation of SPS in higher plants. The enzyme has Km values for UDPG1c and Fru6P of 2.9 mM and 0.22 mM, respectively, with a Vmax of 17 micromol per minute per mg protein and a pH optimum of 8.5. Unlike the higher-plant enzyme, ADPG1c, CDPG1c and GDPG1c can substitute for UDPG1c as the glucosyl donor with Km values of 2.5, 7.2 and 1.8 mM, respectively. The enzyme is activated by Mg2+ but not by G1c6P, and is only weakly inhibited by inorganic phosphate. The purified protein was used to raise a high-titre antiserum, which recognises a low-abundance 81 kDa protein in Synechocystis sp. PCC 6803 extracts. There was no apparent increase in expression of the 81 kDa protein when the cells were exposed to moderate salt stress, and SPS activity was very low in extracts from both unstressed and salt-stressed cells. These results and the lack of evidence for sucrose accumulation in Synechocystis sp. PCC6803 lead to the conclusion that expression of the sps gene plays no obvious role in adaptation to osmotic stress in this species.

Amino Acid Sequence↗
PubMed1999

The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp. PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria.

Random gene tagging was used to obtain new mutants of the marine cyanobacterium, Synechococcus sp. PCC7002, with defects in the CO2-concentrating mechanism (CCM). Two of these mutants, K22 and A41, showed poor growth at limiting CO2. Isolation and sequencing of a 6. 6 kb genomic region revealed the existence of five potential protein-coding regions, all arranged in the same transcriptional direction. These regions code for an RbcR homologue, NdhF3 (subunit 5 of type 1 NAD(P)H dehydrogenase; NDH-1 complex), NdhD3 (subunit 4 of NDH-1), ORF427 and ORF133 (hypothetical proteins). Insertional mutants in ndhD3, ndhF3 and ORF427, like A41 and K22, were all incapable of inducing high-affinity CO2 uptake and were not fully capable of inducing high-affinity HCO3- transport. ndhD3 and ndhF3 mutants displayed P700 re-reduction rates identical to wild-type cells, suggesting that NdhD3 is part of a specific NDH-1 complex that is not involved in photosynthetic cyclic electron transport. Thus, it is feasible that NdhD3, NdhF3 and ORF427 might form part of a novel NDH-1 complex located on the cytoplasmic membrane and involved in tightly coupled energization of high-affinity CO2 transport. The possibility of multiple, functionally distinct NDH-1 complexes in cyanobacteria is discussed.

Carbon Dioxide↗
PubMed1997

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase deficiency delays senescence of ribulose-1,5-bisphosphate carboxylase/oxygenase but progressively impairs its catalysis during tobacco leaf development.

Transgenic tobacco (Nicotiana tabacum L. cv W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) activase grew more slowly than wild-type plants in a CO2-enriched atmosphere, but eventually attained the same height and number of leaves. Compared with the wild type, the anti-activase plants had reduced CO2 assimilation rates, normal contents of chlorophyll and soluble leaf protein, and much higher Rubisco contents, particularly in older leaves. Activase deficiency greatly delayed the usual developmental decline in Rubisco content seen in wild-type leaves. This effect was much less obvious in another transgenic tobacco with an antisense gene directed against chloroplast-located glyceraldehyde-3-phosphate dehydrogenase, which also had reduced photosynthetic rates and delayed development. Although Rubisco carbamylation was reduced in the anti-activase plants, the reduction was not sufficient to explain the reduced photosynthetic rate of older anti-activase leaves. Instead, up to a 10-fold reduction in the catalytic turnover rate of carbamylated Rubisco in vivo appeared to be the main cause. Slower catalytic turnover by carbamylated Rubisco was particularly obvious in high-CO2-grown leaves but was also detectable in air-grown leaves. Rubisco activity measured immediately after rapid extraction of anti-activase leaves was not much less than that predicted from its degree of carbamylation, ruling out slow release of an inhibitor from carbamylated sites as a major cause of the phenomenon. Nor could substrate scarcity or product inhibition account for the impairment. We conclude that activase must have a role in vivo, direct or indirect, in promoting the activity of carbamylated Rubisco in addition to its role in promoting carbamylation.

Carbon Dioxide↗
PubMed1996

Cloning, analysis and inactivation of the ndhK gene encoding a subunit of NADH quinone oxidoreductase from Anabaena PCC 7120.

The function of the type-1 pyridine nucleotide dehydrogenase (NDH-1) in the cyanobacterium Anabaena PCC 7120 was investigated. Immunological analysis with antibodies raised against NdhK from Synechocystis PCC 6803, a subunit of NDH-1, showed that NdhK in Anabaena PCC 7120 is only present on the plasma membrane, which confirms the results of previous studies [Howitt, C.A., Smith, G.D. & Day, D. A. (1993) Biochim. Biophys. Acta 114], 313-320]. Southern analysis with probes from the operon encoding ndhC-K-J from Synechocystis PCC 6803 showed that this operon is also conserved in Anabaena PCC 7120. Part of the operon was amplified using PCR with degenerate primers designed against two sequences encoding regions of NdhC and NdhJ that are conserved between cyanobacteria and chloroplasts. The nucleotide sequence of ndhK encodes a protein of 245 amino acids with a predicted molecular mass of 27.5 kDa. The coding regions of ndhC and ndhK overlap by 7 bp, as found in the chloroplasts of liverwort, maize, and rice. This is markedly different from the case in Synechocystis PCC 6803 where a 71-bp non-coding, intergenic spacer region lies between ndhC and ndhK. The ndhK clone was interrupted by the insertion of a kanamycin-resistance gene and used to transform Anabaena PCC 7120.20 unsegregated transformants were produced, all of which died during attempts to segregate them. This indicates that under the selection conditions used, ndhK is an essential gene in Anabaena PCC 7120.

Amino Acid Sequence↗
PubMed1996

Improved long-term survival after heart transplantation predicted by successful early withdrawal from maintenance corticosteroid therapy.

BACKGROUND: Short-term studies suggest that cardiac transplant immunosuppression without maintenance corticosteroids is feasible in selected patients. However, concern exists as to the long-term effects, specifically the possibility of increased morbidity and mortality because of late allograft rejection and allograft coronary artery disease. METHODS: We retrospectively reviewed the records from 441 consecutive heart transplantation procedures done in 416 patients with use of an immunosuppressive protocol that attempted corticosteroid withdrawal within 2 months of transplantation. forty-two patients died or underwent retransplantation during the first 3 months and were excluded from further analysis. Analysis focused on demographic and long-term outcome variables (including death, rejection, retransplantation, and infection). RESULTS: Thirty percent (111) of eligible patients (374) met the definition of successful early steroid withdrawal. Only male gender independently predicted successful withdrawal. Mortality, both short and long term, was significantly lower in patients in whom successful early withdrawal from corticosteroids was achieved than in patients in whom the early attempts failed (1.7% per year versus 4.7% per year; p < 0.0001). The prevalence of late acute allograft rejection (more than 1 year after transplantation) was lower in patients successfully withdrawn from steroid therapy early after transplantation (0.07 pt-yr of follow-up versus 0.15 pt-yr; p = 0.002). Multivariate analysis of the entire group identified incidence of infection (p = 0.001), older age (p = 0.001), failed early steroid withdrawal (p = 0.006), and female gender (p = 0.016) as independent predictors of mortality. CONCLUSIONS: Successful early corticosteroid withdrawal identifies a subgroup of "immunologically privileged" patients with a low risk for long-term mortality and is not associated with an increased prevalence of late rejection or clinically significant coronary artery disease.

Case-Control Studies↗
PubMed1995

Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants.

The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction (PCR) cycle in the stroma. Glyceraldehyde-3-phosphate dehydrogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enzymes which catalyse this reversible conversion. We report on the engineering of an antisense RNA construct directed against the tobacco (Nicotiana tabacum L.) chloroplast-located GAPDH (A subunit). The construct was integrated into the tobacco genome by Agrobacterium-mediated transformation of leaf discs. Of the resulting transformants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% of wild-type (WT) activities. Segregation analysis of the kanamycin-resistance character in self-pollinated T1 seed from each of the five transformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert. T1 progeny from GAP-R was used to generate plants with GAPDH activities ranging from WT levels to around 7% of WT levels. These were used to study the effect of variable GAPDH activities on metabolite pools for ribulose-1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO2 assimilation and other gas-exchange parameters. The RuBP pool size was linearly related to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO2 assimilation was not affected until RuBP levels dropped to 30-40% of WT levels. That is, the CO2 assimilation rate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol.(mol site)-1 while the ratio for WT plants was 4-5 mol.m(mol site)-1. Leaf conductance was not reduced in leaves with reduced GAPDH activities, resulting in an increase in the ratio of intercellular to ambient CO2 partial pressure. Conductance in plants with reduced GAPDH activities was still sensitive to CO2 and showed a normal decline with increases in CO2 partial pressure. Although PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP. Concomitant gas-exchange and chlorophyll alpha fluorescence measurements indicated that photosynthesis changed from being Rubisco-limited to being RuBP-regeneration-limited at a lower CO2 partial pressure in the antisense plants than in WT plants.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1994

Paternity by cardiac transplant recipients.

To assess whether children fathered by cardiac transplant recipients are at high risk of teratogenicity, cardiac transplant centers listed with the International Society for Heart and Lung Transplantation were surveyed. Paternities after transplantation by heart (n = 35) and heart-lung (n = 1) allograft recipients have resulted in 42 pregnancies (children's age 3.3 +/- 0.3 years). The fathers' age at conception was < 45 years in 40 (95%) and > 45 years in 2 (5%). Most fathers (86%) were enjoying an active and healthy lifestyle at the time of conception; one (2%) was on dialysis and listed for kidney transplantation due to nephrotic syndrome, 1 (2%) had asthma, 4 (10%) had allograft coronary disease (1 died while waiting for second heart transplant when the child was 2 months old), and 2 (5%) were retransplanted after the pregnancies. Immunosuppressive regimens were reported for 37 paternities; drug protocols at the time of conception were as follows: 25 (60%) CsA/prednisone/AZA, 6 (14%) CsA/prednisone, 4 (10%) CsA/AZA, and 2 (5%) AZA/prednisone. Twenty-six (62%) had received treatment for rejection episodes before conception; seven (17%) had received treatment for rejections since conception. Of the 42 children fathered by these recipients, 3 (7%) were preterm, 1 (2%) had a cleft palate and lip that have subsequently been corrected, 1 (2%) died from interruption of umbilical cord circulation at 24 weeks, and 1 (2%) whose father had familial cardiomyopathy was born with a cardiomyopathy that improved with time. Although the numbers are small, the available data suggest that paternity by cardiac transplant recipients may be safe.

Adult↗
PubMed1993

Analysis of a genomic DNA region from the cyanobacterium Synechococcus sp. strain PCC7942 involved in carboxysome assembly and function.

We report on the sequencing and analysis of a 3,557-bp genomic DNA clone that is located between 4.8 and 1.2 kilobase pairs (kb) upstream of the rbcL gene and is capable of complementing a class of cyanobacterium Synechococcus sp. strain PCC7942 mutants requiring a high level of CO2. The upstream 2,704 bp of this sequence is novel, the remaining 852 bp having been reported by other workers. Four new open reading frames (ORFs) have been identified along with putative promoter elements. These ORFs, which could code for proteins of 7, 10.9, 11, and 58 kDa in size, have been named ORF 64, ccmK, ccmL, and ccmM, respectively. The last three have been named ccm genes on the basis that insertional mutagenesis of each produces a phenotype requiring a high level of CO2 (i.e., each produces a lesion in the CO2 concentrating mechanism). The putative gene product for the large ccmM ORF has three internally repeated regions and also has two possible DNA binding motifs. Two defined mutants in the 3,557-bp region, mutants PVU and P-N, have been more fully characterized. The PVU mutant has a drug marker inserted into the ccmL gene, and it possesses abnormal rod-shaped carboxysomes. The P-N mutant is a 2.64-kb deletion of DNA from the same position in ccmL to a region closer to rbcL. This mutant, which has previously been shown to lack carboxysomes and have soluble ribulosebiphosphate carboxylase/oxygenase activity, has now been shown to have a predominantly soluble carboxysomal carbonic anhydrase activity. Both mutants were found to possess carboxysomal carbonic anhydrase activities which are below wild-type levels, and in the P-N mutant this activity appears to be unstable. The results are discussed in terms of the possible interactions of putative ccm gene products in the process of carboxysome assembly and function.

Amino Acid Sequence↗
PubMed1981

Quality personnel.

Assuring the competent function of personnel is a fundamental part of any clinical laboratory quality assurance program. This paper will review some of the problems associated with defining and measuring the competence of laboratory personnel and will suggest a model for a uniform, a nationwide competence measuring system.

Allied Health Personnel↗
PubMed1979

Photoreceptor membrane breakdown in the spider Dinopis: localisation of acid phosphatases.

The ultrastructural localisation of acid phosphatases (AcPhs) during the normal daily breakdown of rhabdomere membrane in Dinopis has been examined using beta-glycerophosphate and p-nitrophenyl phosphate as substrates. Results are related to the classification of organelles in the receptors given by Blest, Powell and Kao (1978). Weak and infrequent reactions are obtained in multivesicular bodies (mvbs) and multilamellar bodies (mlbs) derived from them. Residual bodies (rbs) begin to react strongly as they lyse. Source of AcPhs is endoplasmic reticulum which has barely differentiated towards the GERL configuration; it becomes reactive as it is incorporated into secondary lysosomes. GERL tubules, Y-bodies and vesicles respond erratically and weakly, and are also incorporated into rbs. No evidence was found for a significant participation of Golgi bodies in these processes, and acid phosphatase cytochemistry fails to reveal a topographical relationship between GERL in these cells and Golgi saccules. Coated vesicle clusters found in the predawn receptive segments are AcPh-negative; this implies that their previous identification as GERL-derived "Nebenkerne" carrying hydrolytic enzymes to newly-formed mvbs (Blest, Kao and Powell, 1978) is dubious. Isolation bodies and autophagic vacuoles enclosing other organelles in pathological receptors give strong reactions while adjacent secondary lysosomes derived from rhabdomere membrane and associated GERL give weak ones. It is concluded that rhabdomere-derived rb lysis is more tightly regulated than other autophagic processes, and it is suggested that a high degree of control is necessary in a receptor which may repeat the autophagy of a large mass of transductive membrane at least 60--100 times in the course of its working life.

Acid Phosphatase↗