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P M Finnegan

Publications and source records attributed to P M Finnegan.

16 recordsLinked to original sources
PubMed1999

A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.

Amino Acid Substitution↗
PubMed1999

An alternative oxidase monoclonal antibody recognises a highly conserved sequence among alternative oxidase subunits.

The alternative oxidase is found in the inner mitochondrial membranes of plants and some fungi and protists. A monoclonal antibody raised against the alternative oxidase from the aroid lily Sauromatum guttatum has been used extensively to detect the enzyme in these organisms. Using an immunoblotting strategy, the antibody binding site has been localised to the sequence RADEAHHRDVNH within the soybean alternative oxidase 2 protein. Examination of sequence variants showed that A2 and residues C-terminal to H7 are required for recognition by the monoclonal antibody raised against the alternative oxidase. The recognition sequence is highly conserved among all alternative oxidase proteins and is absolutely conserved in 12 of 14 higher plant sequences, suggesting that this antibody will continue to be extremely useful in studying the expression and synthesis of the alternative oxidase.

Amino Acid Sequence↗
PubMed1999

Phosphoenolpyruvate carboxykinase in the C(4) monocot Urochloa panicoides is encoded by four differentially expressed genes.

Previous screening of a cDNA library of leaf poly(A(+)) RNA from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C(4) monocot, led to the characterization of cDNAs encoding the U. panicoides PCK subunit PCK1. A second PCK sequence, designated PCK2, has now been found by rescreening the library. The deduced PCK2 polypeptide is 626 residues in length, has a predicted molecular mass of 68,686 D, and is 96% identical to the deduced PCK1 sequence. Isolation and characterization of genomic DNA fragments revealed that the PCK1 and PCK2 genes are each closely linked to another PCK gene. These additional genes have been designated PCK3 and PCK4, respectively. In each case, the second gene is located upstream and in the same transcriptional orientation as the gene characterized through cDNA analysis. A reverse transcription-polymerase chain reaction assay was used to demonstrate that PCK1 and PCK2 transcripts predominate in leaves, whereas PCK3 and PCK4 transcripts predominate in roots. Moreover, accumulation of PCK1 and PCK2 transcripts is light dependent. Direct N-terminal sequencing of PCK polypeptides purified from leaves demonstrated that PCK2 is produced. These results strongly suggest that PCK1 and PCK2 are involved in the photosynthetic CO(2)-concentrating mechanism active in U. panicoides.

Amino Acid Sequence↗
PubMed1998

Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules.

Nitrogen-fixing bacteroids in legume root nodules are surrounded by the plant-derived peribacteroid membrane, which controls nutrient transfer between the symbionts. A nodule complementary DNA (GmSAT1) encoding an ammonium transporter has been isolated from soybean. GmSAT1 is preferentially transcribed in nodules and immunoblotting indicates that GmSAT1 is located on the peribacteroid membrane. [14C]methylammonium uptake and patch-clamp analysis of yeast expressing GmSAT1 demonstrated that it shares properties with a soybean peribacteroid membrane NH4 + channel described elsewhere. GmSAT1 is likely to be involved in the transfer of fixed nitrogen from the bacteroid to the host.

Amino Acid Sequence↗
PubMed1997

Differential expression of the multigene family encoding the soybean mitochondrial alternative oxidase.

The alternative oxidase (AOX) of the soybean (Glycine max L.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced for cDNA analysis, were found to be 38.1 and 36.4 Kd, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 KD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein in cotyledons increase upon greening of dark-grown seedlings. These results comprehensively explain the multiple AOX-banding patterns observed on immunoblots of mitochondrial proteins isolated from various soybean tissues by matching protein bands with gene products.

Amino Acid Sequence↗
PubMed1997

Preliminary investigations of Aphodius species activity in cattle faeces treated with ivermectin.

A.sphacelatus at densities of 0.5 and 1.0 beetle/g faeces caused significantly greater median percentage reductions (65.2% and 87.4% respectively) of Pilobolus sporangia than 0.1 beetle/g faeces (31%) in untreated cattle faeces. The median percentage reduction in sporangia due to beetle activity (48.4%) was significantly lower (P < 0.02) in faeces mixed with ivermectin at 1.0 ppm (wet weight) than in untreated faeces (88.4%). After treating a bullock with ivermectin (IvomecR Pour-On), the median percentage reduction in sporangia caused by beetles was significantly less (P < 0.05) on days 9 (78.9%) and 10 (76.9%) than in pre-dose faeces (86.5% and 93.8% respectively). In microcosms without beetles, sporulation of Pilobolus in cattle faeces from a heifer treated with ivermectin was significantly less on days 5, 10 and 15 after dosing. However, this difference was not apparent for days 5 and 10 after storage of faeces at 4 degrees C for 55 and 50 days respectively.

Animals↗
PubMed1995

The mature AEP2 gene product of Saccharomyces cerevisiae, required for the expression of subunit 9 of ATP synthase, is a 58 kDa mitochondrial protein.

The nucleotide sequence of the yeast nuclear AEP2 gene, required for the expression of the mitochondrial DNA-encoded subunit 9 of ATP synthase, predicts a primary translation product of 67.5 kDa. The ATP13 gene is allelic to AEP2 but was reported to encode a protein of about 42 kDa in size. We thus investigated genetically and biochemically the size of the AEP2 gene product. Genetic complementation assays using 3' truncated AEP2 genes, here shows that function is abolished by the removal of only 32 amino acids from the C-terminus of the predicted protein product. Cell-free translation of AEP2 produces a 64 kDa polypeptide (consistent with the AEP2 sequence) which is imported into mitochondria and processed to a 58 kDa product by the removal of a presequence of about 50 amino acids.

Base Sequence↗
PubMed1995

Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides.

A rabbit antiserum was raised against phosphoenolpyruvate carboxykinase (PCK) purified from Urochloa panicoides, a PCK-type C4 monocot. The antiserum was used to screen a cDNA expression library constructed from U. panicoides leaf poly(A)+RNA. Inserts from immunoreactive clones were used to rescreen the library and obtain three overlapping cDNAs comprising a 2220 bp composite sequence. The single complete open reading frame of 1872 bp encodes PCK1, a 624 amino acid polypeptide with a predicted molecular mass of 68,474 Da. Comparison of PCK1 with other ATP-dependent PCKs indicates that PCK1 is significantly larger, mainly due to an N-terminal extension of greater than 65 residues, and reveals high sequence identity across the central portion of the protein, especially over seven sub-sequences. One of these sub-sequences spans motifs common to several ATP-utilising enzymes for phosphate and divalent cation binding. The anti-PCK antiserum recognises a 69 kDa polypeptide on immunoblots of either purified PCK or U. panicoides leaf extracts. However, polypeptides of 63, 62, 61 and 60 kDa are also immunoreactive. Amino terminal sequencing of polypeptides from preparations of purified PCK demonstrates that these smaller polypeptides are related to PCK1, and time course experiments show that these polypeptides arise from the breakdown of PCK during isolation. Northern blot analysis indicates that the 2.7 kb PCK mRNA is abundant in green leaves but not in roots or etiolated shoots. Moreover, PCK mRNA levels increase gradually during greening, reaching maximum levels after about 84 h.

Amino Acid Sequence↗
PubMed1991

Mechanism of misoprostol stabilization in hydroxypropyl methylcellulose.

The stability of misoprostol oil is significantly improved in a hydroxypropyl methylcellulose (HPMC) dispersion (1:100). In order to understand the enhanced stability of misoprostol oil in HPMC, the physical state of misoprostol oil in HPMC films was investigated using differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), and transmission IR (TIR). Further, to determine the effect of polymer structure and the mobility of both water and misoprostol on misoprostol stability, the rate of misoprostol degradation was investigated in the misoprostol/HPMC dispersion (1:100) at 55 degrees C. The water sorption isotherm of the dispersion at 55 degrees C was determined, at seven different relative humidities, ranging from zero to 81%. The DSC and DMA measurements indicated that misoprostol oil, up to 29% in dry weight, is molecularly dispersed in the glassy HPMC. The TIR studies showed no evidence of complexation between misoprostol and HPMC. Stability studies of the misoprostol/HPMC (1:100) dispersion indicated that the first-order rate constants for misoprostol degradation increased in a concave-up fashion as the water content of the dispersion increased. Below two percent water content, the rate of misoprostol degradation was found to be minimal. Overall, it is suggested that misoprostol is stabilized in the dispersion by being molecularly dispersed in HPMC. Further, the glassy state of HPMC should reduce the mobility of misoprostol and water, leading to a minimal rate of degradation for misoprostol at low moisture levels.

Calorimetry, Differential Scanning↗
PubMed1991

Characterization of a yeast nuclear gene, AEP2, required for accumulation of mitochondrial mRNA encoding subunit 9 of the ATP synthase.

The temperature-conditional pet mutant, ts379, of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature due to mutation of a single nuclear locus, AEP2. The inability to synthesize subunit 9 correlates with a lowered accumulation of the cognate oli1 mRNA indicating that the AEP2 product is involved in oli1 transcript maturation or stabilization. The AEP2 gene has been isolated in this study from a wild-type yeast genomic library by genetic complementation of ts379 at the restrictive temperature. A 1,740 nucleotide open-reading frame was observed that encodes a basic, hydrophilic protein of 67,534 Da which possesses a putative mitochondrial address signal. Disruption of chromosomal DNA within this reading frame produced a non-conditional respiratory mutant unable to synthesize subunit 9, identifying the AEP2 gene. Hybridization analyses indicate that AEP2 is located on chromosome XIII and produces a 2.1 kb poly(A)+ transcript. Two additional open-reading frames were found in close proximity to that of AEP2. The three open-reading frames shared no significant homology with entries in several data bases.

Amino Acid Sequence↗
PubMed1990

Identification of a 66 KDa protein associated with yeast mitochondrial ATP synthase as heat shock protein hsp60.

A 66 kDa protein, denoted P66, not hitherto classified as an integral component of yeast mitochondrial ATPase, is often observed in preparations of this enzyme complex. A physical association exists between P66 and the assembled ATPase complex since both components are coimmunoprecipitated by anti-F1 beta monoclonal antibody. Two recombinant clones expressing proteins immunologically similar to P66 were isolated from a yeast genomic library in lambda gt11 by screening with a polyclonal anti-holo-ATPase antibody. Based on restriction site mapping and partial nucleotide sequence analysis, both clones encompass the gene encoding the yeast heat shock protein hsp60. The identification of P66 with hsp60, taken together with its demonstrated association with the mitochondrial ATPase complex, is consistent with recent suggestions that hsp60 is involved in assembly of the ATP synthase complex.

Cloning, Molecular↗
PubMed1990

Physical state of misoprostol in hydroxypropyl methylcellulose films.

Scanning electron (SEM) and light microscopy (LM), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA) techniques were utilized to determine the miscibility of misoprostol and HPMC in the films with a misoprostol content from 0 to 29%, prepared using ethanol and methylene chloride/methanol (MeCl2/methanol, 50:50). Transmission infrared (TIR) analysis was used to look for evidence of any interaction between misoprostol and HPMC. The LM and SEM analysis of the ethanol cast films indicated no oil droplets. The DSC thermograms of the films showed no evidence of a -33 degrees C transition, which is characteristic of pure misoprostol. The DMA showed that the glass-rubber transition temperature (Tg) of the pure HPMC was lowered from 163 to 125-130 and 85-87 degrees C in the presence of 10 and 27-28% misoprostol. Based on these results it is suggested that misoprostol is solubilized in HPMC at concentrations up to 29%. The TIR analysis of the films showed no evidence of interaction between misoprostol and HPMC.

Alprostadil↗
PubMed1989

RNA plasmids.

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Animals↗
PubMed1989

Solid-state interaction of magnesium oxide and ibuprofen to form a salt.

During formulation development work involving ibuprofen, a solid-state interaction between MgO and ibuprofen was observed. In this study the interaction of MgO and ibuprofen was investigated for 1:1 and 2:1 M mixtures of ibuprofen and MgO, which had been stored at 55 degrees C, using the differential scanning calorimetric (DSC), thermogravic analysis (TGA), and multiple internal reflectance infrared (MIR) techniques. Evidence for the reaction was the disappearance of the melting endotherm at 79 degrees C and appearance of a new endotherm at 161 degrees C after less than 1 day of storage at 55 degrees C and, also, the change in the physical appearance of the mixtures. Comparison of the DSC, TGA, and MIR data for the reacted ibuprofen and MgO mixtures and synthetic Mg(ibuprofen)2 indicated that MgO and ibuprofen react to form the Mg salt of ibuprofen. The interaction of ibuprofen and MgO was also studied at 30 and 40 degrees C, using 1:1 M mixtures. At 30 degrees C no significant interaction was observed for up to 80 days; however, at 40 degrees C a reaction was evident on day 1. NaHCO3, K2CO3 1.5H2O, CaO, and Mg(OH)2 also showed solid-state reactions with ibuprofen. MgCl2 and Al(OH)3 did not show this reaction.

Calorimetry, Differential Scanning↗
PubMedDate not supplied

Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae.

Due to mutation in a single nuclear locus, AEP1, the temperature-conditional pet mutant ts1860 of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature of 36 degrees C. The presence at this temperature of near-normal levels of the cognate oli1 mRNA in mutant ts1860 indicates that, as previously shown, the product of the AEP1 gene is required for translation of the mitochondrial oli1 transcript. In this study the AEP1 gene has been cloned from a wild-type yeast genomic library by genetic complementation of a temperature-conditional aep1 strain at the restrictive temperature. A 2,330-bp genomic fragment which restores subunit 9 synthesis in aep1 mutant strains was characterized. This fragment encoded five open reading frames: the longest of these, at 1,554 nucleotides, was identified as the AEP1 gene, since disruption of this reading frame generated a non-conditional pet strain unable to synthesize subunit 9. The predicted product of AEP1 is a basic, hydrophilic protein of 59,571 Da which possesses a putative mitochondrial address sequence. Hybridization studies with AEP1-specific probes indicate that the gene is located on chromosome XIII and produces several poly(A)+ transcripts ranging in size from 0.9 to 2.7 kb. None of the identified reading frames share significant homologies with entries of several data bases.

Amino Acid Sequence↗