RESUMO
AIM: Atrial fibrillation (AF) is a common cardiac arrhythmia, and is associated with worsening quality of life and complications such as stroke. Previous work showed that 8% of patients develop new-onset AF following colonic resection and highlighted factors that might predict the development of postoperative AF. The development of a new arrhythmia may have a negative effect on longer-term quality of life as well as cancer survivorship. The aim of this study is to accurately quantify the incidence of AF following colorectal cancer surgery and to validate a model to predict its development. METHOD: The Atrial Fibrillation After Resection (AFAR) study will recruit 720 patients aged 65 or over undergoing resection of colorectal cancer with curative intent. The primary outcome is development of AF within 90 days of surgery. Assessment of cardiac rhythm will be performed using 24-h Holter monitors at baseline, 30 and 90 days after surgery. An electrocardiogram (ECG) will be performed on the day of discharge. Baseline descriptors including model variables and quality of life will be recorded using EQ-5D-5L. The occurrence of complications and other key surgical outcomes will be recorded. An additional blood test for N-terminal pro B-type natriuretic peptide (NT-proBNP) will be performed prior to surgery. Statistical analysis will validate a previously derived model and will test the incremental value of added variables such as NT-proBNP. Finally, an exploratory analysis will assess whether changes in ECG measures between baseline and postoperative ECG can predict subsequent new-onset AF. CONCLUSION: This study will provide data that may allow us to stratify the risk of developing AF following colorectal cancer surgery. This may inform screening or prophylactic approaches.
Assuntos
Fibrilação Atrial , Fibrilação Atrial/epidemiologia , Fibrilação Atrial/etiologia , Biomarcadores , Humanos , Incidência , Qualidade de VidaRESUMO
AIM: Randomized trials comparing surgical techniques for rectal prolapse are not always feasible. We assessed whether non-randomized comparisons of those who have had surgery with those still waiting would be confounding baseline health status. METHOD: This was a prospective cohort study in seven UK hospitals. Participants were ≥ 18 years and listed for surgical interventions of equivalent intensity for rectal prolapse. They were defined as short or long waiters (≤ 18 or > 18 weeks, respectively). Time on the waiting list was compared with baseline comorbidity (Charlson comorbidity index) and change from baseline in health status (EQ-5D-5L) at the time of surgery. RESULTS: In all, 203 patients were analysed. Median (interquartile range) waiting time was 13.7 weeks (8.1, 20.4) varying across sites. Baseline comorbidity was not an important predictor of waiting time. Median Charlson comorbidity index was 2 (0, 3) for short and 1 (0, 3) for long waiters. A change in waiting time by a week was associated with negligible improvement in the EQ-5D-5L index of 0.001 (95% CI -0.000 to 0.003, P = 0.106). CONCLUSION: Negligible change in patient reported health status while on the waiting list and lack of effect of comorbidities in influencing waiting time support the use of non-randomized pre-/post-studies to compare the effects of surgical interventions for rectal prolapse.
Assuntos
Prolapso Retal , Nível de Saúde , Humanos , Estudos Prospectivos , Qualidade de Vida , Prolapso Retal/cirurgia , Listas de EsperaRESUMO
Medium-chain fatty acids (FAs), found in storage lipids of certain plants, are an important renewable resource. Seeds of undomesticated California bay accumulate laurate (12:0), and a 12:0-acyl-carrier protein thioesterase (BTE) has been purified from this tissue. Sequencing of BTE enabled the cloning of a complementary DNA coding for a plastid-targeted preprotein. Expression of the complementary DNA in the seeds of Arabidopsis thaliana resulted in BTE activity, and medium chains accumulated at the expense of long-chain (greater than or equal to 16) FAs. Laurate became the most abundant FA species and was deposited in the storage triacylglycerols. These results demonstrate a mechanism for medium-chain FA synthesis in plants.
Assuntos
Acetiltransferases/metabolismo , Ácidos Graxos/biossíntese , Ácidos Láuricos/metabolismo , Plantas/metabolismo , Acetiltransferases/genética , Proteína de Transporte de Acila S-Acetiltransferase , Sequência de Aminoácidos , DNA/genética , Ácidos Graxos/isolamento & purificação , Engenharia Genética , Dados de Sequência Molecular , Plantas/genética , Plantas Geneticamente Modificadas , Plasmídeos , Sementes/metabolismoRESUMO
The aggregation of gel-filtered rabbit platelets by 50 microM ADP was inhibited by a labile factor produced by suspensions of cultured bovine pulmonary artery endothelial cells. Inhibition of aggregation occurred when indomethacin-treated endothelial cells (6.10(5) per ml) and rabbit platelets (3.2.10(8) per ml) were incubated together. This anti-aggregatory activity was characterized as similar to endothelium-derived relaxing factor (EDRF) in that it was unstable at neutral pH and by its inhibition by hemoglobin. The activity was unaffected by treatment of the platelets and endothelial cells with the cyclooxygenase inhibitor, indomethacin, and by the lipoxygenase inhibitor, BW755c. In association with the anti-aggregatory activity, the levels of cyclic GMP were elevated 4-fold. The effect of the EDRF-like product on the levels of cyclic nucleotides was mimicked by treatment of platelets with sodium nitroprusside, an activator of soluble guanylate cyclase; sodium nitroprusside had no measurable effect on the levels of cyclic nucleotides of endothelial cells. We conclude that a factor with the properties of EDRF inhibits platelet aggregation, and that this is associated with an activation of guanylate cyclase as in smooth muscle. Thus, EDRF may exert an inhibitory effect on platelets in a manner analogous to its actions on vascular smooth muscle.
Assuntos
Produtos Biológicos/farmacologia , Endotélio Vascular/fisiologia , Guanilato Ciclase/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Animais , Produtos Biológicos/antagonistas & inibidores , Bovinos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Hemoglobinas/farmacologia , Óxido Nítrico , Nucleotídeos Cíclicos/metabolismo , Superóxido Dismutase/metabolismoRESUMO
11(R)-Hydroxyeicosatetraenoic acid [11(R)-HETE] and 12(R)-HETE are biosynthesized by eggs of the sea urchin S. purpuratus. We report here the isolation of the 11(4)- and 12(R)-hydroperoxy-eicosanoids from incubations of the desalted 30-50%(NH4)2SO4 fraction of the egg homogenate; biosynthesis required the addition of calcium but not NADPH. Egg 11- and 12-HETE were formed from octadeuterated arachidonic acid without loss of geminal 2H from C11 or C12, thus revealing that 11- or 12-keto intermediates are not involved in the biosynthesis. The results support the conclusion that egg 11(R)- and 12(R)-HETE are synthesized by a lipoxygenase and not by an NADPH-dependent cytochrome P450 monooxygenase mechanism.
Assuntos
Ácidos Hidroxieicosatetraenoicos/biossíntese , Óvulo/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animais , Ácidos Araquidônicos/metabolismo , Cálcio/farmacologia , Ácidos Hidroxieicosatetraenoicos/isolamento & purificação , Lipoxigenase/metabolismo , NADP/farmacologia , Ouriços-do-Mar , EstereoisomerismoRESUMO
Roberts syndrome (RS) is a rare, autosomal recessive condition characterized primarily by growth retardation, developmental delay, and limb anomalies. Some RS patients (RS+), but not others (RS-), have an abnormality of their constitutive heterochromatin (the "RS effect"). RS+ patients also show a cellular hypersensitivity to DNA damaging agents such as mitomycin C (MMC). Lymphoblastoid cell lines from 2 unrelated RS+ patients were fused and hybrid cells examined for correction of the RS effect and MMC hypersensitivity. Neither cellular defect was corrected in the 2 hybrid cell lines examined, suggesting that these 2 patients represent a single complementation group. Fusions were also performed between one RS+ cell line and 2 different RS- cell lines. In both fusions, the hybrids demonstrated correction of both the heterochromatin abnormality and MMC hypersensitivity. These observations suggest that RS+ and RS- patients belong to different complementation groups and do not arise from the same single gene mutation.
Assuntos
Anormalidades Múltiplas/genética , Heterogeneidade Genética , Linhagem Celular , Deficiências do Desenvolvimento/genética , Feminino , Transtornos do Crescimento/genética , Humanos , Células Híbridas , Recém-Nascido , Deformidades Congênitas dos Membros , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Mitomicina/farmacologia , SíndromeRESUMO
Carrier status determination for Duchenne and Becker muscular dystrophies (D/BMD), disorders caused by mutations in the dystrophin gene at Xp21, is complicated by a number of factors. These include a high mutation rate and a 5-10% recombination frequency across the dystrophin gene. For these reasons, linkage analysis frequently gives an inconclusive result, and a direct mutation detection method for females at risk is desirable. Because 65% of the mutations that cause D/BMD are deletions of one or more exons of the dystrophin gene, diagnosis in most affected males is relatively easy using multiplex polymerase chain reaction (PCR) analysis. However, deletion analysis in females is more difficult because of the interference of the normal X chromosome in the deletion assay. We have developed a quantitative PCR-based analysis designated computer-assisted laser densitometry (CALD), which uses the automated fluorescent fragment analysis application of the Applied Biosystems (Foster City, California) automated sequencer. This method has proved to be 100% accurate in retrospective blind studies analysing a total of 351 samples. Subsequent analysis of more than 800 women from more than 400 D/BMD families has shown that a highly accurate carrier risk can be given in more than 90% of cases.
Assuntos
Triagem de Portadores Genéticos/métodos , Distrofias Musculares/genética , Densitometria/métodos , Feminino , Humanos , Lasers , Masculino , Análise Numérica Assistida por Computador , Reação em Cadeia da Polimerase , Estudos RetrospectivosRESUMO
Tissue-specific variation in (CGG)n repeat size and methylation status of the FMR1 gene was investigated in 17 female premutation carriers. Minor variation in premutation repeat size among leukocyte, lymphoblast, and fibroblast tissues was noted in some subjects. One subject exhibited a premutation size allele of (CGG)64 in leukocyte and fibroblast tissues by polymerase chain reaction analysis but a normal-size allele of (CGG)46 in lymphoblast cells, suggesting low-level mosaicism in blood and clonality of the lymphoblast cell line. Six subjects exhibited differences in methylation pattern between leukocytes and lymphoblasts but not between leukocytes and fibroblasts, whereas 2 subjects showed large differences in methylation pattern between leukocytes and fibroblasts. Cognitive function was studied in 14 subjects using the Wechsler Adult Intelligence Scale-Revised. Mean Verbal and Performance IQs were well within the average range as was the mean Full Scale IQ; nevertheless, a trend toward lower Performance IQ compared with Verbal IQ was observed. No significant correlation was apparent between Full Scale IQ and (CGG)n repeat size; however, a significant positive correlation was observed between Full Scale IQ and the proportion of the active X carrying the normal FMR1 allele in fibroblasts but not in leukocytes or lymphoblasts.
Assuntos
Cognição , Metilação de DNA , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/psicologia , Heterozigoto , Testes de Inteligência , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA , Repetições de Trinucleotídeos , Adulto , Idoso , DNA/análise , DNA/sangue , Feminino , Fibroblastos , Proteína do X Frágil da Deficiência Intelectual , Humanos , Leucócitos/metabolismo , Linfócitos/metabolismo , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Escalas de WechslerRESUMO
The preliminary results of an international collaborative study examining premature menopause in fragile X carriers are presented. A total of 760 women from fragile X families was surveyed about their fragile X carrier status and their menstrual and reproductive histories. Among the subjects, 395 carried a premutation, 128 carried a full mutation, and 237 were noncarriers. Sixty-three (16%) of the premutation carriers had experienced menopause prior to the age of 40 compared with none of the full mutation carriers and one (0.4%) of the controls. Based on these preliminary data, there is a significant association between fragile X premutation carrier status and premature menopause.
Assuntos
Síndrome do Cromossomo X Frágil , Heterozigoto , Insuficiência Ovariana Primária , Adolescente , Adulto , Feminino , Humanos , Cooperação Internacional , Menopausa , Ciclo Menstrual , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
The Fragile X syndrome is a common form of X-linked mental retardation, affecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centers worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Instability of this repeat through successive generations has been demonstrated in many patients and has been associated with numerous factors, including repeat length and molecular structure of the repeat. Nine males with normal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripheral lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecular causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exhibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an interesting molecular phenomenon and raise many questions relating to the factors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.
Assuntos
Deficiências do Desenvolvimento/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA , Alelos , Southern Blotting , Criança , Análise Citogenética , Deficiências do Desenvolvimento/genética , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil/diagnóstico , Humanos , Masculino , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Repetições de TrinucleotídeosAssuntos
Acessibilidade aos Serviços de Saúde , Serviços de Assistência Domiciliar/economia , Medicare/legislação & jurisprudência , Sistema de Pagamento Prospectivo/legislação & jurisprudência , Qualidade da Assistência à Saúde , Idoso , Definição da Elegibilidade , Serviços de Assistência Domiciliar/normas , Humanos , Cobertura do Seguro , Avaliação de Programas e Projetos de Saúde , Estados UnidosRESUMO
Recent work has shown that oocytes of the starfish synthesize (8R)-hydroxyeicosatetraenoic acid and that this eicosanoid has a potent and highly specific action in induction of oocyte maturation. These striking results prompted us to examine the lipoxygenase activity of eggs of the sea urchin Strongylocentrotus purpuratus. Four hydroxyeicosanoids were formed in homogenates of sea urchin eggs; their structures and stereochemistry were characterized by high pressure liquid chromatography, UV spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as (11R)-hydroxy-5,8,12,14-ZZEZ-eicosatetraenoic acid and (12R)-hydroxy-5,8,10,14-ZZEZ-eicosatetraenoic acid (from arachidonic acid) and the corresponding (11R)- and (12R)-hydroxy analogs of eicosapentaenoic acid. The formation of these egg products was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), and their precise structures are consistent with their formation by a lipoxygenase reaction. Eicosapentaenoic acids with a prochiral tritium label in the 10-D or 10-L position were used to investigate the mechanism of biosynthesis. The formation of (12R)-hydroxyeicosapentaenoic acid proceeded with the stereoselective abstraction of the 10-D hydrogen from the substrate. This reaction was shown to be opposite to the (12S) oxygenation catalyzed by porcine leukocyte 12-lipoxygenase. These results with S. purpuratus eggs constitute the first demonstration of (11R)- or (12R)-lipoxygenase activity in any cell type or tissue.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato Lipoxigenases/metabolismo , Lipoxigenase/metabolismo , Oócitos/enzimologia , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Leucócitos/enzimologia , Ouriços-do-Mar , SuínosRESUMO
Recently, oocytes or eggs of two marine invertebrates have been found to metabolize arachidonic acid to specific monohydroxy products. These studies have prompted our examination of the oocytes of higher organisms. In the present study, oocytes of an amphibian, Xenopus laevis, were examined for their capacity to biosynthesize hydroxyeicosatetraenoic acids (HETEs) and related hydroxy fatty acids. Two hydroxyeicosanoids were formed during incubations of oocyte homogenates with [14C]arachidonic acid; their structures and stereochemistry were determined by high-pressure liquid chromatography, uv spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as 15(S)- and 12(S)-hydroxyeicosatetraenoic acids. The synthesis of the two HETEs was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), or by prior exposure of the oocyte homogenates to carbon monoxide, an inhibitor of cytochrome P450. Furthermore, 12(S)- and 15(S)-hydroperoxyeicosatetraenoic acids were isolated from brief incubations of gel-filtered ammonium sulfate fraction of frog oocyte homogenates; isolation of the hydroperoxide is further support for the existence of 12(S)- and 15(S)-lipoxygenase activities in the oocytes of X. laevis. Other polyunsaturated acids, including C18.2, C18.3, C20.3, C20.5, and C22.6 were also substrates for the lipoxygenase, and in each case the major product was formed by omega 6 oxygenation.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Lipoxigenase/metabolismo , Oócitos/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Monóxido de Carbono/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Hidroxieicosatetraenoicos/metabolismo , Indometacina/farmacologia , Oogênese , Xenopus laevisRESUMO
Acyl-acyl-carrier protein (ACP) thioesterases are, at least in part, responsible for the fatty acyl chain length composition of seed storage oils. Acyl-ACP thioesterases with specificity for each of the saturated acyl-ACP substrates from 8:0 through 16:0 have been cloned, with the exception of 18:0, and are members of the FatB class of thioesterases. The authors have determined that the tropical tree species mangosteen (Garcinia mangostana) stores 18:0 (stearate) in its seed oil in amounts of up to 56% by weight. Acyl-ACP thioesterase activity as measured in crude mangosteen seed extracts showed a preference for 18:1-ACP substrates, but had significant activity with 18:0 relative to that with 16:0-ACP, suggesting a thioesterase might be involved in the production of stearate. Three distinct acyl-ACP thioesterases were cloned from mangosteen seed cDNA; two representative of the FatA class and one representative of the FatB class. When expressed in vitro, the enzyme encoded by one of the FatAs (Garm FatA1) while preferring 18:1-ACP showed relatively low activity with 16:0-ACP as compared to 18:0-ACP, similar to the substrate preferences shown by the crude seed extract. Expression of Garm FatA1 in Brassica seeds led to the accumulation of stearate up to 22% in seed oil. These results suggest that Garm FatA1 is at least partially responsible for determining the high stearate composition of mangosteen seed oil and that FatA as well FatB thioesterases have evolved for specialized roles.
Assuntos
Frutas/enzimologia , Frutas/genética , Ácidos Esteáricos/metabolismo , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Sequência de Aminoácidos , Brassica/enzimologia , Brassica/genética , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Escherichia coli/genética , Dados de Sequência Molecular , Óleos de Plantas/química , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Sementes/química , Sementes/enzimologia , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Cholera toxin catalyzed the transfer of radioactive label from [adenine-2,8-3H2]NAD+ or ((32P]NAD+ to rat C6 glioma cell membrane and cytosolic proteins. Labeled proteins were resolved by polyacrylamide-NaDodSO4 gel or two-dimensional gel electrophoresis and stained with Coomassie blue, and the gels were subjected to fluorography or autoradiography. Autoradiograms of gels revealed labeled Mr 42000 and 46000-48000 membrane proteins that are putative subunits of the regulatory component (G/F) of the C6 cell hormone-sensitive adenylate cyclase. Cholera toxin also catalyzed the labeling of several cytosolic proteins including a Mr 54000 protein that was observed in autoradiograms of two-dimensional gels to migrate as an acidic satellite relative to Coomassie-stained C6 cell tubulin. Tubulin modified by ADP-ribosylation would undergo an acid shift relative to the stained unmodified tubulin in two-dimensional gels. The data led us to postulate that tubulin undergoes cholera toxin catalyzed ADP-ribosylation. Bovine brain tubulin prepared by three cycles of warm/cold polymerization/depolymerization was incubated with [32P]NAD+, GTP, and cholera toxin and then subjected to two-dimensional gel electrophoresis. Autoradiograms of the gels revealed the presence of [32P]ADP-ribosylated proteins that migrated as acidic satellites relative to the Coomassie-stained brain alpha and beta tubulin. Peptide maps of bovine brain tubulin and the associated [32P]ADP-ribosylated proteins showed a correspondence between the autoradiographic images and the stained peptide fragments. The data demonstrate that cholera toxin catalyzes the ADP-ribosylation of tubulin.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Toxina da Cólera/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Química Encefálica , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Glioma , Guanosina Trifosfato/farmacologia , NAD/metabolismo , RatosRESUMO
We have studied the effects of activators of the Ca++- and phospholipid-dependent enzyme protein kinase C on isometric tension development by both intact and skinned coronary artery strips. The intact strips contracted upon incubation with 12-O-tetradecanoylphorbol-13-acetate. 12-O-tetradecanoylphorbol-13-acetate produced a leftward shift in the concentration-response relationship for contraction of the tissues by K+, histamine and norepinephrine. Phorbol-12,13-dibutyrate elicited contraction of detergent-skinned artery strips when the free Ca++ concentration in the bathing media was 0.1 microM or greater. This effect was diminished greatly in the presence of polymyxin B, a putative inhibitor of protein kinase C. Phorbol-12,13-dibutyrate shifted the Ca++ concentration-tension response relationship for the skinned tissue to the left. These results are consistent with a role for protein kinase C in regulating the contractile responses of coronary arterial smooth muscle to a variety of stimuli, at least in part by increasing the sensitivity of the contractile apparatus to Ca++.
Assuntos
Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Animais , Cálcio/metabolismo , Sinergismo Farmacológico , Histamina/farmacologia , Norepinefrina/farmacologia , Dibutirato de 12,13-Forbol , Polimixina B/farmacologia , Potássio/farmacologia , Proteína Quinase C/metabolismo , Suínos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.
Assuntos
Plantas/enzimologia , Engenharia de Proteínas , Tioléster Hidrolases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Brassica/genética , Brassica/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Lauratos/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plantas/genética , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/metabolismo , Sementes/enzimologia , Sementes/genética , Especificidade por Substrato/genética , Tioléster Hidrolases/genéticaRESUMO
A new method was developed for analyzing the steric configuration of hydroxyeicosatetraenoates (HETEs) and other hydroxy fatty acids. Racemic HETE methyl esters were reacted with either benzoyl or naphthoyl chloride in pyridine and the resulting aromatic ester derivatives purified by reversed phase HPLC and subsequently chromatographed on a chiral stationary phase HPLC column [(R)-(-)-N-3,5-dinitrobenzoyl-alpha-phenylglycine)]. In contrast to the enantiomers of the underivatized HETE methyl esters which were only partially resolved, the enantiomers of their aromatic ester derivatives were completely separable on this chiral phase. Chiral HETEs can be retrieved from the aromatic derivatives by alkaline hydrolysis. Thus, this method has both analytical and preparative applications.