RESUMEN
OBJECTIVE: Epilepsy causes measurable irregularity over a range of brain signal frequencies, as well as autonomic nervous system functions that modulate heart and respiratory rate variability. Imaging dynamic neuronal signals utilizing simultaneously acquired ultra-fast 10â¯Hz magnetic resonance encephalography (MREG), direct current electroencephalography (DC-EEG), and near-infrared spectroscopy (NIRS) can provide a more comprehensive picture of human brain function. Spectral entropy (SE) is a nonlinear method to summarize signal power irregularity over measured frequencies. SE was used as a joint measure to study whether spectral signal irregularity over a range of brain signal frequencies based on synchronous multimodal brain signals could provide new insights in the neural underpinnings of epileptiform activity. METHODS: Ten patients with focal drug-resistant epilepsy (DRE) and ten healthy controls (HC) were scanned with 10â¯Hz MREG sequence in combination with EEG, NIRS (measuring oxygenated, deoxygenated, and total hemoglobin: HbO, Hb, and HbT, respectively), and cardiorespiratory signals. After pre-processing, voxelwise SEMREG was estimated from MREG data. Different neurophysiological and physiological subfrequency band signals were further estimated from MREG, DC-EEG, and NIRS: fullband (0-5â¯Hz, FB), near FB (0.08-5â¯Hz, NFB), brain pulsations in very-low (0.009-0.08â¯Hz, VLFP), respiratory (0.12-0.4â¯Hz, RFP), and cardiac (0.7-1.6â¯Hz, CFP) frequency bands. Global dynamic fluctuations in MREG and NIRS were analyzed in windows of 2â¯min with 50% overlap. RESULTS: Right thalamus, cingulate gyrus, inferior frontal gyrus, and frontal pole showed significantly higher SEMREG in DRE patients compared to HC. In DRE patients, SE of cortical Hb was significantly reduced in FB (pâ¯=â¯.045), NFB (pâ¯=â¯.017), and CFP (pâ¯=â¯.038), while both HbO and HbT were significantly reduced in RFP (pâ¯=â¯.038, pâ¯=â¯.045, respectively). Dynamic SE of HbT was reduced in DRE patients in RFP during minutes 2 to 6. Fitting to the frontal MREG and NIRS results, DRE patients showed a significant increase in SEEEG in FB in fronto-central and parieto-occipital regions, in VLFP in parieto-central region, accompanied with a significant decrease in RFP in frontal pole and parietal and occipital (O2, Oz) regions. CONCLUSION: This is the first study to show altered spectral entropy from synchronous MREG, EEG, and NIRS in DRE patients. Higher SEMREG in DRE patients in anterior cingulate gyrus together with SEEEG and SENIRS results in 0.12-0.4â¯Hz can be linked to altered parasympathetic function and respiratory pulsations in the brain. Higher SEMREG in thalamus in DRE patients is connected to disturbances in anatomical and functional connections in epilepsy. Findings suggest that spectral irregularity of both electrophysiological and hemodynamic signals are altered in specific way depending on the physiological frequency range.
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Circulación Cerebrovascular/fisiología , Epilepsia Refractaria/fisiopatología , Hemodinámica/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Neuroimagen/métodos , Adulto , Epilepsia Refractaria/diagnóstico por imagen , Electroencefalografía/métodos , Entropía , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Espectroscopía Infrarroja Corta/métodos , Adulto JovenRESUMEN
PURPOSE: Wavefront analysis has demonstrated that refractive surgery-induced corneal first surface aberrations are large, are dominated by symmetric aberrations (spherical-like aberrations), and are correlated to measures of visual performance. It is not clear whether the correlation between corneal first surface aberrations and visual performance can be generalized to other corneal conditions where large asymmetric aberrations (coma-like aberrations) may dominate the aberration structure. The purpose of the research reported here was to determine the general utility of corneal first surface wavefront analysis in predicting visual performance. METHODS: Patients were 13 normals and 78 patients with a variety of corneal conditions including surgically removed pterygia, penetrating keratoplasty, keratoconus, radial keratotomy, laser in situ keratomileusis, and others. Videokeratographs were taken for all patients and used to calculate corneal first surface wavefront variance for 3 and 7 mm pupils. Similarly, visual performance was quantified by measurements of contrast sensitivity and high and low contrast acuities through both 3 and 7 mm pupils. RESULTS: Statistically significant correlations existed between all three measures of visual performance and the corneal wavefront variance. All relationships were stronger for the 7 mm diameter-pupil condition than the 3 mm pupil. CONCLUSION: Regardless of the cause, corneas with increased wavefront variance showed a quantifiable decrease in visual performance that was pupil size dependent.
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Córnea/fisiopatología , Enfermedades de la Córnea/fisiopatología , Agudeza Visual/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sensibilidad de Contraste , Enfermedades de la Córnea/cirugía , Topografía de la Córnea , Humanos , Persona de Mediana Edad , Procedimientos Quirúrgicos Oftalmológicos , Pupila , Refracción OcularRESUMEN
Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylmethionine serves as the methyl donor. In humans, the electron is thought to be provided by methionine synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and methionine synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and methionine synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of methionine synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of methionine synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and methionine synthase.
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5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Flavodoxina/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Reactivos de Enlaces Cruzados/metabolismo , Activación Enzimática/genética , Escherichia coli/enzimología , Escherichia coli/genética , Etildimetilaminopropil Carbodiimida/metabolismo , Flavodoxina/química , Lisina/química , Lisina/genética , Lisina/metabolismo , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Succinimidas/metabolismo , Vitamina B 12/química , Vitamina B 12/metabolismoAsunto(s)
Fibrinólisis/efectos de los fármacos , Activadores Plasminogénicos/uso terapéutico , Complicaciones Posoperatorias/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéutico , Extracción de Catarata/efectos adversos , Humanos , Hipema/tratamiento farmacológico , Hipema/etiología , Inyecciones , Proteínas RecombinantesRESUMEN
The differential diagnosis of cavities in the ascending aorta includes pseudoaneurysms, intimal flaps, and abscesses. We describe the transesophageal echocardiographic and pathologic appearance of a fusiform ascending aortic aneurysm that contained atypical outpouchings that were initially confused with an intimal flap. Awareness of this unreported abnormality and its echocardiographic features will avoid the misdiagnosis of more serious aortic pathology such as acute aortic dissection or infective endocarditis.
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Aneurisma de la Aorta/diagnóstico por imagen , Ecocardiografía Transesofágica , Absceso/diagnóstico por imagen , Anciano , Disección Aórtica/diagnóstico por imagen , Aneurisma Falso/diagnóstico por imagen , Aneurisma Infectado/diagnóstico por imagen , Aneurisma de la Aorta/patología , Arteriosclerosis/patología , Calcinosis/patología , Diagnóstico Diferencial , Ecocardiografía Doppler en Color , Ecocardiografía Doppler de Pulso , Endocarditis Bacteriana/diagnóstico por imagen , Femenino , Humanos , Trombosis/patología , Túnica Íntima/diagnóstico por imagenAsunto(s)
Enfermedades de la Retina/inducido químicamente , Tioridazina/efectos adversos , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Persona de Mediana Edad , Retinitis Pigmentosa/inducido químicamente , Esquizofrenia Paranoide/tratamiento farmacológico , Tioridazina/administración & dosificación , Trastornos de la Visión/inducido químicamenteRESUMEN
Tissue plasminogen activator (tPA) has been used to treat severe postcataract and vitrectomy fibrinous membranes, but intraocular bleeding has occurred with doses of 25 micrograms or higher. We report three patients, one with nonclearing total hyphema and uncontrollable intraocular pressure and two with severe fibrinous membrane formation, who had treatment with low-dose (4 micrograms to 6 micrograms) intraocular tPA. Although the fibrinous membranes or hyphema resolved in all three patients, they recurred and bleeding that required additional treatment occurred in one patient. Intraocular low-dose tPA may minimize the risk of corneal and retinal toxicity and may be considered an alternative treatment in intractable cases. However, secondary intraocular hemorrhage can occur, and the timing between the initial vascular injury, treatment with tPA, and subsequent bleeding may reduce the risk of further hemorrhaging.
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Extracción de Catarata/efectos adversos , Lesiones de la Cornea , Oftalmopatías/terapia , Lesiones Oculares Penetrantes/complicaciones , Hipema/terapia , Queratoplastia Penetrante/efectos adversos , Activador de Tejido Plasminógeno/uso terapéutico , Adulto , Anciano , Cámara Anterior , Niño , Córnea/patología , Oftalmopatías/etiología , Oftalmopatías/patología , Lesiones Oculares Penetrantes/patología , Fibrina , Humanos , Hipema/etiología , Hipema/patología , Inyecciones , Masculino , Complicaciones Posoperatorias/terapia , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Activador de Tejido Plasminógeno/administración & dosificaciónRESUMEN
The principles for management of acute ocular trauma are also applicable to the subsequent reconstruction of the anterior segment. As with the primary repair of ocular trauma, meticulous anatomical restoration during reconstructive surgery minimizes secondary complications and enhances the visual prognosis. Anterior segment reconstruction may then involve procedures such as stripping of corneal pannus, removal of lens and vitreous remnants, iris and angle reconstruction, intraocular lens implantation, and penetrating keratoplasty. A total of 39 consecutive cases of severe ocular trauma, which had undergone penetrating keratoplasty and anterior segment reconstruction, were evaluated for visual outcome, graft survival, and secondary complications. Post-operatively, 49% of eyes achieved > 20/100 as compared with 10% before surgery, and 72% improved by at least two Snellen lines. In all, 31 (80%) initial keratoplasties remained clear, as did all 4 subsequently regrafted corneas, for an overall keratoplasty success rate of 90%. Elevated intraocular pressure occurred postoperatively in 18 eyes (46%), and among these, 10 of 13 eyes (77%) had preoperative glaucoma, whereas 8 of 26 (31%) did not (P < 0.015). Peripheral anterior synechiae could be anatomically corrected at surgery in 80% of cases (24 of 30 eyes). Thus, despite major trauma and a high prevalence of glaucoma, both the visual and the anatomical improvements were highly satisfactory and without severe complications.
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Segmento Anterior del Ojo/cirugía , Lesiones Oculares Penetrantes/cirugía , Queratoplastia Penetrante , Adolescente , Adulto , Anciano , Segmento Anterior del Ojo/lesiones , Niño , Preescolar , Lesiones Oculares Penetrantes/etiología , Femenino , Supervivencia de Injerto , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Estudios Retrospectivos , Esclerótica/lesiones , Agudeza VisualRESUMEN
Apolipoprotein J (apoJ), a glycoprotein associated with subclasses of plasma high density lipoproteins (HDL), was found to accumulate in aortic lesions in a human subject with transplantation-associated arteriosclerosis and in mice fed a high-fat atherogenic diet. Foam cells present in mouse aortic valve lesions expressed apoJ mRNA, suggesting local synthesis contributes to apoJ's localization in atherosclerotic plaque. As a prerequisite for elucidating the physiological function of apoJ by using a mouse model, cDNA clones representing the mouse homolog of apoJ were isolated, characterized, and sequenced. The nucleotide sequence predicts a 448 amino acid, 50,260 dalton protein. There was 81% nucleotide sequence similarity between mouse and human apoJ, and 75% similarity at the amino acid level. Mouse apoJ contains six potential N-glycosylation sites, a potential Arg-Ser cleavage site to generate alpha and beta subunits, a cluster of five cysteine residues in each subunit, three putative amphipathic helices, and four potential heparin-binding domains. Southern blot analysis indicates that the gene encompasses approximately 23 kb of DNA. Recombinant inbred strains were used to map apoJ to mouse chromosome 14, tightly linked to Mtv-11. All of the transcribed portions of the gene were cloned and analyzed, and all intron-exon boundaries were defined. The first of the 9 exons is untranslated. Single exons encode the signal peptide, the cysteine-rich domain in the alpha subunit, two potential amphipathic helices flanking a heparin-binding consensus sequence, and a potential amphipathic helix overlapping a heparin-binding domain, supporting their potential functional significance in apoJ. A variety of mouse tissues constitutively express a 1.9 kb apoJ mRNA, with apparently identical transcriptional start sites utilized in all tissues tested. ApoJ mRNA was most abundant in stomach, liver, brain, and testis, with intermediate levels in heart, ovary, and kidney. The high degree of similarity between mouse and human apoJ, in structure and distribution of the gene product, gene structure, and deposition in atherosclerotic plaques, suggests that the mouse is an ideal model with which to elucidate the role of apoJ in HDL metabolism and atherogenesis.
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Arteriosclerosis/genética , Glicoproteínas/genética , Chaperonas Moleculares , Secuencia de Aminoácidos , Animales , Arteriosclerosis/diagnóstico , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , Clusterina , Vasos Coronarios/patología , ADN Complementario/análisis , Modelos Animales de Enfermedad , Marcadores Genéticos , Genoma , Glicoproteínas/análisis , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Datos de Secuencia Molecular , ARN Mensajero/análisisRESUMEN
Apolipoprotein J (apoJ)-containing high-density lipoproteins (HDL), isolated from human plasma by immunoaffinity chromatography, are associated with apoAI and a protein of approximately 44 kDa. In order to advance our understanding of apoJ's role in the vasculature, a comprehensive investigation was performed to identify and characterize this 44-kDa protein and to study its interaction with apoJ. The 44-kDa protein, a monomeric glycoyslated polypeptide, was identified by N-terminal sequencing as serum paraoxonase. Paraoxonase exists in two oxidation states: one contains all free cysteines while the other has one disulfide bond between Cys42 and Cys284. Northern analysis of eight human tissues shows paraoxonase message present only in the liver. The majority of apoJ/paraoxonase-HDL are 90-140 kDa; however, not all of the plasma paraoxonase is associated with apoJ. The specificity of the apoJ/paraoxonase interaction, inferred by the constant mole ratio of the two proteins in affinity-purified apoJ-HDL, is confirmed in direct binding assays. For purified proteins, there is more than a 5-fold increase in the apparent affinity of apoJ for immobilized paraoxonase as the paraoxonase coating concentration is increased from 0.5 to 2.0 micrograms/mL. Both oxidation states of paraoxonase bind to apoJ with equal affinity. Our data combined with other evidence suggest that the plasma link of apoJ with paraoxonase will be implicated as a predictor of vascular damage.
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Esterasas/sangre , Glicoproteínas/sangre , Chaperonas Moleculares , Secuencia de Aminoácidos , Arildialquilfosfatasa , Clusterina , ADN Complementario/genética , Disulfuros/química , Esterasas/química , Esterasas/genética , Esterasas/metabolismo , Glicoproteínas/metabolismo , Humanos , Isoenzimas , Hígado/enzimología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Conformación Proteica , ARN Mensajero/análisis , Análisis de SecuenciaRESUMEN
The authors reviewed 39 consecutive cases of severe ocular trauma that had undergone penetrating keratoplasty plus anterior segment reconstruction and evaluated visual outcome, graft survival, and secondary complications. Postoperatively, 49% of eyes achieved visual acuity of 20/100 or better compared with 10% before surgery, and 72% improved at least 2 Snellen lines. With a mean follow-up of 23 months, 31 (80%) initial keratoplasties remained clear, and all 4 subsequently re-grafted corneas maintained clarity, for an overall keratoplasty success rate of 90%. Elevated intraocular pressure occurred postoperatively in 18 eyes (46%), and, among these, 10 of 13 eyes (77%) had preoperative glaucoma, whereas 8 of 26 (31%) did not (P less than or equal to 0.015). Preoperative anterior synechiae could be anatomically corrected at surgery in 80% of cases (24 of 30 eyes). In 7 cases with persistent synechiae, 6 (86%) were associated with glaucoma (P less than or equal to 0.025). Retinal detachment occurred in one case. Thus, despite major trauma and a high prevalence of glaucoma, both the visual restoration and anatomic reconstruction achieved were satisfactory and without severe complications.
Asunto(s)
Segmento Anterior del Ojo/cirugía , Lesiones Oculares Penetrantes/cirugía , Queratoplastia Penetrante , Adolescente , Adulto , Anciano , Segmento Anterior del Ojo/lesiones , Niño , Preescolar , Lesiones de la Cornea , Femenino , Estudios de Seguimiento , Supervivencia de Injerto , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Pronóstico , Estudios Retrospectivos , Esclerótica/lesiones , Agudeza VisualRESUMEN
Two hundred twenty in-use medications from 101 patients with nonmicrobial ocular surface disease were studied by culturing the bottle caps, a drop produced by simple inversion, and the interior contents removed sterilely. Conjunctival cultures were taken from these patients and 50 age-matched controls. Pathogenic organisms were harvested from conjunctivae significantly more frequently (P less than .01) from cases (34 of 101) than from controls (five of 50). Sixty-four medications (29%) had microorganisms cultured from at least one medication site. Gram-negative organisms were significantly more likely (P less than .00001) to be isolated from all medication sites than gram-positive organisms. Additionally, when isolated from medication sites, the gram-negative organisms were highly likely to be cultured from the conjunctiva as well. This was not true for pathogenic gram-positive organisms. We conclude that a cycle of contamination between in-use medications and conjunctivae may represent an important risk factor for microbial keratitis in patients with ocular surface disease.
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Contaminación de Medicamentos , Oftalmopatías/microbiología , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/aislamiento & purificación , Soluciones Oftálmicas , Conjuntiva/microbiología , Infecciones del Ojo/microbiología , Femenino , Hongos/aislamiento & purificación , Humanos , Masculino , Técnicas Microbiológicas , Persona de Mediana Edad , PomadasRESUMEN
Lattice corneal dystrophy associated with familial systemic amyloidosis (Meretoja syndrome) has rarely been described other than in patients of Finnish origin. The authors report two North American patients with this disease who manifest blepharochalasis, lattice corneal dystrophy, open-angle glaucoma, and cranial neuropathy. In one patient, a corneal intraepithelial and subepithelial pseudodendrite was managed by superficial keratectomy, and this same patient benefited from surgical brow suspension for facial muscular weakness. In the second patient, penetrating keratoplasty was complicated by a neurotrophic persistent epithelial defect. Corneal tissue from both superficial keratectomy and penetrating keratoplasty exhibited ultrastructurally characteristic amyloid filaments and associated elastoid material. Transmission electron microscopy of conjunctiva and skin biopsies similarly revealed amyloid deposits associated with most basement membranes, the perineurium and endoneurium of most peripheral nerves, and the intima and media of arteries. By immunoperoxidase staining, the corneal amyloid deposits were positive for the amyloid P-component protein but negative for the nonimmunoglobulin amyloid A protein and prealbumin. Serum prealbumin and amyloid A related protein were normal.
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Amiloidosis/patología , Distrofias Hereditarias de la Córnea/patología , Enfermedades de los Nervios Craneales/patología , Anciano , Amiloidosis/genética , Enfermedades de los Nervios Craneales/genética , Femenino , Humanos , Queratoplastia Penetrante , Enfermedades de la Piel/genética , Enfermedades de la Piel/patología , SíndromeRESUMEN
The surgical technique and postoperative problem management of conjunctival autograft transplantation for advanced primary and recurrent pterygium are reviewed. Problems such as graft edema, corneoscleral dellen, and epithelial inclusion cysts infrequently occur. Corneal astigmatism, Tenon's granuloma, retraction and/or necrosis of the graft, and muscular disinsertion are even less frequently encountered. Limbal-conjunctival autograft for recalcitrant recurrent cases is proposed.
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Conjuntiva/trasplante , Complicaciones Posoperatorias/terapia , Pterigion/cirugía , Astigmatismo/etiología , Enfermedades de la Conjuntiva/etiología , Enfermedades de la Córnea/etiología , Quistes/etiología , Edema/etiología , Granuloma/etiología , Hematoma/etiología , Humanos , Necrosis/etiología , Recurrencia , Trasplante AutólogoRESUMEN
Mucolipidosis IV is a lysosomal storage disease characterized by prominent involvement of the corneal epithelium. A 5-year-old boy with mucolipidosis IV experienced recurrent episodes of severe ocular pain, tearing, and ipsilateral facial flushing. This was suggestive of reflex sympathetic dystrophy, a syndrome of pain and sympathetic hyperactivity. The examination revealed marked corneal surface irregularities, corresponding to massive accumulations of intracytoplasmic storage material in the epithelium. Episodic pain in patients with mucolipidosis IV is an important symptom, presumably reflecting the distinctive corneal ultrastructural abnormality in this disease.
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Córnea/patología , Mucolipidosis/patología , Dolor/etiología , Extracción de Catarata , Preescolar , Conjuntiva/patología , Epitelio/patología , Humanos , Masculino , Lágrimas/metabolismoRESUMEN
Pseudomonas mevalonii (formerly designated Pseudomonas sp. M (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001; Gill, J. F., Jr., Beach, M.J., and Rodwell, V. W. (1985) J. Biol. Chem. 260, 9393-9398] 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), overexpressed in Escherichia coli (1), has been purified to electrophoretic homogeneity in 75% yield (final specific activity 48 mumols of NAD+ reduced per min/mg protein). The enzyme catalyzes its normal catabolic reaction (mevalonate + 2 NAD+ + CoASH----HMG-CoA + 2NADH + 2H+), and two half-reactions which involve mevaldehyde, the postulated intermediate in the aforementioned reactions and mevaldehyde + NADH + H+----mevalonate + NAD+). The rates of all four reactions and the Michaelis constants for all substrates were measured. Coenzyme A decreased the KM for mevaldehyde reduction 12-fold and stimulated VMAX 2-3 fold. CoASH thus may remain bound throughout the catalytic cycle. Dithiothreitol and analogs of CoASH were tested for their ability to reproduce the CoASH stimulation. Pantetheine, but not dithiothreitol, pantothenate, or desulfo-CoA mimicked CoASH stimulation. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) indicated two sulfhydryl groups per subunit. Both groups remained accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of mevalonate and/or NAD+ but only one group in the presence of HMG-CoA. N-Ethylmaleimide inhibited all the aforementioned reactions. HMG-CoA, but not mevalonate, afforded protection completely and irreversibly inactivated the enzyme. The reactive sulfhydryl group thus may not be a catalytic residue, but may be involved in a conformational change.
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Hidroximetilglutaril-CoA Reductasas/metabolismo , Pseudomonas/enzimología , Cromatografía por Intercambio Iónico , Clonación Molecular , Ácido Ditionitrobenzoico/farmacología , Escherichia coli/genética , Etilmaleimida/farmacología , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Cinética , Plásmidos , Pseudomonas/genéticaRESUMEN
Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001). Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T. C., and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17913-17918). We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues. Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active. Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents. Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate. The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase. Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate). By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme. Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme. Neither mutant C156A nor C156/296A was affected by this reagent. We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156. However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine. Possible roles of cysteine in maintaining structural stability are discussed.