The peroxisomal protein import machinery--a case report of transient ubiquitination with a new flavor.

The peroxisomal protein import machinery displays remarkable properties. Be it its capacity to accept already folded proteins as substrates, its complex architecture or its energetics, almost every aspect of this machinery seems unique. The list of unusual properties is still growing as shown by the recent finding that one of its central components, Pex5p, is transiently monoubiquitinated at a cysteine residue. However, the data gathered in recent years also suggest that the peroxisomal import machinery is not that exclusive and similarities with p97/Cdc48-mediated processes and with multisubunit RING-E3 ligases are starting to emerge. Here, we discuss these data trying to distill the principles by which this complex machinery operates.

Complementary DNA sequences of the 24 kDa and 21 kDa subunits of complex I from Neurospora.

We have cloned and sequenced cDNAs coding for two subunits of the peripheral arm of Neurospora crassa complex I. The two polypeptides are synthesized as precursor proteins which are processed to mature forms with predicted molecular masses of 24331 and 20982 Da.

Identification of a 24 kDa intrinsic membrane protein from mammalian peroxisomes.

A 24 kDa protein from rat liver peroxisomal membrane was isolated and subjected to Edman degradation. Using the N-terminal sequence of this polypeptide we have identified several rat and human expressed sequence tags in the GenBank Database. The complete sequence of a human cDNA clone was determined. The open reading frame encodes an extremely basic protein 212 amino acid residues long. A high similarity between this mammalian protein and hypothetical proteins from Caenorhabditis elegans and Neurospora crassa was found. Hydropathy analysis reveals the existence of two putative membrane-spanning domains in conserved regions of the three homologous proteins.

Primary structures of two subunits of NADH: ubiquinone reductase from Neurospora crassa concerned with NADH-oxidation. Relationship to a soluble NAD-reducing hydrogenase of Alcaligenes eutrophus.

The primary structures of the nuclear-encoded 51 kDa and 78 kDa subunits of the respiratory chain NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria were determined by sequencing cDNA and the N-terminus of the mature proteins. Both subunits are related to the soluble NAD-reducing hydrogenase of the bacterium Alcaligenes eutrophus. Sequence comparison between these subunits, the corresponding subunits of the bovine complex I and the bacterial NAD-reducing hydrogenase further confirms the binding sites of NAD(H), FMN and three iron-sulfur clusters.

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation.

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.

Primary structure and in vitro expression of the N. crassa phosphoglycerate kinase.

The primary structure of 3-phosphoglycerate kinase (PGK) from Neurospora crassa was determined by sequencing a full-length cDNA. The deduced 418 amino acids protein shows a considerable identity to PGKs of other organisms with all the residues thought to be important for the function of the yeast enzyme conserved. The cloned PGK cDNA could be efficiently expressed in vitro resulting in a product with the expected molecular weight.

[Characterization of Doppler flow of the left atrial appendage and its relation to thrombi. A transesophageal echocardiographic study]. / Caracterização do fluxo Doppler do apêndice auricular esquerdo e sua relação com trombos. Estudo ecocardiográfico transesofágico.

OBJECTIVE: The characteristics and clinical implications associated with Left Atrial Appendage (LAA) flow have not been clearly analyzed. The purpose of this transesophageal pulsed doppler study was to evaluate the importance of rhythm and echo doppler parameters on LAA thrombus formation. DESIGN: Prospective study. SETTING: Patients in a follow-up transesophageal echocardiographic study at Gregorio Maranon General Hospital, Madrid. PATIENTS: Through a transesophageal echocardiographic color Doppler prospective study we evaluated 44 consecutive patients. INTERVENTIONS: In each patient we measured presence of atrial fibrillation, presence of LAA thrombi and LAA doppler flow pattern parameters (peak velocity and area enclosed by the pulsed doppler filling and emptying waves). MEASUREMENTS: We identified three types of LAA doppler flow: Type I, clearly defining biphasic flow, was present only in sinus rhythm patients and not associated to LAA thrombus; Type II saw-fish active flow pattern was detected only in atrial fibrillation patients not associated also with LAA thrombus. Type III low flow pattern occurred only in atrial fibrillation patients and is associated to the majority of LAA thrombus. CONCLUSIONS: These findings document the importance of LAA doppler factors when transesophageal echocardiography is used to study LAA thrombus formation.

[Echocardiographic quantification of mitral regurgitation. Comparative study with transthoracic and transesophageal Doppler color echocardiography]. / Quantificação ecocardiográfica da regurgitação mitral. Estudo comparativo Doppler codificado a cor transtorácico e transesofágico.

The quantification of mitral regurgitation by means of colour coded Doppler echocardiography still is a controversial issue. A prospective study on 86 consecutive patients with mitral regurgitation diagnosis was carried out by means of conventional transthoracic colour coded Doppler echocardiography in order to make a comparative study with results obtained by means of transoesophageal colour coded Doppler echocardiography. A systematic measurement of the following parameters was carried out in each patient: Colour coded area or mitral regurgitation jet, auricular depth of mitral regurgitation jet, severity degree of mitral regurgitation, left auricular area, mitral regurgitation ratio and transthoracic crossection plan of largest regurgitation area. Both echocardiographic techniques had the first three parameters in common. The mitral regurgitation area by transthoracic echocardiography was 370 +/- 41 mm2 and by transoesophagial technique 558 +/- 43 mm2 (p less than or equal to 0.001 - r = 0.71). The depth of mitral regurgitation by transthoracic path was 31 +/- 2.7 mm and by transoesophageal path 37 +/- 25 mm (p less than or equal to 0.01 - r = 0.74). The existing correlation between the mitral regurgitation area obtained by both techniques improved (r = 0.84) when a four camera apical transthoracic plan was used and worsened (r = 0.45) when the longitudinal parasternal path was used. We came to the conclusion that the valorization of mitral regurgitation made by both techniques is not accurately comparable, existing undervaluation in transthoracic echocardiography. The discrepancies between the two techniques are more patent and statistically more significant when longitudinal parasternal plan is used for transthoracic quantification of severity degree of mitral regurgitation.(ABSTRACT TRUNCATED AT 400 WORDS)

[Assessment of the degree of severity of mitral regurgitation by the systolic inversion of the pulmonary venous flow. Transesophageal echocardiography study]. / Quantificação do grau de gravidade da regurgitação mitral através de inversão sistólica do fluxo venoso pulmonar. Estudo ecocardiográfico transesofágico.

It is controversial the value of an echocardiographic mitral regurgitation evaluation based on planimetric patterns color codification area. We made this study using as alternative methods of quantification, the parameter of pulmonary venous flow, concretly maximum velocity and area of pulmonary systolic inversion venous flow. Considered the values of this related parameters to 3 crescent degrees on mitral regurgitation severity and obtained the following results: Lightness mitral insufficiency with maximum inversion area, 10 +/- 21 mm2 and maximum inversion velocity, 3.2 +/- 6 cm/seg. Moderate mitral insufficiency with maximum inversion area, 171 +/- 140 mm2 and maximum moderate velocity, 21 +/- 13 cm/seg. Several mitral insufficiency with maximum inversion area, 648 +/- 615 and maximum inversion velocity, 50 +/- 20 cm/seg. We conclude that systolic parameters of pulmonary venous flow evaluated by transesophagic echocardiography constitute considerable sensibility rules in the evaluation of severity degree in echocardiographic mitral regurgitation.

[Regurgitation jets in mitral prosthesis studied using transesophageal echocardiography]. / Estudos dos fluxos de regurgitação de próteses mitrais obtidos por ecocardiografia transesofágica.

STUDY OBJECTIVE: To analyze the regurgitant jets of mitral valve prostheses studied by transesophageal echocardiography. DESIGN: Prospective study of patients with mitral prostheses with or without suspicion of dysfunction. SETTING: Patients referred to the Echocardiology Department of the Instituto de Cardiologia in Madrid, some as outpatients. PATIENTS AND INTERVENTIONS: Twenty five patients with mitral valve prostheses (5 biological, 7 St Jude, 12 Bjork Shiley and 1 Hall Kaster). Ten male patients and fifteen female, mean age 50 years. Five also had aortic valve prostheses. In only 5 there was a suspicion of dysfunction. The transthoracic echos were done with phased array transducers and the transthoracic with 2.5 (18 patients) or 5 MHz (7 patients) transducers coupled to a commercial echocardiographer. The presence of regurgitant jets was looked for, as well as their origin, maximal area and length, their type and degree of turbulence. MAIN RESULTS: Only 3 mitral regurgitations were noticed by transthoracic echocardiography. All the mechanical prostheses and 80% of the biological ones had regurgitant jets by transesophageal echocardiography. The tilting disc prostheses had two physiological jets, the St. Jude 2, 3, or exceptionally only one central jet. The biological prostheses had one central regurgitant jet, that was smaller than those from the mechanical prostheses (p less than 0.05). There were ten malfunctioning prostheses (5 Bjork Shiley, 3 St Jude and 2 biological). The diagnosis of dysfunction was based on the abnormal origin of the jet (9 cases) or just simply on the characteristics of the regurgitation (turbulence). Four patients were submitted to surgery, all with dysfunctioning prostheses. CONCLUSIONS: Transthoracic echocardiography has a low sensitivity for detecting the regurgitant jets of mitral prostheses. All mechanical prostheses and 80% of biological ones have regurgitation by transesophageal echocardiography. The area and length of the jets are not so different between normal and malfunctioning prostheses in order to separate them. The abnormal jets are recognised because of their abnormal origin and/or their characteristics, with formation of mosaic in color coded Doppler echocardiography.

[Transesophageal Doppler echocardiography in the evaluation of cardiac pathology]. / A ecocardiografia Doppler transesofágica na avaliação da patologia cardíaca.

Transesophageal echocardiography is one of the most important innovations that have lately turned up for the evaluation of the cardiac patient. This technical approach consists not only of the study of the two dimensional image but of the cardiac flows obtained with pulsed and color Doppler as well, using a gastroscopic probe to which an ultrasonic transducer has been adapted. In this paper we make a revision of the methodology used in our laboratory. We discuss the utility of this technical approach not only for the study of the ambulatory patient but also for the critically ill patient.

[Meaning of mosaic color area in mitral regurgitation. Echocardiographic transesophageal study]. / Significado da área de mosaico cor na regurgitação mitral. Estudo ecocardiográfico transesofágico.

OBJECTIVE: The purpose of our study was to analyse the meaning of total and mosaic color Doppler area of the mitral regurgitation jet, in terms of the degree of mitral regurgitation severity. PATIENTS: In and out patients referred to the Echocardiographic Laboratory of Gregorio Marañon General Hospital, Madrid. SETTING: Transesophageal echocardiographic prospective study MATERIAL AND METHODS: By pulsed and color Doppler transesophageal approach we studied 94 consecutive patients with mitral regurgitation diagnosis. We divided the entire population in three groups according to the degree of transthoracic mitral regurgitation severity and mitral regurgitation color area index (Groups I, II and III). In each patient we systematically measured the regurgitant maximal area (AT) and of the aliasing color area, as well as maximal peak velocity (VIS) and area (AIS) of the reversed pulmonary venous pulsed Doppler flow obtained at the level of the left upper pulmonary vein. RESULTS: For the group I, color Doppler AT was 411 +/- 315 mm2 and AN was 204 +/- 123 mm2 (R = 0.25), pulmonary venous pulsed Doppler VIS was 4 +/- 8 cm/sec (R = NS for AT and 0.79 for AN) and AIS was 9 +/- 6 mm2 (R = NS for AT and 0.82 for AN). In the group II, color Doppler AT was 802 +/- 447 mm2, AN was 671 +/- 307 mm2 (R = 0.42). the pulmonary venous pulsed Doppler VIS was 22 +/- 12 cm/sec (R = NS for AT and 0.66 for AN). In the group III we obtained an AT value of 1174 +/- 462 mm2 and an AN value of 1092 +/- 417 mm2 (R = 0.62). In this group the pulmonary venous pulsed Doppler VIS was 50 +/- 13 cm/sec (R = 0.57 for AT and 0.76 for AN) and the correspondent AIS was 671 +/- 570 mm2 (R = 0.38 for AT and 0.91 for AN). CONCLUSIONS: Mosaic transesophageal echocardiographic color Doppler area of mitral regurgitant jets has a direct relationship with the reversal criteria of pulsed Doppler pulmonary venous flow. This relationship is greater than the total color Doppler area of the same regurgitant jet. The mosaic color Doppler area of mitral regurgitant jets is a more correct estimation of the systolic variation of left atrial pressure, when compared with the total color area of mitral regurgitation.

[Transesophageal echocardiography study with dynamic spontaneous contrast of the left atrial appendage, its relationship with the anatomy and thrombus formation]. / Estudo ecocardiográfico transesofágico do contraste espontâneo dinâmico no apêndice auricular esquerdo, sua relação com a anatomia e formação de trombos.

OBJECTIVE: Transesophageal two-dimensional echocardiographic study of anatomical characteristics of the left atrial appendage and its relation to spontaneous dynamic echocardiographic contrast. DESIGN: Outpatients undergoing a prospective two-dimensional transesophageal echocardiographic study. SETTING: Consecutive outpatients studied at the Echocardiographic Laboratory of Gregorio Marañon General Hospital, Madrid. MATERIAL AND METHODS: In each patient at the level of the left atrial appendage we calculated the following transesophageal echocardiographic parameters: end-systolic and end-diastolic maximal longitudinal and transversal diameters, total systolic and diastolic areas, percentage of systolic fractional shortening, presence of left atrial appendage thrombus and spontaneous dynamic echo-contrast. MAIN RESULTS: Left atrial appendage spontaneous dynamic contrast was observed in 48% of the total population. In the group of patients with left atrial spontaneous echo-contrast we observed larger longitudinal systolic (44 +/- 14 mm vs 28 +/- 13 mm, p = 0.01) and diastolic (52 +/- 16 mm vs 38 +/- 12 mm, p = 0.005) diameters, larger transversal systolic (25 +/- 10 mm vs 19 +/- 6 mm, p = 0.03) and diastolic (28 +/- 8 mm vs 25 +/- 9 mm, p = NS) diameters and also larger systolic (601 +/- 204 mm2 vs 337 +/- 110 mm2, p < 0.0001) and diastolic (715 +/- 230 mm2 vs 507 +/- 184 mm2, p = 0.001) areas, compared to the group without this dynamic echocardiographic phenomena. Left atrial appendage percentage of fractional shortening was considerably reduced in patients with spontaneous dynamic echo-contrast (15 +/- 14% vs 39 +/- 18%, p = 0.001) and related to local thrombus formation (13% vs 1%, p < 0.001). CONCLUSIONS: Left atrial spontaneous dynamic echo-contrast is more common in patients with enlarged left atrial appendage systo-diastolic diameters and areas. In this group of patients the presence of left atrial spontaneous echo-contrast is related to a significant reduction in left atrial appendage contractile function and thrombus formation. Parameter analysis of left atrial appendage anatomy by two-dimensional transesophageal echocardiography may have clinical relevance in the assessment of patients with high risk for left atria thromboembolic phenomena.

[Transesophageal echocardiography study of mitral valve prosthesis]. / Estudo ecocardiográfico transesofágico de próteses valvulares em posição mitral.

OBJECTIVE: Transesophageal (TEE) and Transthoracic (TTE) comparative echocardiographic analysis of patients with mitral valve prosthesis. DESIGN: Prospective study. SETTING: In hospital and out patients with mitral prosthesis in a follow-up study by the Cardiology Department and referred to the echocardiographic laboratory of Gregorio Marañon General Hospital, Madrid. PATIENTS: We studied 90 consecutive patients with mitral prosthesis diagnosis. INTERVENTIONS: Comparative and prospective echocardiographic study of transthoracic and transesophageal techniques in mitral prosthesis pathology. RESULTS: TTE diagnosed 18% of dysfunctioning mitral prosthesis patients compared to 56% of cases identified by TEE. TTE observed 1% of mitral prosthesis vegetation compared to 8.8% by TEE. TEE diagnosed left atrial thrombosis in 2.2% and TEE in 10%. Left atrial spontaneous dynamic contrast was identified exclusively by TEE in 55% of all cases. Paravalvular mitral prosthesis leak was correctly identified exclusively by TEE in 20% of cases. CONCLUSIONS: TEE has a greater diagnostic capacity compared to TTE in mitral prosthesis patients. This technique can give a greater security in evaluating mitral prosthesis thrombi, vegetations and leak, establishing a more precise diagnosis of mitral prosthesis dysfunction. The authors concluded that TEE is the technique of choice in noninvasive evaluation of patients admitted with the suspicion of mitral prosthesis dysfunction.

[Changes in the physiologic pattern of mitral prosthesis regurgitation in the presence of a prosthetic dysfunction]. / Alteração do padrão fisiológico de regurgitação protésica mitral em presença de disfunção.

AIM OF STUDY: Evaluation by transesophageal echocardiography of the effect on the characteristics of physiological regurgitant jets (JF) resulting from prosthetic disfunction due to pathologic regurgitation (JF). PATIENTS AND METHODS: We studied 69 consecutive patients with the diagnosis of prosthesis in mitral position using transesophageal echocardiography and color doppler codification. The patients were divided in two groups (N and D groups) according to the presence of prosthesis disfunction by pathologic regurgitation. In each patient we determined planimetric areas and atrial peak depth of each JF and also the sum of JF planimetric areas of each mitral prosthesis. When pathological regurgitation was present we calculated the highest planimetric area, severity degree and atrial peak depth in each JP. RESULTS: The planimetric area in each JF of group N was 330 +/- 167 mm2 and in group D 117 +/- 116 mm2 (p less than 0.001). The sum of the areas of JF in group N was 474 +/- 204 mm2 and in group D 254 +/- 176 mm2 (p less than 0.01). The atrial depth of JF in group was 32 +/- 15 mm and in group D 26 +/- 18 mm (p less than 0.01). In group D 29% of the patients had mild pathological regurgitation, 10% moderate and 61% severe. The maximum planimetric area of JP in group D was 1078 +/- 1007 mm2 with atrial depth of 37 +/- 28 mm. CONCLUSION: The pathological regurgitation in disfunction prosthesis in mitral position has a significant reduction effect in the dimension of prosthesis physiologic regurgitation jets. This transesophageal echocardiographic observation makes it possible to characterize and clarify more precisely the different types of mitral prosthesis jets.

[Acute hemodynamic effects of ibopamine, captopril, and placebo in patients with severe heart failure]. / Efeitos hemodinâmicos agudos da ibopamina, captopril e placebo em pacientes com insuficiência cardíaca grave.

PURPOSE: To evaluate the acute hemodynamic effects of ibopamine (IBO), captopril (CAP) and placebo (PLA) in patients with severe congestive heart failure at rest. METHODS: Twelve male patients in sinus rhythm with dilated cardiomyopathy and NYHA class IV were studied with Swan-Ganz hemodynamics. Drugs were given in a blinded fashion. Rest, 30 min and every hour for 5 h measurements were made after oral ingestion of 100 mg IBO, 25 mg CAP or PLA. Prior to the study, patients were on diuretics as the only medication for at least 48 h. Comparisons were made with analysis of variance of repeated measurements and Duncan's multiple comparisons procedure. RESULTS: Significant increase in cardiac index and stroke volume index and reduction in systemic vascular resistance were observed with IBO and CAP for 2 h after ingestion. IBO however increased right and left filling pressures in the first hour after its administration. Ventricular tachycardia occurred in 2 patients 1 h after IBO administration. CONCLUSION: Both IBO and CAP improved hemodynamic parameters in the first two hours after oral ingestion in patients with dilated cardiomyopathy in class IV.

[Comparative study of transthoracic Doppler echocardiogram, computed tomography and aortography in the diagnosis of thoracic aortic dissection]. / Estudo comparativo do ecocardiograma Dopller transtorácico, tomografia computadorizada e aortografia no diagnóstico de dissecção da aorta torácica.

PURPOSE: Comparative and prospective evaluation of three methods (transthoracic echo-Doppler (TTE), computerized tomography (CT) and aortography (AORT) utilized for aortic dissection diagnosis. METHODS: The 39 patients with confirmed aortic dissection (surgery or autopsy) underwent, within a few hours of each other, all three methods scrutinized. There were 19 cases of type A and 20 of type B dissection. RESULTS: In type A dissection the methods were equivalent (TTE = 73.7%, CT = 84.2%, AORT = 73.7% p = NS) but for type B, TTE was significantly inferior to the other two methods (TTE = 60%, TC 90%, AORT = 80%, p < 0.05 for TTE, for TC and AORT p = NS). In three occasions, even though all three methods were performed, the diagnosis was not obtained. CONCLUSION: The methods which were evaluated make the diagnosis in the majority of cases. In type A all methods are similar, however, in type B, TC and AORT are superior to TTE. Even performing all three methods in each patient, in three instances the diagnosis was not made.

Characterization of a membrane fragment of respiratory chain complex I from Neurospora crassa. Insights on the topology of the ubiquinone-binding site.

1. A membrane fragment of complex I from the fungus Neurospora crassa was isolated by immunoprecipitation from alkaline-extracted mitochondrial membranes. 2. Analysis of the polypeptide composition of this hydrophobic domain of complex I has brought insights on the topology of two subunits of the enzyme, namely the 20.8 and 9.3 kDa components. 3. Our results indicate that the ubiquinone-binding site of complex I resides in the interface of the peripheral and membrane arms of the enzymes. The significance of these findings are discussed.

Two nuclear-coded subunits of mitochondrial complex I are similar to different domains of a bacterial formate hydrogenlyase subunit.

A computer comparison of protein sequences revealed similarity between the 30.4 kDa subunit of complex I from the fungus Neurospora crassa and the ORF5 subunit of formate hydrogenlyase from Escherichia coli. The ORF5 protein was previously known to be homologous to the 49 kDa component of the mitochondrial enzyme. We show that the 30.4 kDa corresponds to the N-terminal part while the 49 kDa subunit corresponds to the C-terminal portion of the bacterial protein. Thus, this bacterial protein represents a fusion of the two mitochondrial polypeptides suggesting that the two complex I genes arose from a single ancestor. Our results indicate that the 30.4 kDa and 49 kDa subunits are part of a structural and functional unit in complex I.