Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis.

This study was conducted to evaluate monthly changes in the ram seminal plasma protein profile using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with a polyacrylamide linear gradient gel. Likewise, comparative analyses of the protein composition of ovine seminal plasma (SP) from ejaculates obtained along the year, and its relationship with sperm motility, viability and concentration of ejaculate were carried out. Western-blot analysis was performed to specifically detect P14, a ram SP protein postulated to be involved in sperm capacitation and gamete interaction [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49], and its variations along the year have also been established. The experiment was carried out from May 2003 to April 2004, with nine Rasa Aragonesa rams. Ejaculates obtained every 2 days were pooled and used for each assay, to avoid individual differences, and three two-dimensional SDS-PAGE gels were run for each month. The high resolution of the gradient gel allowed the image analysis software to detect around 252 protein spots, with pIs ranging from 4.2 to 7.6, and molecular weight (M(r)) from 12.5 to 83.9 kDa. Four protein spots (1, 2, 3 and 4) of low M(r) (15.1, 15.7, 15.9 and 21.0 kDa) and acidic pI (5.9, 5.3, 5.7 and 6.6), respectively, had the highest relative intensity in the SP map (11.2, 9.3, 4.7 and 7.7%, respectively). Spot 3 was more abundant (P<0.05) from May to December, and negatively correlated (P<0.05, r=-0.34) with sperm viability and concentration (P<0.05, r=0.36). Another 12 protein spots also had significant quantitative differences (P<0.05) along the year, and 17 protein spots, which correlated with some seminal quality parameter, did not show quantitative monthly changes. Western-blot analysis indicated that spots 1 and 2 reacted with the anti-P14 antibody, raised against the P14 band (approximate M(r) 14 kDa) of ram SP. This indicates that spots 1 and 2 are similar to RSP15 [Bergeron A, Villemure M, Lazure C, Manjunath P. Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev 2005;71:461-70], bovine PDC-109 [Esch FS, Ling NC, Bohlen P, Ying S, Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun 1983;113:861-7] (also called BSP A1/A2 [Manjunath P, Sairam MR. Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. Biochem J 1987;241:685-92]) and goat GSP-14/15 kDa [Villemure M, Lazure C, Manjunath P. Isolation and characterization of gelatine-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 2003;1:39], based on our previous results on the P14 amino acid sequence [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539-49].

Sperm survival and heterogeneity are correlated with fertility after intrauterine insemination in superovulated ewes.

Efficient animal production involves accurate estimations of fertilizing ability. One key factor is the plasma membrane of the sperm cell, which is actively involved in the cascade of events before oocyte fusion. Many methods are used to analyze the characteristics of this membrane, including partition in aqueous two-phase systems which is an efficient method to analyze sperm surface changes accounting for loss of viability and different functional states. Centrifugal countercurrent distribution (CCCD) analysis can also be used in an aqueous two-phase system to determine the relationship between sperm parameters and in vivo fertility in ewes. In a previous work, we found a significant correlation between two post-CCCD parameters (heterogeneity and recovered viability) and field fertility when the same sample was used after cervical AI. The present study was intended to find out whether the control of several external factors that affect reproductive efficiency is able to increase the correlation coefficient between post-CCCD parameters and fertility. Thus, 90 Rasa aragonesa ewes were controlled on the same farm and received intrauterine inseminations using the same technical equipment. The fertilizing ability of the raw semen and sperm samples selected by a dextran/swim-up process was compared using a low number of spermatozoa per insemination (7 x 10(7)) to enhance possible fertility differences. A new post-CCCD parameter was considered; the loss of viability (LV) occurred during the CCCD process. This variable denotes the sperm surviving ability and corresponds to the difference between the total number of viable cells loaded and recovered after the CCCD run. The mean fertility of eight sperm control samples was 60% (range: 25-76%), and there was no significant correlation between standard parameters and in vivo fertility. LV ranged from 2 to 69% (average 27%) and was negatively correlated with fertility (r = -0.914, P < 0.01). Ejaculate heterogeneity (H) ranged from 20 to 47% and was positively, but not significantly, correlated with fertility (r = 0.391). A predictive equation for fertility was deduced by multiple analysis with a very high correlation coefficient (r = 0.967), and level of significance (P < 0.005): predictive fertility PF = 52.546 - 0.594 LV + 0.665 H. The mean fertility of 13 swim-up selected samples was 63% (range: 25-86%). Again, only parameters derived from the CCCD analysis were highly correlated with fertility, especially LV and H (P < 0.05).

Ram sperm selection by a dextran/swim-up procedure increases fertilization rates following intrauterine insemination in superovulated ewes.

The objective of this study was to compare the efficacy of 2 dextran/swim-up media to increase the sperm quality parameters and the maintenance of these parameters at 15 degrees C and 30 degrees C over 6 hours. Additionally, this study examined whether differences in sperm quality reflect different reproductive efficiencies following intrauterine insemination in superovulated ewes. The study involved 2 selected samples (SS) obtained by dextran/swim-up, performed either with (SS+) or without (SS-) capacitating compounds, and a control sample consisting of raw semen diluted in the same medium. The efficacies of the swim-up sperm selection procedures were similar in both media, and no significant differences were found among the evaluated parameters. Conversely, we found important differences between selected and control samples. Sperm motility, viability (as assessed by carboxifluorescein diacetate/propidium iodide [PI] staining), and mitochondrial activity (as assessed by rhodamine 123/PI) were significantly higher in the selected samples than in the control. Additionally, following incubation at 15 degrees C, the preservation of sperm quality was significantly better in the selected samples than in the control samples. After 6 hours of incubation at 15 degrees C, selected samples had a motility value of 46%, which was significantly (P < .001) higher than the value observed in control samples (27%). The percentage of viable cells observed after 6 hours of incubation at 15 degrees C was significantly (P < .0001) higher in selected samples than in the control samples. Furthermore, after 2 hours of incubation at 30 degrees C, swim-up samples had viability values that were significantly (P < .0001) higher than those of the control samples. SS+ and SS- samples did not differ significantly in spermatozoa yield, sperm quality, or survival. Differences between selected samples and controls were reflected in the fertilization rate obtained following intrauterine insemination in superovulated ewes that experienced a 52-hour interval between progestagen removal and artificial insemination. A restricted criterion for fertilization rate evaluation was established, and only the percentage of embryos recovered from the uterine horns 6 days after insemination was considered with respect to the total number of corpora lutea counted in the ovaries. The fertilization rate of SS- samples (50%) was significantly higher (P > .001) than those of the SS+ (2%) and control samples (5%).

[Applications of nuclear magnetic resonance in the diagnosis of cardiopathies]. / Aplicaciones de la resonancia magnética nuclear en el diagnóstico de las cardiopatías.

Applications of nuclear magnetic resonance imaging in cardiovascular diagnosis. The role of magnetic resonance imaging in the evaluation of cardiovascular system is still evolving. Nevertheless, this technique is of great promise for cardiac patient management in the near future. Magnetic resonance imaging provides a high contrast between the blood pool and myocardial function that has been shown to be effective for the evaluation of a wide variety of anatomic abnormalities as well as to assess cardiac contractile function or myocardial perfusion. Moreover, magnetic resonance spectroscopy has provided a new research tool for the evaluation of myocardial metabolism. Such performances indicate the potential of magnetic resonance techniques to establish the link between myocardial function and metabolism. In this presentation we will review the current status of magnetic resonance imaging for the diagnosis and evaluation of a wide variety of cardiovascular diseases and discuss its future potential.

Diagnostic accuracy of predicting coronary artery disease related to patients' characteristics.

Patients' demographic and clinical characteristics may affect diagnostic accuracy of cardiologists. We asked a group of experienced cardiologists from three institutions to estimate the pretest probability of coronary artery disease in 257 patients referred for diagnostic coronary angiography and with no history of previous myocardial infarction nor valvular heart disease. Physicians pretest estimates were compared with the diagnostic findings of coronary angiography. We tested the influence of five variables on the accuracy of the pretest estimates: age, sex, chest pain characteristics, rest electrocardiogram and electrocardiographic exercise test result. Cardiologists tended to overestimate the presence of coronary artery disease and this tendency was particularly remarkable in the group of patients showing a negative exercise test. Pretest diagnostic accuracy was 0.72 when the test result was negative and 0.85 when the test result was positive (95% confidence interval of the difference 0.03 to 0.23; p < 0.001). The diagnosis of coronary artery disease was also more accurate for male than for female patients (0.81 vs 0.70; 95% confidence interval of the difference 0.02 to 0.21; p < 0.02). Characteristics of chest pain, age and rest electrocardiogram did not affect the level of pretest diagnostic accuracy. Cardiologists should be cognizant of correctly interpreting a negative exercise test and the clinical data of female patients; in both cases, they should move circumspect of the diagnosis of coronary artery disease.

How the exercise electrocardiogram is used in clinical practice in patients with suspected coronary artery disease.

Clinical use of a test should be evaluated not only on the change of pretest probability but also on the increased confidence in the judged probability, and on the decision of recommending additional tests or therapy after test results. Before and after the test, cardiologists referring a patient for exercise electrocardiogram for suspected coronary artery disease were asked to estimate the probability of coronary artery disease and the minimal and maximal value of their estimate (plausible range), to judge whether to refer the patient for coronary angiography, and to specify patient's treatment. After the test, the percentage of patients with estimated probability < or = 20% increased (from 53 to 69%; p = 0.005) and the percentage of patients with intermediate probability (between 21 and 80%) decreased (from 37 to 16%; p = 0.0001). The mean plausible range decreased from pre- to post-test assessment (31 +/- 23 to 15 +/- 16%; p = 0.0001), indicating an increase in cardiologists' confidence in their estimates. After the test, the percentage of cardiologists who wished to either refer or not refer patients for coronary angiography increased (from 4 to 13%, p < 0.005; and from 37 to 65%, p < 0.0001, respectively). Test results enable cardiologists to change the assessment of disease probability, the confidence in their own estimates, and their judgment of whether to refer patients for coronary angiography.

[Non-invasive study of myocardial ischemia. Alternatives to the conventional stress test]. / Estudio no invasivo de la isquemia miocárdica. Alternativas a la prueba de esfuerzo convencional.

Stress techniques for inducing myocardial ischemia in patients with coronary atherosclerosis have been widely studied. The most frequently used are those requiring physical effort, increasing their diagnostic efficiency with radioisotopes and echocardiographic monitoring. Patients unable to make physical exercise and with suspected ischemic heart disease may undergo nonexercise stress test. Transesophageal atrial stimulation and pharmacological stress tests with dipyridamol, dobutamine and adenosine are good alternatives, provided a strict methodology is used.

[Acute non-lethal complete occlusion of the left main coronary artery]. / Oclusión aguda completa no letal del tronco común de la coronaria izquierda.

Myocardial infarction secondary to total obstruction of the left main coronary artery is an infrequent finding in clinical practice. Even though this disease usually results in high mortality rates, various cases have been described documenting survival after complete occlusion of the left main trunk. We present a patient who survived after a massive myocardial infarction due to total obstruction of the left main coronary artery. Orthotopic cardiac transplantation was subsequently performed with good results.

[Non-invasive diagnosis of rejection in heart transplant: usefulness of Doppler echocardiography]. / Diagnóstico no invasivo del rechazo en el trasplante cardiaco: utilidad de la ecocardiografía-Doppler.

Cardiac transplantation is a valid therapeutic option for advanced chronic heart failure, with a one year survival of 60-80%. Although the introduction of cyclosporine has markedly improved survival in heart transplant recipients, early detection of cardiac allograft rejection remains the major problem to be solved. At present, the diagnosis of cardiac rejection has been based on the results of endomyocardial biopsy, which remains actually as the gold standard for rejection surveillance. However, ideally the detection of cardiac rejection should be noninvasive to allow frequent follow-up of the patients. Several attempts have been made to find a noninvasive alternative to replace endomyocardial biopsy, doppler-echocardiography being the most promising technique. A review of current noninvasive methods for graft rejection detection will be discussed emphasising the importance of doppler-echocardiography.

[Changes in diastolic function in heart rejection: role of Doppler echocardiography]. / Alteraciones de la función diastólica en el rechazo cardíaco: papel del ecocardiograma-Doppler.

In acute cardiac rejection, left ventricular diastolic function is altered. To study these abnormalities and their utility in cardiac allograft rejection, we studied 56 cardiac transplant recipients. All patients were assessed with endomyocardial biopsy and Doppler echocardiography done in the same day. A total of 163 Doppler studies were recollected. Cardiac transplant recipients were excluded during the early 6 weeks postoperative period. Totally, 100 biopsies were normal, 48 positive for mild rejection (Billingham Gde I-II) and 15 positive for moderate or severe rejection (Billingham Gde III-IV). Compared to negative biopsies, during acute rejection left ventricular wald thickness significantly increased (p < 0.05); isovolumic relaxation period and pressure half-time significantly decreased (p < 0.05, p < 0.001 respectively). Nevertheless, increase in peak early mitral flow velocity was only significantly associated with severe rejection (p < 0.001). Correlating only progressive shortening of isovolumic relaxation period parameter in the diagnosis of graft rejection, we forward a high sensibility (85%) and low specificity (57%). Thus, Doppler echocardiographic evaluation of left ventricular diastolic function provides a non-invasive tool for early detection of acute rejection monitoring after the early postoperative period.

[Ambulatory heart catheterization. Initial experience]. / Cateterismo cardiaco ambulatorio. Experiencia inicial.

Development of cardiovascular surgical techniques and more recently, balloon angioplasty, has resulted in a dramatic increase in the number of cardiac catheterizations performed. Previously, the usual policy was to admit the patient to the hospital for several days. Various series of patients submitted to ambulatory cardiac catheterizations have revealed that this procedure con be performed safely, with a potential reduction in hospital and patients costs, better utilization of medical beds and good image quality. In this study we present our initial experience in this kind of outpatient procedure.

Clinical assessment of the probability of coronary artery disease: judgmental bias from personal knowledge.

Probability estimates of angiographic coronary artery disease made by experienced, board-certified staff cardiologists were compared with those of cardiologists in training (fellows). In addition, estimates made before coronary angiography were compared with those made several months later based on written clinical summaries of 15 items of objective clinical and test data. Cardiologists were asked to estimate the probabilities of coronary artery disease, multivessel disease, and triple-vessel or left main disease. The study population consisted of 510 consecutive patients without valvular disease referred for the first time for coronary angiography to three hospitals. Both staff and fellows consistently overestimated the pre-angiographic probability of coronary artery disease. The probabilities estimated from patient summaries were always significantly lower than the pre-angiographic assessments. Only staff cardiologists reliably assessed the probabilities of coronary artery disease during the second assessment (p less than 0.05). Thus, estimates of disease probability based on clinical judgment vary according to the source of information, and these estimates are more accurate when physicians have objective data on hand and do not know the identities of the patients.

[Dysautonomic syncope. Diagnosis and treatment]. / Síncopes disautonómicos. Diagnóstico y tratamiento.

Inappropriate activation or disbalance of vasomotor reflexes may have a close relationship with the pathogenesis of disautonomic syndromes, a frequent underlying cause of recurrent syncope. A combined approach of meticulous historical data, physical examination and selected laboratory procedures may delineate the most common causes of recurrent syncope. Tests of autonomic function may be particularly helpful in the diagnosis of this entity. They include some non-invasive maneuvers such as stimulation of carotid sinus, Valsalva maneuver or tilt-table test. Therapy for this syndrome includes pharmacologic agents, surgical and radiotherapeutic maneuvers and atrioventricular sequential pacing. In this work we will present an overall formulation of the diagnostic evaluation and a therapy approach of the patient presenting with this complaint.

[Aneurysm of interauricular septum associated with mesosytolic click]. / Aneurisma del septo interauricular asociado a chasquido mesosistólico.

An echocardiographic study was performed to a patient with symptoms of cardiac failure and midsystolic click confirmed by phonocardiography. The echocardiograms suggested an incipient dilated myocardiopathy and also atrial septal aneurysm. We report the relationship between midsystolic click and atrial septal aneurysm.

[Left ventricular diverticulum at the subaortic level]. / Divertículo ventricular esquerdo a nível subaórtico.

We report a case of a left ventricular diverticulum located immediately under the aortic valve ring. The patient presented both aortic insufficiency and stenosis, and was submitted to valve replacement surgery and closure of the diverticulum. The peculiarity of this case relies on its non atherosclerotic etiology, its unusual location and also because of the calcification of the diverticulum's wall.

Complementary mechanisms of atenolol and diltiazem in the clinical improvement of patients with stable angina.

The combination of atenolol with diltiazem has been shown to be useful in the treatment of patients with coronary artery disease. Eighteen patients with proven coronary artery disease, stable angina, and no previous myocardial infarction were studied before and after treatment with atenolol (100 mg/day) (9 patients) or diltiazem (180 mg/day) (9 patients). Ischemic threshold at stress test, pressure-rate product at ischemic threshold, direct oxygen consumption at ischemic threshold, and exercise ejection fraction were determined. There was a slight increase in the duration of exercise, maximal oxygen consumption, and ischemic threshold after treatment with each drug. Double product at ischemic threshold decreased from 20.9 to 19.8 (p = NS) with atenolol but increased from 20.1 to 21.9 (p = NS) with diltiazem. Conversely oxygen consumption at ischemic threshold increased with atenolol to nearly significant values from 17.2 to 23.6 (p = 0.067) but not with diltiazem (16.2 to 22.3; p = 0.16). Before treatment, exercise ejection fraction increased less than 10% or decreased from its resting values in all patients but 1 with atenolol and 1 with diltiazem, but exercise ejection fraction increased significantly after treatment with atenolol (60.6 to 67.5; p = 0.02) but not with diltiazem. This improvement was due to a significant reduction in end systolic volume (103.8 to 78.6; p = 0.019), despite a similar increase in heart rate and blood pressure in all patients.(ABSTRACT TRUNCATED AT 250 WORDS)