Conformational changes and stabilization induced by phosphate binding to 5'-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus.

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.

Does a circadian variation occur in myocardial ischemia over 48 hours in patients with unstable angina?

OBJECTIVE: To study the incidence of and variation in myocardial ischemia over 48 hours in patients with unstable angina. METHODS: Thirty-nine patients with unstable angina underwent long-term electrocardiography for 48 hours. The number of events and the period of time of ischemia (in minutes) were analyzed for the 48 hours, in two periods of 24 hours, and in periods of 4 hours. RESULTS: We analyzed 1755.8 hours of monitoring tapes, and ischemic episodes were detected in 18 (46.2%) patients, corresponding to 173 ischemic episodes, allowing the evaluation of 1304 minutes of ischemia.only 4 of which were (2.2%) symptomatic, Considering the entire period of time of recording and the predetermined time intervals, we observed a higher number of ischemic episodes (38) and a longer duration of ischemia (315.4 minutes) between 11:00 am and 3:00 pm. However, no significant differences occurred among the values in the different intervals. CONCLUSION: Long-term electrocardiography over 48 hours showed a high incidence (97.8%) of silent ischemic episodes in patients with unstable angina. No evidence of a circadian variation of myocardial ischemia in unstable angina was observed.

A rolling-circle plasmid from Psychrobacter sp. TA144: evidence for a novel rep subfamily.

In this paper we report the cloning and sequencing of two small plasmids, pTAUp and pTADw, from the Antarctic Gram-negative Psychrobacter sp strain TA144. The observation that pTAUp contains a putative Rep-coding gene (Psyrep) suggested that its duplication occurs via a rolling-circle replication mechanism. This hypothesis was confirmed by the identification of the pTAUp single-stranded DNA form. The putative pTAUp plus origin of replication was found at the 3' end of the Psyrep by using an in vivo complementation assay. Structural similarities at the level of (i) gene organization, (ii) protein sequence, and (iii) nick site sequences strongly suggest that the psychrophilic enzyme belongs to a new subfamily of replication enzymes.

[When should we stop cardiopulmonary resuscitation in patients brought to the emergency room in cardiac arrest without pre-hospital care?]. / Quando interromper, na sala de emergência, as manobras de ressuscitação cardiopulmonar na parada cardiorrespiratória sem atendimento pré-hospitalar?

PURPOSE: The decision of stopping cardiopulmonary resuscitation (CPR) in patients brought to emergency room in arrest remains a challenge. Such decision is even more difficult when someone is brought by bystanders, after an acute loss of consciousness without any out-of-hospital care. To evaluate the probability of survival of these patients we reviewed retrospectively charts in our institution, during a period of five years. METHODS: One hundred and one patients that fulfilled these characteristics came to our emergency in arrest. The time to arrival since symptoms started, cardiac rhythm at first electrocardiogram (EKG), age, gender, initial CPR success, late outcomes and previous diseases were obtained. Patients were divided in two groups regarding which cardiac rhythms they had at first EKG: A-patients arriving in asystole; and VF-patients arriving in ventricular fibrillation. To evaluate time to arrival, we arbitrarily choose 15 min as a reference point. RESULTS: In these 101 subjects the mean age was 62 +/- 13.7 years and 63 (62.3%) were men. Previous heart disease was documented in 74 [dilated cardiomyopathy in 22 (21.7%), coronary heart disease in 41 (40.6%), arterial hypertension in 25 (24.7%) and others in 6 (5.6%)]. In 66 episodes we were sure of the time patients spent before arrival (mean 2.5 +/- 11 min). Only in 63 subjects we had no doubts about the rhythm at entrance: VF in 37 (58.7%), A in 22 (34.9%) and an accelerated idioventricular rhythm (AIR) in four (6.3%). Time to arrival was 18.6 +/- 10.6 in VF vs 32.5 +/- 11.7 min in A (p = 0.012). Fourteen (13.8%) subjects resumed a supraventricular rhythm with systolic pressure > or = 90 mmHg after CPR and all of them were in VF (13) or AIR (one). Nine patients (8.9%) evolved in coma. Only five (4.9%) were discharged from the hospital without any neurological disturbance and their time to arrival ranged from one to 15 (9 +/- 5.8) min. CONCLUSION: Delayed arrival to the emergency room (> 15 min) associated with asystole were predictors of unsuccessful CPR, and both data are helpful in deciding when to stop CPR in subjects arriving at the emergency department with no out-of-hospital care.

[The relationship between conventional ECG and the culprit coronary artery in acute myocardial infarct]. / Relação entre o ECG convencional e a artéria coronária responsável no infarto agudo do miocárdio.

PURPOSE: To evaluate electrocardiogram (ECG) in detecting acute myocardial infarction (AMI) during the first 12 hours of symptoms and its relationship to the culprit coronary artery. METHODS: We studied 68 patients aged 55.6 (30 to 76) years, 61 males, with AMI confirmed by elevated CKMB isoenzyme and cinecoronariography (CINE). In all of them we obtained two ECG: first (i), with < 12 hours of symptoms and a second, > or = 5 days during evolution. ECG were analyzed in order to disclose up and downward ST-T segments > or = 1 mm, new Q waves > or = 0.04 s and R/S > or = 1 plus downward ST-T segment in leads V1 and V2. Then we have done correlation between these and the culprit coronary lesions at CINE. RESULTS: The culprit coronary lesions were: right coronary artery (RCA) in 16, left circumflex (LC) in 26 and left anterior descending (LAD) in 31 cases. According to the ECG, the RCA determined inferior AMI in all patients and the LC only in 62% of cases. Posterior AMI due to LC was seen in 81% of cases at ECG and, associated with lateral AMI, in 52%. Lone lateral AMI was seen in 5% and true posterior in 14% of cases, all of them due to LC. CONCLUSION: False negative ECG (i) in AMI is in fact due to LC occlusion which, frequently, causes posterior wall more then inferior wall myocardial infarction at ECG.

[Multiple myocardial coronary bridges]. / Pontes miocárdicas múltiplas.

White woman, 31 years old, complaint pain in the left arm, at rest, and few months later she had pain in the precordial region with dyspnea, diaphoresis and dizziness without any inducing factor. One hour later, symptoms ceased spontaneously, remaining daily precordial pain. Subsidiary examinations as cineangiocoronariography revealed 100% of arterial lumen constriction in the middle portion of the descending coronary artery, during systole, as well as in the diagnoalis and marginal branches. Multiple myocardial coronary bridges has been described in necropsy, but during cinecoronarioangiography, this case appeared to be the first one.