Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild-type protein

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two active-site cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild-type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred chi 1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild-type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild-type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pKa of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild-type thioredoxin. The pKa is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild-type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.     

A technique for performing transesophageal echocardiography safely in patients with Zenker's diverticulum

Transesophageal echocardiography was indicated for evaluation of mitral valve pathology in a patient with a Zenker's diverticulum. However, transesophageal echocardiography is potentially dangerous and therefore relatively contraindicated in such patients. Our gastroenterologist directly intubated the esophagus with a fiberoptic endoscope and introduced an overtube through which transesophageal echocardiography was performed without incident.     

Energy balance in endurance-trained female cyclists and untrained controls

Reports of low energy intakes in trained female athletes imply they have an increased energetic efficiency. To address this question, we determined how energy balance was achieved in endurance-trained females cyclists and lean controls (n = 5 in each group). Daily energy expenditure was measured by using standardized physical activity protocols in a whole room calorimeter on two separate occasions: a cycling day and a noncycling day. Energy intake for weight maintenance was determined by a period of controlled feeding 5 days before and the day of each energy expenditure measurement. Energy balance was achieved in the cyclists on the cycling day while they consumed 2,900-3,000 kcal (their usual condition) and in controls on the noncycling day while they consumed 2,100-2,200 kcal (their usual condition). Total daily energy expenditure was not significantly different between the cyclists and controls on the noncycling day with both groups performing similar levels of activity. On the cycling day, daily energy expenditure was significantly greater in the cyclists vs. controls (P < 0.03) as a result of their greater amount of cycling activity. Components of daily energy expenditure, i.e., resting metabolic rate and thermic effect of food and activity (noncycling), were not significantly different between groups. Overall, we found no significant increase in the energetic efficiency of endurance-trained female cyclists compared with controls.     

Use of neurosurgical techniques for removal of a cardiac tumor

A 6-week-old male infant with a capillary hemangioma of the right atrioventricular groove adjacent to the right coronary artery and conduction system underwent successful resection with the aid of microneurosurgical instrumentation. The technical challenge was thought to be analogous to that encountered by microneurosurgeons in their dissection of brain tumors.     

Quality-of-life following thoracotomy for lung cancer

Contrary to the issues of perioperative morbidity and survival following surgery for lung cancer, little attention has been given to quality-of-life. To address this, quality-of-life was assessed preoperatively and 1, 3, 6 and 9 months postoperatively in a cohort of 117 consecutive subjects who underwent thoracotomy with a certain or presumptive diagnosis of lung cancer. Those with cancer (n = 91) confirmed at thoracotomy were contrasted to those without (n = 26). Moderate to severe dyspnea, reported in 14% preoperatively, increased to 34% at 1 and 3 months (p < 0.005) but returned to approximately 10% at 6 and 9 months. Similarly, activities of daily living were impaired in 11% preoperatively; this disability increased to 21% at 1 month (p < 0.005), and returned to baseline at 6 and 9 months. Those with cancer compared to those without a postoperative diagnosis of cancer had similar quality-of-life preoperatively but deteriorated more in the postoperative period. This study demonstrates that important deterioration in quality-of-life occurs during the first 3 months postoperatively in those with a final diagnosis of cancer but improvement back to baseline can be expected thereafter.     

Axon-sparing lesions of the medial nucleus of the amygdala decrease affiliative behaviors in the prairie vole (Microtus ochrogaster): Behavioral and anatomical specificity.

The neural basis of affiliative behavior was examined in the prairie vole, a rodent that exhibits high levels of social contact and paternal behavior. In the 1st study, the axon-sparing excitotoxin N-methyl-D-aspartate (NMDA) produced lesions in the basolateral nucleus of the amygdala or the corticomedial amygdala. Males with corticomedial lesions showed significantly less contact with a familiar adult female and a pup when compared with males with lesions of the basolateral nucleus or controls. This behavioral change was not associated with changes in exploratory behavior, motor function, performance in an olfactory task, fearfulness, physical well-being, or body temperature. In a 2nd study, NMDA lesions restricted to the medial nucleus also decreased paternal behavior. Neurons in the medial nucleus of the amygdala appear to be essential for the normal expression of paternal care in this species. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Management of postoperative heparin rebound following cardiopulmonary bypass

Postoperative heparin rebound was investigated in 50 adult patients undergoing cardiopulmonary bypass with the use of the Hepcon heparin analyzer. Prior to bypass each patient received 2 mg/kg of heparin. During bypass, the activated clotting time (ACT) was utilized to assess the need for additional heparin to maintain the value between 300 and 400 seconds. The average amount of heparin given was 160 mg. Once cardiopulmonary bypass was terminated the Hepcon unit was employed to determine the actual amount of active circulating heparin and to calculate the dose of protamine sulfate. The average amount of protamine administered intraoperatively was 200 mg. The overall mean ratio of protamine-to-heparin was 1.25 : 1. Once hemostasis was achieved, no circulating heparin was measured with the Hepcon unit, and the ACT value had returned to its baseline, the incisions were closed and the patients were transferred to the intensive care unit. One hour later a blood sample was obtained and analyzed by the Hepcon unit for any heparin rebound. We found that 26 patients (52%) had circulating heparin and required an additional dose of protamine, averaging 70 mg. Drainage from the thoracotomy tubes averaged 400 cc in the first 24 hours, and a mean of 2 units of packed cells was infused. Three patients (6%) did not require any blood transfusions. The use of the Hepcon unit has produced a safe and expedient method of analyzing and neutralizing active circulating heparin in patients following cardiopulmonary bypass. It is a useful adjunct in blood conservation because it reduces excessive postoperative blood loss associated with heparin rebound.     

Apoptosis as a mechanism of keratinocyte death in toxic epidermal necrolysis

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are life-threatening diseases characterized by extensive epidermal destruction. The aim of our study was to investigate apoptosis in keratinocytes of patients with TEN and TEN/SJS overlap syndrome. Keratinocytes from TEN patients were found to undergo extensive apoptosis. These results suggest that cell destruction in TEN occurs as a result of apoptosis. Our findings suggest that apoptosis inhibitory agents may play an important part in the therapeutic strategy of TEN.     

A comparison of the surface activity of the fungal hydrophobin SC3p with those of other proteins

The fungal hydrophobin SC3p, a protein secreted by Schizophyllum commune, has become known to form SDS-insoluble layers and to change the physico-chemical properties of an interface. In this study, the surface activity of SC3p was studied by determining the interfacial tensions gamma iv and gamma sl during adsorption of SC3p at both the liquid-vapour and the solid-liquid interface utilizing the in situ technique axisymmetric drop-shape analysis by profile. To this end, protein solution droplets were put on the solid fluoroethylene-propylene-Teflon. At the liquid-vapour interface, SC3p caused a large decrease of gamma iv from 72 to 43 mJ m-2 at the concentration of 0.1 mg ml-1. At the solid-liquid interface, gamma sl was slightly decreased, whereas the contact angle theta increased, indicating an increase in hydrophobicity of FEP-Teflon, which is unique among the proteins studied so far. Earlier findings indicated a decrease in hydrophobicity of Teflon upon adsorption of SC3p, but this was after a washing and drying step. In order to reconcile these findings with those of the present study, adsorption of SC3p to hydrophobic surfaces is suggested to occur in bilayers. The second layer is supposed to be less strongly adsorbed than the first layer and can be easily removed by washing.