Assembly and full functionality of recombinantly expressed dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex

The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior pyruvate dehydrogenase (E1) binding domain and two lipoyl domains. Mature human E2, expressed in a protease-deficient Escherichia coli strain at 27 degrees , was prepared in a highly purified form. Purified E2 had a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1. Electron micrographs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with E1 binding around the inner core periphery. With saturating E1 and excess dihydrolipoyl dehydrogenase (E3) but no E3-binding protein (E3BP), the recombinant E2 supported the overall PDC reaction at 4% of the rate of bovine E2.E3BP subcomplex. The lipoates of assembled human E2 or its free bilipoyl domain region were reduced by E3 at rates proportional to the lipoyl domain concentration, but those of the E2.E3BP were rapidly used in a concentration-independent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby. Given this restriction and the need for E3BP for high PDC activity, directed channeling of reducing equivalents to bound E3 must be very efficient in the complex. The recombinant E2 oligomer increased E1 kinase activity by up to 4-fold and, in a Ca2+-dependent process, increased phospho-E1 phosphatase activity more than 15-fold. Thus the E2 assemblage fully provides the molecular intervention whereby a single E2-bound kinase or phosphatase molecule rapidly phosphorylate or dephosphorylate, respectively, many E2-bound E1. Thus, we prepared properly assembled, fully functional human E2 that mediated enhanced regulatory enzyme activities but, lacking E3BP, supported low PDC activity.     

Development and factor analytic validation of the SPANAS among women in Spain: (more) cross-cultural convergence in the structure of mood

We reported our findings on the development and preliminary validation of a Spanish-language measure of positive and negative affect. Using confirmatory factor analytic techniques on data generated by 708 women in northern Spain, we obtained reasonable construct validity and reliability data for the measure. Consistent with past cross-cultural studies, a two-factor Positive Affect-Negative Affect (PA-NA)structure emerged, with PA and NA as relatively independent entities. The structure in this sample converged with that reported for a culturally separate group of participants. This factor structure has therefore revealed invariance across a number of cultural groups in Asia, Europe, and North America.     

Does type of colostomy influence outcome of colostomy closure?

A retrospective analysis of 81 patients who had closure of colostomy over a 32-month period was carried out to establish factors affecting the outcome of this operation. Their ages averaged 27 years and there were 69 male patients. The sigmoid colon was the most common site and the loop colostomy was most frequently performed. The majority were closed 3 or more months after construction. Loop colostomy took significantly less time to close and patients were fed significantly earlier compared with the other types. Patients who underwent closure after Hartmann's procedure had the longest hospital stay. The complication rate was 12% and there was no mortality. Colostomy closures in this study had minimal complications and no mortality. The loop colostomy is as easy to close as it is to perform and results in shorter hospital stay.     

The relationship between hepatitis C virus and schistosomiasis: histopathologic evaluation of liver biopsy specimens

The reported high incidence of anti-HCV seropositivity in the Egyptian population seems surprising. Some suggest that schistosomiasis is the responsible factor, either by producing false positivity for HCV antibodies or by predisposing to actual HCV infection in some way. In an attempt to investigate this unclear relationship on a histological level, we performed a thorough semiquantitative morphological study of liver biopsy specimens from 44 anti-HCV-positive Egyptian patients with chronic liver disease. More than half of these patients (23) had serological evidence of schistosomiasis. The results have shown that all 44 liver biopsy specimens demonstrated the histopathological features known to be characteristic of chronic HCV hepatitis. Statistical analysis showed no significant difference between the schistosomal and nonschistosomal groups regarding the semiquantitative histological scores of these features. This study confirms the presence of definite HCV-induced hepatic pathology in all anti-HCV seropositive cases. More importantly, it shows the lack of enhancement of this pathology in the schistosomal patients.     

Gas exchange and gas transport in rats during warming after deep immersion hypothermia

Gas exchange, oxygen transport and utilisation were shown to be limited at body temperature 17.5 degrees C in rats. Disturbances in the cardio-vascular system seem to be the main limiting factors telling on the above parameters in the course of spontaneous rewarming.     

Kinetic studies of Bacillus polymyxa nitrogenase

Nitrogenase from the facultative anaerobe Bacillus polymxa was separated into its component proteins, which were recombined in the ratio that produced optimal specific activity (125 to 175 nmol of C2H2 reduced/min per mg of total protein). The apparent Michaelis constants (Km)for the magnesium adenosine triphosphate complex, reducible substrates azide, acetylene, and N2 and the nonphysiological electron donor hydrosulfite (S2O42-) were determined to be 0.7, 0.7, 0.2, 0.06, and 0.03 MM, respectively. These apparent Km values are in reasonable agreement with those reported for the nitrogenases of Azotobacter vinelandii and Klebsiella pneumoniae. Either a total lack of cooperativity between binding sites or a single binding site for reducible substrates is indicated by analysis of Hill plots. Hill plot slopes of approximately 1.7 suggest that multiple binding sites exist for both ATP and S2O42-.