Redox properties of flavocytochrome c3 from Shewanella frigidimarina NCIMB400

The thermodynamic and catalytic properties of flavocytochrome c3 from Shewanella frigidimarina have been studied using a combination of protein film voltammetry and solution methods. As measured by solution kinetics, maximum catalytic efficiencies for fumarate reduction (kcat/Km = 2.1 x 10(7) M-1 s-1 at pH 7.2) and succinate oxidation (kcat/Km = 933 M-1 s-1 at pH 8.5) confirm that flavocytochrome c3 is a unidirectional fumarate reductase. Very similar catalytic properties are observed for the enzyme adsorbed to monolayer coverage at a pyrolytic graphite "edge" electrode, thus confirming the validity of the electrochemical method for providing complementary information. In the absence of fumarate, the adsorbed enzyme displays a complex envelope of reversible redox signals which can be deconvoluted to yield the contributions from each active site. Importantly, the envelope is dominated by the two-electron signal due to FAD [E degrees ' = -152 mV vs the standard hydrogen electrode (SHE) at pH 7.0 and 24 degrees C] which enables quantitative examination of this center, the visible spectrum of which is otherwise masked by the intense absorption bands due to the hemes. The FAD behaves as a cooperative two-electron center with a pH-dependent reduction potential that is modulated (pKox at 6.5) by ionization of a nearby residue. In conjunction with the kinetic pKa values determined for the forward and reverse reactions (7.4 and 8.6, respectively), a mechanism for fumarate reduction, incorporating His365 and an anionic form of reduced FAD, is proposed. The reduction potentials of the four heme groups, estimated by analysis of the underlying envelope, are -102, -146, -196, and -238 mV versus the SHE at pH 7.0 and 24 degrees C and are comparable to those determined by redox potentiometry.     

The Power of Contexts: Teaching and Learning in Recently Desegregated Schools

The following critical ethnography interrogates what it means for urban students to learn in multicultural ways, given the oppressive historical and present contexts of their newly desegregated urban district. By retelling events that occurred in the district and the classroom, I present a picture of urban students who are willing to learn and engage in classroom activities when the activities do not threaten their emotional safety. Although their actions are understandable, the students' conscious decisions to disengage from school stifles learning opportunities that would allow them to empathize and connect with other students as a move toward individual and group empowerment. Using critical race theory, I problematize the possibilities for successful multicultural classrooms in urban districts with complex legacies of injustice and racial hostility.     

Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.     

Altered substrate specificity in flavocytochrome b2: structural insights into the mechanism of L-lactate dehydrogenation

Flavocytochrome b(2) from Saccharomyces cerevisiae is a l-lactate/cytochrome c oxidoreductase belonging to a large family of 2-hydroxyacid-dependent flavoenzymes. The crystal structure of the enzyme, with pyruvate bound at the active site, has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863]. The authors indicate that the methyl group of pyruvate is in close contact with Ala198 and Leu230. These two residues are not well-conserved throughout the family of (S)-2-hydroxy acid oxidases/dehydrogenases. Thus, to probe substrate specificity in flavocytochrome b(2), these residues have been substituted by glycine and alanine, respectively. Kinetic studies on the L230A mutant enzyme and the A198G/L230A double mutant enzyme indicate a change in substrate selectivity for the enzyme toward larger (S)-2-hydroxy acids. In particular, the L230A enzyme is more efficient at utilizing (S)-2-hydroxyoctanoate by a factor of 40 as compared to the wild-type enzyme [Daff, S., Manson, F. D. C., Reid, G. A., and Chapman, S. K. (1994) Biochem. J. 301, 829-834], and the A198G/L230A double mutant enzyme is 6-fold more efficient with the aromatic substrate l-mandelate than it is with l-lactate [Sinclair, R., Reid, G. A., and Chapman, S. K. (1998) Biochem. J. 333, 117-120]. To complement these solution studies, we have solved the structure of the A198G/L230A enzyme in complex with pyruvate and as the FMN-sulfite adduct (both to 2.7 A resolution). We have also obtained the structure of the L230A mutant enzyme in complex with phenylglyoxylate (the product of mandelate oxidation) to 3.0 A resolution. These structures reveal the increased active-site volume available for binding larger substrates, while also confirming that the integrity of the interactions important for catalysis is maintained. In addition to this, the mode of binding of the bulky phenylglyoxylate at the active site is in accordance with the operation of a hydride transfer mechanism for substrate oxidation/flavin reduction in flavocytochrome b(2), whereas a mechanism involving the formation of a carbanion intermediate would appear to be sterically prohibited.     

Men in committed, romantic relationships have lower testosterone

Variation in human male testosterone levels may reflect, and effect, differential behavioral allocation to mating and parenting effort. This proposition leads to the hypothesis that, among North American men, those involved in committed, romantic relationships will have lower testosterone levels than men not involved in such relationships. Our study is the first to examine whether being in such a relationship (rather than being married) is the meaningful predictor of male testosterone levels. To test this hypothesis, 122 male Harvard Business School students filled out a questionnaire and collected one saliva sample (from which testosterone level was measured). Results revealed that men in committed, romantic relationships had 21% lower testosterone levels than men not involved in such relationships. Furthermore, the testosterone levels of married men and unmarried men who were involved in committed, romantic relationships did not differ, suggesting that, at least for this sample, male pair bonding status is the more significant predictor of testosterone levels than is marital status.     

Differing cardioprotective efficacy of the Na+/Ca2+ exchanger inhibitors SEA0400 and KB-R7943

KB-R7943 and SEA0400 are Na(+)/Ca(2+) exchanger (NCX) inhibitors with differing potency and selectivity. The cardioprotective efficacy of these NCX inhibitors was examined in isolated rabbit hearts (Langendorff perfused) subjected to regional ischemia (coronary artery ligation) and reperfusion. KB-R7943 and SEA0400 elicited concentration-dependent reductions in infarct size (SEA0400 EC(50): 5.7 nM). SEA0400 was more efficacious than KB-R7943 (reduction in infarct size at 1 microM: SEA0400, 75%; KB-R7943, 40%). Treatment with either inhibitor yielded similar reductions in infarct size whether administered before or after regional ischemia. SEA0400 (1 microM) improved postischemic recovery of function (+/-dP/dt), whereas KB-R7943 impaired cardiac function at >/=1 microM. At 5-20 microM, KBR-7943 elicited rapid and profound depressions of heart rate, left ventricular developed pressure, and +/-dP/dt. Thus the ability of KB-R7943 to provide cardioprotection is modest and limited by negative effects on cardiac function, whereas the more selective NCX inhibitor SEA0400 elicits marked reductions in myocardial ischemic injury and improved +/-dP/dt. NCX inhibition represents an attractive approach for achieving clinical cardioprotection.     

High-fat diet effects on gut motility,hormone, and appetite responses to duodenal lipid in healthy men

There is evidence that gastrointestinal function adapts in response to a high-fat (HF) diet. This study investigated the hypothesis that an HF diet modifies the acute effects of duodenal lipid on appetite, antropyloroduodenal pressures, plasma CCK and plasma glucagon-like peptide-1 (GLP-1) levels in humans. Twelve healthy men were studied twice in randomized, crossover fashion. The effects of a 90-min duodenal lipid infusion (6.3 kJ/min) on the above parameters were assessed immediately following 14-day periods on either an HF or a low-fat (LF) diet. After the HF diet, pyloric tonic and phasic pressures were attenuated, and the number of antropyloroduodenal pressure-wave sequences was increased when compared with the LF diet. Plasma CCK and GLP-1 levels did not differ between the two diets. Hunger was greater during the lipid infusion following the HF diet, but there was no difference in food intake. Therefore, exposure to an HF diet for 14 days attenuates the effects of duodenal lipid on antropyloroduodenal pressures and hunger without affecting food intake or plasma hormone levels.