Exploring and embracing complexity in a distance-learning curriculum for physicians.

The recent pressures on clinical medicine such as the attention to medical error and the challenges of interdisciplinary care have also exerted pressure on health professions education. Educators must now gauge how to redesign education systems to adapt quickly to these disruptions. Sometimes disruptions can be self-inflicted, such as the VA National Quality Scholars Fellowship's decision to use interactive video (IV) as its primary medium for delivering the curriculum to its six sites around the nation. The authors describe how this disruption to their education system helped to fashion a learning environment that is adaptable. Along the journey from a classroom-based curriculum to an IV-based curriculum, the authors and others involved in the program learned the basic tenets of IV sessions, redefined the roles of the teachers and learners, and discovered an IV environment that functions as a complex adaptive learning system. This distance-learning curriculum can be a model for other health professions education, since it starts with simple rules, changes from within, has a tolerance for unpredictability, and continually moves forward and transforms itself despite tension.     

Changes in human muscle transverse relaxation following short-term creatine supplementation

The rapid increase in body mass that often occurs following creatine (Cr) supplementation is believed to be due to intracellular water retention. The purpose of this study was to determine whether Cr consumption alters the magnetic resonance (MR) transverse relaxation (T(2)) distribution of skeletal muscle. Transverse relaxation can be used to model water compartments within a cell or tissue. In this double-blind study, subjects were asked to supplement their normal diet with creatine monohydrate (20 g day(-1) for 5 days) mixed with a grape drink (Creatine group, n = 7), or the grape drink alone (Placebo group, n = 8). Phosphorous MR spectroscopy was used to determine the effectiveness of the supplementation protocol. Subjects that responded to the Cr supplementation (i.e. showed a > 5 % increase in the ratio of the levels of phosphocreatine (PCr) and ATP) were placed in the Creatine group. Both proton MR imaging and spectroscopy were used to acquire T(2) data, at 1.89 T, from the flexor digitorum profundus muscle of each subject before and after supplementation. Following the supplementation period, the Creatine group showed a gain in body mass (1.2 +/- 0.8 kg, P < 0.05, mean +/- S.D.), and an increase in PCr/ATP ratio (23.8 +/- 16.4 %, P < 0.001). Neither group showed any changes in intracellular pH or T(2) calculated from MR images. However, the spectroscopy data revealed at least three components (> 5 ms) at approximately 20, 40 and 125 ms in both groups. Only in the Creatine group was there an increase in the apparent proton concentration of the two shorter components combined (+5.0 +/- 4.7 %, P < 0.05). According to the cellular water compartment model, the changes observed in the shorter T(2) components are consistent with an increase in intracellular water.     

Staphylococcus aureus aconitase inactivation unexpectedly inhibits post-exponential-phase growth and enhances stationary-phase survival

Staphylococcus aureus preferentially catabolizes glucose, generating pyruvate, which is subsequently oxidized to acetate under aerobic growth conditions. Catabolite repression of the tricarboxylic acid (TCA) cycle results in the accumulation of acetate. TCA cycle derepression coincides with exit from the exponential growth phase, the onset of acetate catabolism, and the maximal expression of secreted virulence factors. These data suggest that carbon and energy for post-exponential-phase growth and virulence factor production are derived from the catabolism of acetate mediated by the TCA cycle. To test this hypothesis, the aconitase gene was genetically inactivated in a human isolate of S. aureus, and the effects on physiology, morphology, virulence factor production, virulence for mice, and stationary-phase survival were examined. TCA cycle inactivation prevented the post-exponential growth phase catabolism of acetate, resulting in premature entry into the stationary phase. This phenotype was accompanied by a significant reduction in the production of several virulence factors and alteration in host-pathogen interaction. Unexpectedly, aconitase inactivation enhanced stationary-phase survival relative to the wild-type strain. Aconitase is an iron-sulfur cluster-containing enzyme that is highly susceptible to oxidative inactivation. We speculate that reversible loss of the iron-sulfur cluster in wild-type organisms is a survival strategy used to circumvent oxidative stress induced during host-pathogen interactions. Taken together, these data demonstrate the importance of the TCA cycle in the life cycle of this medically important pathogen.     

Selectivity of angiotensin II antisera

A significant problem in the immunoassay of angiotensin II is the cross-reactivity of most available antisera with the peptide's metabolic products, (des-Asp1)-angiotensin II and (des-Asp1.Arg2)-angiotensin II. In order to attempt to generate antisera of greater selectivity, a variety of conjugates between angiotensin II or derivative peptides and carrier proteins were examined as immunogens with the aim of generating antisera that would selectively identify the amino terminal region of the peptide. Selectivity for the amino terminus was achieved by either (1) immunization with N-acetylated angiotensin II-amide which had been coupled to rabbit serum albumin by its carboxy terminus, or (2) immunization with angiotensin-(1-7)-heptapeptide which was randomly coupled to thyroglobulin. The antisera produced with the N-acetylated immunogen cross-reacted with the unacetylated ligand (Asn1-Val5)-angiotensin, but did not recognize the human hormone (Asp1,Ile5)-angiotensin. Carboxy-terminal coupling of angiotensin without N-acetylation did not induce selectivity for the amino terminus, nor did a conjugate which was linked to the carrier protein via a diazo bond to His6 of the peptide. These findings may be explained by the fact that N-acetylated angiotensin II resists degradation by amino peptidases and thus retains its structure in the immunogen and by the fact that the (1-7)-heptapeptide has lost the immunodominant carboxy-terminal epitope, thus emphasizing the desired amino terminal determinant.     

Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching

Switching of Saccharomyces mating type by replacement of sequences at the MAT locus involves a choice between two donors, HML and HMR. MATα cells inhibit recombination along the entire left arm of chromosome III, including HML, whereas MATa cells activate this same region. MATa-dependent activation of HML depends on a small, cis-acting DNA sequence designated the recombination enhancer (RE), located 17 kb centromere-proximal to HML. A comparison of RE sequences interchangeable between Saccharomyces cerevisiae and Saccharomyces carlsbergensis defines a minimum RE of 244 bp. RE activity is repressed in MATα cells by binding of the Matα2–Mcm1 corepressor to a site within the RE. Mutation of the two Matα2 binding sites removes most, but not all, of this repression, and RE chromatin structure in MATα cells becomes indistinguishable from that seen in MATa. Surprisingly, a 2-bp mutation in the Mcm1 binding site completely abolishes RE activity in MATa cells; moreover, RE chromatin structure in the MATa mutant becomes very similar to that seen in MATα cells with a normal RE, displaying highly ordered nucleosomes despite the absence of Matα2. Further, a mutation that alters the ability of Mcm1 to act with Matα2 in repressing a-specific genes also alters donor preference in either mating type. Thus, Mcm1 is critically responsible for the activation as well as the Matα2-Mcm1-mediated repression of RE activity.     

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.