Susan E. Shadle

Daunorubicin Cardiotoxicity EVIDENCE FOR THE IMPORTANCE OF THE QUINONE MOIETY IN A FREE-RADICAL-INDEPENDENT MECHANISM

Anthracyclines, such as daunorubicin (Daun), and other quinone-containing compounds can stimulate the formation of toxic free radicals. The present study tests the hypothesis that the quinone moiety of Daun, by increasing free-radical production, disrupts sarcoplasmic reticulum (SR) function and thereby inhibits myocardial contractility in vitro. We compared Daun with its quinone-deficient analogue, 5-iminodaunorubicin (5-ID), using experimental interventions to produce various contractile states that depend on SR function. At concentrations of Daun or 5-ID that did not alter contractility (dF/dt) of steady-state contractions (1 Hz) in electrically paced atria isolated from adult rabbits, only Daun significantly attenuated the positive inotropic effects on dF/dt of increased rest intervals (PRP; post-rest potentiation) or increased stimulation frequencies. Attenuation was to 98+/-6% at 1 Hz, and 73+/-8 and 67+/-8% for 30 and 60 sec PRP, respectively, and 73+/-3 and 63 +/-3% at 2 and 3 Hz, respectively, for 88 microM Daun (P<0.05, vs pre-drug baseline values, mean +/- SEM). These effects of Daun were similar to those of caffeine (2 mM), an agent well known to deplete cardiac SR calcium. We also examined the effect of Daun in isolated neonatal rabbit atria, which lack mature, functional SR; Daun did not alter the force-frequency relationship or PRP contractions. Additional studies in Ca(2+)-loaded SR microsomes indicated that both Daun and 5-ID opened Ca(2+) release channels, with Daun being 20-fold more potent than 5-ID in this respect. Neither anthracycline, however, induced free-radical formation in SR preparations (assayed via nicking of supercoiled DNA) prior to stimulating Ca(2+) release. Thus, our results indicate that Daun impairs myocardial contractility in vitro by selectively interfering with SR function; the quinone moiety of Daun appears to mediate this cardiotoxic effect, acting through a mechanism that does not involve free radicals.

Doxorubicin cardiac dysfunction

Utilizing a model of chronic doxorubicin cardiomyopathy, this study examines the relationship between changes in expression and function of calcium handling proteins and contractile dysfunction. Apossible mechanism to account for this relationship is suggested. New Zealand white rabbits were injected with either doxorubicin (1 mg/kg, twice weekly for 8 wk) or 0.9% NaCl. Gene transcript, protein levels, and the function of several proteins from the left ventricle were assessed. Protein levels of sarcoplasmic reticulum (SR) Ca2+ transporting ATPase (SERCA2a and b), Ca2+ release channel (RYR2), calsequestrin, Na/Ca exchanger, mRNA levels of RYR2, and [3H]-ryanodine binding (Bmax) to RYR2 were significantly decreased in doxorubicin-treated rabbits; protein levels of phospholamban, dihydropyridine receptor α2 subunit, and SR Ca2+ loading rates were not decreased. However, only protein levels of SERCA2 and RYR2, mRNA levels of RYR2, and Bmax of RYR2 significantly regressed with left-ventricular fractional shortening. Analysis of contractile function of atrial preparations isolated from doxorubicin-treated rabbits revealed that doxorubicin diminished contractility (dF/dt) of rest-potentiated contractions consistent with SR dysfunction. Serum concentrations of free triiodothyronine (T3) decreased in doxorubicin-treated rabbits. Our results suggest that chronic doxorubicin administration in the rabbit causes a SR-dependent contractile dysfunction that may result, in part from decreased T3.

Structural model of the amino propeptide of collagen XI α1 chain with similarity to the LNS domains

Fibrillar collagens are the principal structural molecules of connective tissues. The assembly of collagen fibrils is regulated by quantitatively minor fibrillar collagens, types V and XI. A unique amino‐terminal propeptide domain of these collagens has been attributed this regulatory role. The structure of the amino terminal propeptide has yet to be determined. Low sequence similarity necessitated a secondary structure‐based method to carry out homology modeling based upon the determined structure of LNS family members, named for a common structure in the laminin LG5 domain, the neurexin 1B domain and the sex hormone binding globulin. Distribution of amino acids within the model suggested glycosaminoglycan interaction and calcium binding. These activities were tested experimentally. Sequence analyses of existing genes for collagens indicate that 16 known collagen α chains may contain an LNS domain. A similar approach may prove useful for structure/function studies of similar domains in other collagens with similar domains. This will provide mechanistic details of the organization and assembly of the extracellular matrix and the underlying basis of structural integrity in connective tissues. The absolute requirement for collagen XI in skeletal growth is indicated by collagen XI deficiencies such as chondrodystrophies found in the cho/cho mouse and in humans with Stickler syndrome.

Reduction of 13-deoxydoxorubicin and daunorubicinol anthraquinones by human carbonyl reductase.

Carbonyl reductase (CR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of several carbonyls. Anthracyclines used to treat cancer are reduced by CR at the C13 carbonyl and the resulting metabolites are implicated in the cardiotoxicity associated with anthracycline therapy. CR also is believed to have a role in detoxifying quinones, raising the question whether CR catalyzes reduction of anthracycline quinones. Steadystate kinetic studies were done with several anthraquinone-containing compounds, including 13-deoxydoxorubicin and daunorubicinol, which lack the C13 carbonyl, thus unmasking the anthraquinone for study. kcat and kcat/Km values for 13-deoxydoxorubicin and daunorubicinol were nearly identical, indicating that that the efficiency of quinone reduction was unaffected by the differences at the C13 position. kcat and kcat/Km values were much smaller for the analogs than for the parent compounds, suggesting that the C13 carbonyl is preferred as a substrate over the quinone. CR also reduced structurally related quinone molecules with less favorable catalytic efficiency. Modeling studies with doxorubicin and carbonyl reductase revealed that methionine 234 sterically hinder the rings adjacent to the quinone, thus accounting for the lower catalytic efficiency. Reduction of the anthraquinones may further define the role of CR in anthracycline metabolism and may influence anthracycline cytotoxic and cardiotoxic mechanisms.

Faculty Drivers and Barriers: Laying the Groundwork for Undergraduate STEM Education Reform in Academic Departments

BackgroundCalls to improve student learning and increase the number of science, technology, engineering, and math (STEM) college and university graduates assert the need for widespread adoption of evidence-based instructional practices in undergraduate STEM courses. For successful reforms to take hold and endure, it is likely that a significant shift in culture around teaching is needed. This study seeks to describe the initial response of faculty to an effort to shift teaching norms, with a long-term goal of altering the culture around teaching and learning in STEM. While the effort was envisioned and led at the institutional level, dialog about the proposed change and actions taken by faculty was emergent and supported within departments.ResultsFaculty identify a variety of barriers to proposed changes in teaching practice; however, faculty also identify a variety of drivers that might help the institution alter teaching and learning norms. Analysis of faculty responses reveals 18 categories of barriers and 15 categories of drivers in faculty responses. Many of the barrier and driver categories were present in each department’s responses; however, the distribution and frequency with which they appear reveals departmental differences that are important for moving forward with strategies to change teaching practice.ConclusionsAddressing faculty’s barriers to change is essential, but identifying and leveraging faculty’s drivers for the change is potentially equally important in efforts to catalyze changes that are supported or constrained by the local context. Further, the collection of faculty perspectives opens a dialog around the current and future state of teaching, an important step in laying the groundwork for change. Departmental differences in barriers and drivers make clear the importance of “knowing” the local contexts so strategies adopted by departments can be appropriately tailored. Results are discussed in light of what kind of strategies might be employed to effect changes in STEM education.

Study of Calsequestrin Aggregation by Flow Field‐Flow Fractionation with Light Scattering Detection

Abstract Flow field‐flow fractionation (FIFFF) with multi‐angle light scattering detection indicates that calsequestrin forms even numbered aggregates, supporting the view that this Ca2+ binding protein aggregates through the interaction of dimers. Contrary to previous reports based on size exclusion chromatography, FlFFF further indicates that the dimer is the stable species, with very little monomer present under the conditions analyzed in this study. Increasing the concentration of K+ (100–700 mM) causes the dimer to be the increasingly dominant species over monomer, tetramer, and other aggregate species. Increasing the concentration of Ca2+ (3–10 mM) causes increased aggregation of dimers into higher order species. Finally, addition of small amounts of the anthracycline analog trifluoperazine (0.10–0.50 mM), which is known to disrupt calsequestrin function, induces severe aggregation.

Real-world industry collaboration within a mechatronics class

This paper describes the implementation and assessment of an innovative senior/graduate level mechatronics (robotics) module that integrated structured and unstructured learning experiences, in collaboration with an industry partner. With real-world constraints and expectations, students designed and delivered a product as the final project. In fall 2007, the corporate partner provided state-of-the-art, programmable robotic kits with a user-friendly programming environment. The assigned project was to design a biomedical robot to work in a hospital intensive care unit (ICU) to perform tasks such as transporting supplies or delivering paperwork. Students with diverse skills and majors were grouped in ten teams, two to three students each. Student learning activities included designing a robot from a box of FisherTechnik materials, without the aid of instruction manuals; writing program code using the PCS environment; and integrating hardware and software. After four weeks of building, training, and testing, each teampsilas robot was unique. In the final competition, each robot was assigned to a particular room in the ICU to perform a specific task. Overall, the results indicated that the students gained hands-on experience with the state-of-art technology and effectively applied the conceptual course content to a real application.

Assessing the STEM landscape: the current instructional climate survey and the evidence-based instructional practices adoption scale

BackgroundThe efficacy of active learning within STEM education is clear, and many institutions are working to help faculty adopt evidence-based instructional practices (EBIPs) which can promote active learning. In order to know the current status of our campus regarding these goals, measures of current instructional climate and the adoption of evidence-based instructional practices (EBIPs) are desired.ResultsUsing a campus-wide online survey approach with remuneration for faculty participants, the 28-item current instructional climate survey (CICS) and the 6-item EBIP adoption scale were developed. When CICS and EBIP adoption scale outcomes are compared, patterns emerge which reflect the climate, conditions, and personal characteristics of faculty at different stages of EBIP adoption.ConclusionsAlthough not causal relationships, understanding both climate and personal change characteristics can be helpful to campus change agents in assessing the current STEM landscape of faculty practices.