Ca-binding and Ca-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca²⁺. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca²⁺ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10⁶ M⁻¹, and 21.58·10⁻⁶ moles·g⁻¹. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca²⁺ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca²⁺. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.     

Localization of the azoreductase ofClostridium perfringens by immuno-electron microscopy

An antibody againstClostridium perfringens azoreductase was used with protein A (gold-labeled) to locate the site of synthesis of extracellular azoreductase in this bacterium. Electron microscopy of immunogold-stained thin sections ofC. perfringens cells showed an average of 134 gold particles per cell, distributed throughout the cytoplasm and not associated with any organized structures.     

Inhibitors of lipoxygenase products improve survival in traumatic shock

We have used three selective inhibitors of arachidonic acid metabolism in order to investigate the role of lipoxygenase metabolites in the pathogenesis of traumatic shock (LD₉₀). The following inhibitors were used: CGS-5391B (2.5 mg/kg), a cyclooxygenase and lipoxygenase inhibitor, CGS-5677 (2.0 mg/kg), a selective lipoxygenase inhibitor, and U-60, 257 (0.3 mg/kg), a putative inhibitor of glutathione-s-transferase. These inhibitors did not alter arterial blood pressure or heart rate when given to sham shock rats. The traumatic shock model was characterized by a 4.5-fold increase in plasma cathepsin D activity, a 4-fold increase in plasma myocardial depressant factor (MDF) activity, and a mean survival time of 1.5 ± 0.2 h. Only the dual inhibitor significantly blunted the accumulation of cathepsin D in the plasma (7.5 ± 0.8 vs 11.3 ± 0.8 U/ml, p<0.01). However, all three inhibitors significantly suppressed plasma MDF accumulation by 50–60%: CGS-5391B, CGS-5677, and U-60,2257 (p<0.01). Moreover, these three agents significantly improved survival time in traumatic shock. The increased survival time and reduced MDF activity afforded by these inhibitors suggest a significant role for lipoxygenase metabolites, particularly LTC₄ and LTD₄, in the pathogenesis of traumatic shock.     

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.     

Fractures of the Calcaneus—Diagnosis and Treatment

Good lateral, Anthonsen oblique, anterior-posterior, and axial roentgenograms are necessary to define the extent of fractures of the calcaneus. Roentgenograms of the lumbar spine, including the thoracolumbar junction, are recommended whenever a fall has occurred sufficient to fracture the calcaneus.The great majority of calcaneal fractures are compression shearing fractures. Significant disruption of articular surfaces and spreading of the body beneath the fibula are major surgical problems and require careful treatment. In a series of 50 fractures treated by four methods, open reduction with stabilization by bone grafts, followed by active motion at six to eight weeks, gave the best results.     

A bioluminescent reporter for the halophilic archaeon Haloferax volcanii

Haloarchaea have evolved to thrive in hypersaline environments. Haloferax volcanii is of particular interest due to its genetic tractability; however, few in vivo reporters exist for halophiles. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesophilic reporter proteins lack these characteristics. Mesophilic proteins that acquire salt-stabilizing mutations, however, can lead to proper function in haloarchaea. Using laboratory-directed evolution, we developed and demonstrated an in vivo luciferase that functions in the hypersaline cytosol of H. volcanii.

     

Challenges and advice for MD/PhD applicants who are underrepresented in medicine

The importance of diversity is self-evident in medicine and medical research. Not only does diversity result in more impactful scientific work, but diverse teams of researchers and clinicians are necessary to address health disparities and improve the health of underserved communities. MD/PhD programs serve an important role in training physician-scientists, so it is critical to ensure that MD/PhD students represent diverse backgrounds and experiences. Groups who are underrepresented in medicine and the biomedical sciences include individuals from certain racial and ethnic backgrounds, individuals with disabilities, individuals from disadvantaged backgrounds, and women. However, underrepresented students are routinely discouraged from applying to MD/PhD programs due to a range of factors. These factors include the significant cost of applying, which can be prohibitive for many students, the paucity of diverse mentors who share common experiences, as well as applicants’ perceptions that there is inadequate support and inclusion from within MD/PhD programs. By providing advice to students who are underrepresented in medicine and describing steps programs can take to recruit and support minority applicants, we hope to encourage more students to consider the MD/PhD career path that will yield a more productive and equitable scientific and medical community.