Patricia S. Sexton

Inhibition of B16 melanoma metastasis by overexpression of the cysteine proteinase inhibitor cystatin C.

Progression to metastasis has been correlated with increased cysteine proteinase activity for a number of tumour types. One mechanism of cysteine proteinase regulation in normal cells is by natural protease inhibitors, the cystatins. Here we further characterize a transfected cell line showing increased cystatin C transcription driven by cytomegalovirus (CMV) promoter/enhancer sequences. Properties of this cystatin C altered cell line such as growth in vitro, lung colonization after tail vein injection in mice, production of cystatin, and cysteine proteinase inhibitor activities were examined. Although there was no difference between the growth rate of the cystatin transfected cell line and that of the control, there was a substantial difference in metastatic ability. No increase was noted in cystatin C secretion into the media for the cystatin C transfected cell line compared with the control transfected cell line. There was, however, a difference in cysteine protease inhibitor activity in the cell-free extracts. These results show that alteration of cystatin C levels by overexpression in B16 melanoma alters properties associated with metastasis.

Inhibition of motility and invasion of B16 melanoma by the overexpression of cystatin C.

Although increased expression of cysteine proteinases has been shown to be correlated with increased metastasis for a wide variety of tumours, the contribution of cysteine proteinases to the metastatic spread of tumour cells is not well understood. In order to examine this question we have overexpressed a specific cysteine proteinase inhibitor, cystatin C, by stable transfection of B16F10 melanoma. Increased expression of cystatin C inhibited motility and in vitro invasiveness of B16 melanoma by 50% in both stimulated (autocrine motility factor, laminin) and unstimulated cells. These results suggest that cysteine proteinases are involved in B16 melanoma motility and invasion.

Discordant expression of the sterol pathway in lens underlies simvastatin-induced cataracts in Chbb Thom rats

Simvastatin rapidly induced cataracts in young Chbb:Thom (CT) but not Sprague Dawley (SD) or Hilltop Wistar (HW) rats. Oral treatment for 14 but not 7 days committed CT rat lenses to cataract formation. The cholesterol to phospholipid molar ratio in lenses of treated CT rats was unchanged. Differences between strains in serum and ocular humor levels of simvastatin acid poorly correlated with susceptibility to cataracts. No significant differences were found between rat strains in the capacity of simvastatin acid to inhibit lens-basal sterol synthesis. Prolonged treatment with simvastatin comparably elevated HMG-CoA reductase protein and enzyme activity in lenses of both cataract resistant and sensitive strains. However, in contrast to SD and HW rats, where sterol synthesis was markedly increased, sterol synthesis in CT rat lenses remained at baseline. Discordant expression of sterol synthesis in CT rats may be due to inadequate upregulation of lens HMG-CoA synthase. HMG-CoA synthase protein levels, and to a much lesser extent mRNA levels, increased in lens cortex of SD but not CT rats. Because upregulation of the sterol pathway may result in increased formation of isoprene-derived anti-inflammatory substances, failure to upregulate the pathway in CT rat lenses may reflect an attenuated compensatory response to injury that resulted in cataracts.

Immunomagnetic capture of lens membrane fractions containing steroid binding protein.

This study describes the use of magnetic Dynabeads to purify microsomes from a crude microsomal fraction. A 28 kDa membrane-associated protein is proposed to mediate the binding of progesterone and other steroid hormones to ocular lens membranes and the rapid-nongenomic actions of these steroids. The subcellular location of this membrane steroid binding protein (MSBP) was probed by capture of organelles containing MSBP by magnetic beads displaying an antibody to a cytoplasmic domain of the protein. The beads were exposed to a crude microsomal fraction from lens epithelia. Western blotting was used to identify captured organelles and confirm the presence of MSBP. Microsomes and trace fiber cell plasma membrane were captured. Microsomes contained the 28 kDa MSBP. Lens fiber cell membrane contained a 55 kDa immunoreactive protein. The role of this serendipitously recognized protein in binding of steroids is unknown.

Comparison of effects of U18666A and enantiomeric U18666A on sterol synthesis and induction of apoptosis

Treatment of animals or cells with the amphipathic tertiary amine U18666A {3β-[2-(diethylamino) ethoxy]androst-5-en-17-one} provides models for several human diseases (e.g., cataracts, Niemann-Pick disease, and epilepsy). Although U18666A can inhibit several enzymes in the cholesterol synthesis pathway, we hypothesized that induction of these varied conditions was due to physical effects of the amine rather than to inhibition of specific proteins. To test this possibility we compared the capacity of U18666A and its enantiomer, ent-U18666A, to inhibit net sterol synthesis and induce apoptosis in cultured bovine lens epithelial cells. Nonenantiospecific actions dependent on the physical properties of these mirror image molecules would be identical, but effects dependent upon enantiospecific interactions would be different for the enantiomers. At the same concentrations, both forms of the compound equally inhibited sterol synthesis and induced apoptosis. These observations supported a generalized mechanism of enzyme inhibition such as perturbation of the microenvironment of endoplasmic enzymes and alteration of membrane order, perhaps of the mitochondrial membrane, to explain induction of apoptosis.

Calbindin Immunoreactivity in Purkinje Cells of the Bullfrog Cerebellum during Thyroxine-Induced Metamorphosis

Calbindin-immunoreactive Purkinje cells were identified in the cerebella of frog tadpoles that had been treated with thyroxine to accelerate metamorphosis. The dorsal part of the cerebellar plate contained the full complement of Purkinje cells which were all CaBP-immunoreactive, while in the ventral part of the cerebellum Purkinje cells acquired CaBP-immunoreactivity only after several days of thyroxine treatment. The ventral group of Purkinje cells was separated from the dorsal group by a distinct gap, which is the site of a shallow sulcus in adult frogs. Additionally, following thyroxine treatment, the numbers of CaBP-immunoreactive Purkinje cells in the ventral group were only half the numbers seen in frogs that metamorphosed spontaneously. We suggest that the variation in the CaBP-immunoreactivity of the dorsal and ventral groups of Purkinje cells, along with the gap in the Purkinje cell layer between the two groups, may be indicative of two distinct populations of Purkinje cells, with distinct patterns of generation, maturation, and perhaps, origin and connectivity, in the cerebellum of frogs.

American sign language and deaf culture competency of osteopathic medical students.

The study examined the effectiveness of a workshop on Deaf culture and basic medical American Sign Language for increasing osteopathic student physicians’ confidence and knowledge when interacting with ASL-using patients. Students completed a pretest in which they provided basic demographic information, rated their confidence levels, took a video quiz on basic medical signs, and experienced a practical standardized encounter with a Deaf patient. They then attended a 4-hour workshop and, 2 weeks later, completed a posttest. Thirty-three students completed the pretest; 29 attended the workshop; 26 completed the posttest. Video quiz scores increased significantly from pretest to posttest, as did scores for the standardized patient encounter after completion of the workshop. Students also reported increased levels of confidence in interactions with the Deaf community. The results suggest that a single workshop was effective in increasing both confidence and short-term knowledge in interactions with Deaf patients.

Entrustable Professional Activities for Entering Residency: Establishing Common Osteopathic Performance Standards in the Transition From Medical School to Residency

Entrustable professional activities (EPAs) are measurable units of observable professional practice that can be entrusted to an unsupervised trainee. They were first introduced as a method of operationalizing competency-based medical education in graduate medical education. The American Association of Medical Colleges subsequently used EPAs to establish the core skills that medical students must be able to perform before they enter residency training. A recently published guide provides descriptions, guidelines, and rationale for implementing and assessing the core EPAs from an osteopathic approach. These osteopathically informed EPAs can allow schools to more appropriately assess a learners whole-person approach to a patient, in alignment with the philosophy of the profession. As the single accreditation system for graduate medical education moves forward, it will be critical to integrate EPAs into osteopathic medical education to demonstrate entrustment of medical school graduates. The authors describe the collaborative process used to establish the osteopathic considerations added to EPAs and explores the challenges and opportunities for undergraduate osteopathic medical education.