Teaching physiology online: successful use of case studies in a graduate course.

To address the need for greater flexibility in access to higher education, an online graduate course in physiology using case studies was developed and offered in summer 2012. Topics in both animal and human physiology were organized as modules that contained a case study with questions, a prerecorded online lecture, and three research journal articles. We followed best practices for teaching and learning in distance education, including the preparation of materials before the course starting date, a discussion board for responding to pre- and postcase discussion questions, and prompt reply to student queries. For exams, students generated their own questions based on new cases and developed their own case study for the final project. Although only 20% of students had previously taken an online course, all students stated that they would recommend this course to others. Postcase assessment indicated that students found the cases interesting, informative, and presented at the appropriate level. Most students said that the online course took them more time but that they learned more content and used the primary literature more than in a traditional class. Our results indicate that a well-organized physiology course using a case study format is a very effective model for online learning.

Molecular weight of different angiotensin II polymers directly determines: density of endothelial membrane AT1 receptors and coronary vasoconstriction.

We have shown that angiotensin II (Ang II) does not diffuse across the vessel wall, remaining intravascularly confined and acting solely on the coronary endothelial luminal membrane (CELM) receptors. A sustained intracoronary infusion of Ang II causes transient coronary vasoconstriction (desensitization) due to membrane internalization of CELM Ang II type 1 receptors (CELM-AT1R). In contrast, sustained intracoronary infusion of a non-diffusible polymer of Ang II (Ang II-Pol, 15,000 kDa) causes a sustained vasoconstriction by preventing CELM-AT1R internalization. In addition, a sustained intracoronary infusion of Ang II leads to a depressed response following a secondary Ang II administration (tachyphylaxis) that is reversed by Ang II-Pol. These findings led us to hypothesize that the rate of desensitization, tachyphylaxis, and AT1R internalization were dependent on Ang II-Pol molecular weight. To test this hypothesis, we synthesized Ang II-Pols of the following molecular weights (in kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15,000. Vasoconstriction was measured following intracoronary infusion of Ang II-Pols in Langendorff-perfused guinea pig hearts at constant flow. The CELM protein fraction was extracted using the silica pellicle technique at different time points in order to determine the rate of AT1R internalization following each Ang II-Pol infusion. CELM-AT1R density was quantified by Western blot. We found that the rate of desensitization and the tachyphylaxis effect varied inversely with the molecular weight of the Ang II-Pols. Inversely proportional to the molecular weight of Ang II-Pol the CELM-AT1R density decreases over time. These results indicate that the mechanism responsible for the decreased rate of desensitization and tachyphylaxis by higher molecular weight Ang II polymers is due to reduction in the rate of CELM-AT1R internalization. These Ang II polymers would be valuable tools for studying the relationship between AT1R internalization and physiological effects.

Role of adherens junction proteins in differential herpes simplex virus type 2 infectivity in communication-competent and -deficient cell lines.

BACKGROUND: Gap junctional intercellular communication decreases with HSV-2 infection. To determine the importance of functional gap junctions for infectivity, we compared HSV-2 growth in communication-competent and -deficient cell lines. METHODS: HSV-2 infectivity was tested in five cell lines: WB rat liver epithelial cells (communication-competent), WB-aB1 (communication-deficient), WB-a/32-10 (communication-rescued), HeLa (communication-deficient), and Cx43-transfected HeLa (communication-rescued) cells. HSV-2 growth curves and indirect immunofluorescence labeling of viral and cell proteins were performed in wild-type and mutant WB cells. RESULTS: Although wild-type WB cells were highly permissive for HSV-2 infection, virus production was significantly attenuated in communication-deficient and -rescued mutant WB cells. HeLa exhibited no difference in infectivity between communication-competent and -deficient cell lines. Tight and adherens junction proteins, including zonula occludens-1 and nectin-1, were not different in the WB cell lines. However, E-cadherin levels were elevated and ß-catenin was found to co-localize with glycoprotein E, a viral glycoprotein associated with cell-to-cell spread, in the mutant WB cells. CONCLUSIONS: These results suggest that attenuated viral production in mutant WB cells is due to viral protein co-localization with adherens junction proteins rather than the loss or restoration of functional gap junctions.

Herpes simplex virus-type 2 infectivity and agents that block gap junctional intercellular communication.

In rat liver epithelial (WB) cells, the protein kinase C inhibitor H7 blocked gap junctional intercellular communication (GJIC) and reduced virus infectivity. Octanol, 18-beta-glycyrrhetinic acid, and staurosporine, agents that reduce GJIC, had no effect upon virus infectivity. Previous studies demonstrated that herpes simplex virus-type 2 (HSV-2) infection was accompanied by attenuated GJIC. Of agents tested, only H7 reduced plaque forming unit (pfu) ability in a dose-dependent manner with 100% plaque reduction at 40 microM without evidence of cytotoxicity. Dye transfer indicated that H7 decreased GJIC, although Western blotting revealed that it did not alter phosphorylation of the gap junction protein, connexin 43 (Cx43). Using indirect immunofluorescence, Cx43 was found to localize in membrane plaques in uninfected cells and H7 did not alter this distribution. However, Cx43 was lost from the membrane at 24h in both H7-treated and untreated cells infected with HSV-2. Viral infection increased serine phosphorylation, particularly in the nuclear region, and this effect was reduced following H7 treatment. Thus, H7 attenuated both GJIC and infectivity of HSV-2 in WB cells but the anti-viral effects were due to reduced nuclear protein phosphorylation rather than alterations in phosphorylation or localization of Cx43.