Regional variation of the inwardly rectifying potassium current in the canine heart and the contributions to differences in action potential repolarization.

The inward rectifier potassium current, IK1, contributes to the terminal phase of repolarization of the action potential (AP), as well as the value and stability of the resting membrane potential. Regional variation in IK1 has been noted in the canine heart, but the biophysical properties have not been directly compared. We examined the properties and functional contribution of IK1 in isolated myocytes from ventricular, atrial and Purkinje tissue. APs were recorded from canine left ventricular midmyocardium, left atrial and Purkinje tissue. The terminal rate of repolarization of the AP in ventricle, but not in Purkinje, depended on changes in external K(+) ([K(+)]o). Isolated ventricular myocytes had the greatest density of IK1 while atrial myocytes had the lowest. Furthermore, the outward component of IK1 in ventricular cells exhibited a prominent outward component and steep negative slope conductance, which was also enhanced in 10 mM [K(+)]o. In contrast, both Purkinje and atrial cells exhibited little outward IK1, even in the presence of 10 mM [K(+)]o, and both cell types showed more persistent current at positive potentials. Expression of Kir2.1 in the ventricle was 76.9-fold higher than that of atria and 5.8-fold higher than that of Purkinje, whereas the expression of Kir2.2 and Kir2.3 subunits was more evenly distributed in Purkinje and atria. Finally, AP clamp data showed distinct contributions of IK1 for each cell type. IK1 and Kir2 subunit expression varies dramatically in regions of the canine heart and these regional differences in Kir2 expression likely underlie regional distinctions in IK1 characteristics, contributing to variations in repolarization in response to in [K(+)]o changes.

Student Perceived Value of Anatomy Pedagogy, Part I: Prosection or Dissection?

A series of three annual surveys of David Geffen School of Medicine (DGSOM) at UCLA students and UCR/UCLA Thomas Haider Program in Biomedical Sciences students were administered from 2010 to 2012 to ascertain student perceptions of which anatomy pedagogy-prosection or dissection-was most valuable to them during the first year of preclinical medical education and for the entire medical school experience in general. Students were asked, "What value does gross anatomy education have in preclinical medical education?" We further asked the students who participated in both prosection and dissection pedagogies, "Would you have preferred an anatomy curriculum like the Summer Anatomy Dissection during your first year in medical school instead of prosection?" All students who responded to the survey viewed anatomy as a highly valued part of the medical curriculum, specifically referring to four major themes: Anatomy is (1) the basis for medical understanding, (2) part of the overall medical school experience, (3) a bridge to understanding pathology and physiology, and (4) the foundation for clinical skills. Students who participated in both prosection and dissection pedagogies surprisingly and overwhelmingly advocated for a prosection curriculum for the first year of medical school, not a dissection curriculum. Time efficiency was the dominant theme in survey responses from students who learned anatomy through prosection and then dissection. Students, regardless of whether interested in surgery/radiology or not, appreciated both pedagogies but commented that prosection was sufficient for learning basic anatomy, while dissection was a necessary experience in preparation for the anatomical medical specialties. This suggests that anatomy instruction should be integrated into the clinical years of medical education.

Student Perceived Value of Anatomy Pedagogy, Part II: Clinical Practice and Assessment.

We describe student beliefs of how anatomy education influenced their preparation for standardized clinical assessments and clinical skills. We conducted three annual surveys of students of the David Geffen School of Medicine (DGSOM) at the University of California Los Angeles (UCLA) and students of the University of California, Riverside (UCR)/UCLA Thomas Haider Program in Biomedical Sciences from 2010 to 2012. Students were asked, "What specific knowledge or skills did you learn from your gross anatomy experience that helped you prepare for USMLE board exams, third-year clerkships, and physical examination skills?" All students who responded to the survey viewed anatomy as a highly valued part of the medical curriculum. Almost all students felt that anatomy knowledge in general was useful for their success with United States Medical Licensing Examination (USMLE) exams, how they perceived their physical exam skills, and how they perceived their preparation for third- or fourth-year clerkships. On the other hand, when asked about how the anatomy curriculum helped prepare students for fourth-year clerkships, there was a downward trend over a three-year period with each subsequent class. Although anatomy is a highly valued part of the medical school experience, students value integration of the anatomical and clinical sciences, as evidenced by a perceived diminishing value of anatomy pedagogy taught outside of clinical context with subsequent classes over the course of three years.

Progressive visceral leishmaniasis is driven by dominant parasite-induced STAT6 activation and STAT6-dependent host arginase 1 expression.

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

LPS-induced CCL2 expression and macrophage influx into the murine central nervous system is polyamine-dependent.

Increased polyamine production is observed in a variety of chronic neuroinflammatory disorders, but in vitro and in vivo studies yield conflicting data on the immunomodulatory consequences of their production. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in endogenous polyamine production. To identify the role of polyamine production in CNS-intrinsic inflammatory responses, we defined CNS sites of ODC expression and the consequences of inhibiting ODC in response to intracerebral injection of LPS±IFNγ. In situ hybridization analysis revealed that both neurons and non-neuronal cells rapidly respond to LPS±IFNγ by increasing ODC expression. Inhibiting ODC by co-injecting DFMO decreased LPS-induced CCL2 expression and macrophage influx into the CNS, without altering LPS-induced microglial or macrophage activation. Conversely, intracerebral injection of polyamines was sufficient to trigger macrophage influx into the CNS of wild-type but not CCL2KO mice, demonstrating the dependence of macrophage influx on CNS expression of CCL2. Consistent with these data, addition of putrescine and spermine to mixed glial cultures dramatically increased CCL2 expression and to a much lesser extent, TNF expression. Addition of all three polyamines to mixed glial cultures also decreased the numbers and percentages of oligodendrocytes present. However, in vivo, inhibiting the basal levels of polyamine production was sufficient to induce expression of apolipoprotein D, a marker of oxidative stress, within white matter tracts. Considered together, our data indicate that: (1) CNS-resident cells including neurons play active roles in recruiting pro-inflammatory TREM1-positive macrophages into the CNS via polyamine-dependent induction of CCL2 expression and (2) modulating polyamine production in vivo may be a difficult strategy to limit inflammation and promote repair due to the dual homeostatic and pro-inflammatory roles played by polyamines.

Identification and characterization of a diamine exporter in colon epithelial cells.

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.

Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells.

Alpha-difluoromethylornithine (DFMO) inhibits the proto-oncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G1 cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G1 cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27Kip1. In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G1/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation.

A stable, inducible, dose-responsive ODC overexpression system in human cell lines.

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media.

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]

Polyamines modulate the interaction between nuclear receptors and vitamin D receptor-interacting protein 205.

Nuclear receptors (NR) activate transcription by interacting with several different coactivator complexes, primarily via LXXLL motifs (NR boxes) of the coactivator that bind a common region in the ligand binding domain of nuclear receptors (activation function-2, AF-2) in a ligand-dependent fashion. However, how nuclear receptors distinguish between different sets of coactivators remains a mystery, as does the mechanism by which orphan receptors such as hepatocyte nuclear factor 4alpha (HNF4alpha) activate transcription. In this study, we show that HNF4alpha interacts with a complex containing vitamin D receptor (VDR)-interacting proteins (DRIPs) in the absence of exogenously added ligand. However, whereas a full-length DRIP205 construct enhanced the activation by HNF4alpha in vivo, it did not interact well with the HNF4alpha ligand binding domain in vitro. In investigating this discrepancy, we found that the polyamine spermine significantly enhanced the interaction between HNF4alpha and full-length DRIP205 in an AF-2, NR-box-dependent manner. Spermine also enhanced the interaction of DRIP205 with the VDR even in the presence of its ligand, but decreased the interaction of both HNF4alpha and VDR with the p160 coactivator glucocorticoid receptor interacting protein 1 (GR1P1). We also found that GR1P1 and DRIP205 synergistically activated HNF4alpha-mediated transcription and that a specific inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), decreased the ability of HNF4alpha to activate transcription in vivo. These results lead us to propose a model in which polyamines may facilitate the switch between different coactivator complexes binding to NRs.