Autotaxin inhibition: development and application of computational tools to identify site-selective lead compounds.

Autotaxin (ATX) catalyzes the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). Both ATX and LPA have been linked to pathophysiologies ranging from cancer to neuropathic pain. Inhibition of LPA production by ATX is therefore of therapeutic interest. Here we report the application of previously-developed, subsite-targeted pharmacophore models in a screening workflow that involves either docking or binary QSAR as secondary filters to identify ATX inhibitors from previously unreported structural types, four of which have sub-micromolar inhibition constants. Cell-based assays demonstrate that ATX inhibition and cytotoxicity structure-activity-relationships (SAR) exhibit selectivity cliffs, characterized by structurally similar compounds exhibiting similar biological activities with respect to ATX inhibition but very different biological activities with respect to cytotoxicity. Thus, general cytotoxicity should not be used as an early filter to eliminate candidate ATX inhibitor scaffolds from further SAR studies. Assays using two substrates of vastly different sizes demonstrate that the tools developed to identify compounds binding outside the central core of the active site did identify compounds acting at an allosteric site. In contrast, tools developed to identify active-site directed compounds did not identify active-site directed compounds. The stronger volume overlap imposed when selecting screening candidates expected to bind outside the active site is likely responsible for the stronger match between intended and actual target site.

Localization of sulphonylurea receptor proteins SUR2A and SUR2B and/or SUR1 in rat kidney.

To further explore precise expression and localization of sulphonylurea receptor isoforms SUR2A and SUR2B (SUR1) in rat kidney, total RNA was isolated from the kidney tissue using the TRIzol kit. Three different primer sets designed against SUR isoforms were used in reverse transcriptase reactions. Western blotting was done on membrane fractions obtained from kidney tissues using the primary antisera for SUR2A and SUR2B (SUR1). Paraformaldehyde fixed kidney sections were immunostained with SUR2A and SUR2B (SUR1) primary antisera. Sections were developed with DAB as a chromogen. RT-PCR results demonstrated mRNA consistent with SUR1 isoform to be the only identifiable transcript. Western blotting could not identify any protein consistent with SUR2A or SUR2B (SUR1) but recognized instead a smaller 55kD protein of unknown identity. Immunohistochemistry demonstrated a differential staining pattern whereby SUR2A was localized to the mesangial cells, intra- and extrarenal blood vessels and smooth muscles. In contrast, SUR2B (SUR1) was localized only to distal nephron epithelia. Intense immunoreactivity was localized to the thick ascending limb and as well as in the outer and inner medullary collecting ducts, both. Our results demonstrate differential and highly localized expression pattern of sulphonylurea receptor proteins SUR2A and 2B (SUR1) in rat kidney with implications for drug design.

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1.

The zebrafish, Danio rerio, is emerging as an important model organism for the pathophysiological study of some human kidney diseases, but the sites of expression and physiological roles of a number of protein orthologues in the zebrafish nephron remain mostly undefined. Here we show that a zebrafish potassium channel is orthologous to the mammalian kidney potassium channel, ROMK. The cDNA (kcnj1) encodes a protein (Kcnj1) that when expressed in Xenopus laevis oocytes displayed pH- and Ba2+-sensitive K+-selective currents, but unlike the mammalian channel, was completely insensitive to the peptide inhibitor tertiapin-Q. In the pronephros, kcnj1 transcript expression was restricted to a distal region and overlapped with that of sodium­chloride cotransporter Nkcc, chloride channel ClC-Ka, and ClC-Ka/b accessory subunit Barttin, indicating the location of the diluting segment. In a subpopulation of surface cells, kcnj1 was coexpressed with the a1a.4 isoform of the Na+/K+-ATPase, identifying these cells as potential K+ secretory cells in this epithelium. At later stages of development, kcnj1 appeared in cells of the developing gill that also expressed the a1a.4 subunit.Morpholino antisense-mediated knockdown of kcnj1 was accompanied by transient tachycardia followed by bradycardia, effects consistent with alterations in extracellular K+ concentration in the embryo.Our findings indicate that Kcnj1 is expressed in cells associated with osmoregulation and acts as a K+ efflux pathway that is important in maintaining extracellular levels of K+ in the developing embryo.

Association Between Learning Approaches and Medical Student Academic Progression During Preclinical Training.

PURPOSE: Students have diverse learning preferences that can impact the achievement of learning outcomes. However, there is a lack of unequivocal evidence for an association between the learning preferences and academic success. The purpose of the study was to examine the association between learning approaches and academic success of medical students during the basic science curriculum. METHODS: In this cross-sectional comparative study, low-achieving (n=80) and high-achieving students (n=50) from semesters 1 through 3 of the Ross University School of Medicine's basic science curriculum completed the Approaches and Study Skills Inventory for Students (ASSIST) short-form to provide data on their learning approaches. Student's-t test was applied to find statistical differences in learning approaches of low and high achievers, and point-biserial was used to analyze the correlation between academic performance and learning approaches. Mean sum scores with standard deviation on ASSIST short-form scales (deep, surface, and strategic approaches) as well as subscales are reported. RESULTS: High-achieving students reported a preference for using deep and strategic learning approaches compared to low-achieving students (p < 0.05). Low achievers indicated that they predominantly used the surface approach to learning (p<0.05). Yet, "fear of failure," a subscale of the surface approach, was greater among high achievers. Additionally, significant gender differences were found on subscales of "lack of purpose," "syllabus bound," "unrelated memorization" (surface approach), and "time management" and "organized studying" (strategic approach). CONCLUSION: Our results suggest that low-achieving medical students' predominant reliance on the surface approach to learning may affect their academic success and that it may be worthwhile to help medical students become aware of the effectiveness of their individual preferred learning approaches early on in their training. Identification and adoption of optimal learning approaches should increase the achievement of successful learning outcomes.

Intraepithelial gammadelta+ lymphocytes maintain the integrity of intestinal epithelial tight junctions in response to infection.

BACKGROUND & AIMS: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. METHODS: Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. RESULTS: In the absence of gammadelta+ iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in gammadelta+ iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of gammadelta T-cell receptor (TCR-Vgamma7+) iIELs that, after reconstituting gammadelta iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. CONCLUSIONS: These findings identify a novel role for gammadelta+ iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.

Renal effects of glibenclamide in cystic fibrosis mice.

In vitro studies have shown that glibenclamide sensitivity is conferred upon Kir 1.1 K(+) channels when they are co-expressed with the cystic fibrosis transmembrane conductance regulator (CFTR). In rats, glibenclamide acts as a K(+)-sparing diuretic by a mechanism that involves blockade of Kir 1.1 channels in the distal nephron. To test whether interaction between Kir 1.1 and CFTR is required to mediate the renal effects of glibenclamide (15 mg/kg), clearance experiments were performed comparing wild type (WT) and Cftr(tm2cam) Delta F508 cystic fibrosis (CF) mice. Glibenclamide treatment was associated with an equivalent diuresis in both WT and CF mice. Glibenclamide was K(+)-sparing in both genotypes with no significant change in urinary K(+) excretion observed. That glibenclamide was an effective K(+)-sparing diuretic in CF animals suggests that CFTR expression is not a requirement to mediate its renal actions in mice.

Evidence for endocytosis of ROMK potassium channel via clathrin-coated vesicles.

ROMK channels are present in the cortical collecting ducts of kidney and are responsible for K(+) secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K(+) secretion in cortical collecting ducts. We investigated the molecular mechanisms for endocytosis of ROMK channels expressed in Xenopus laevis oocytes and cultured Madin-Darby canine kidney cells. When plasma membrane insertion of newly synthesized channel proteins was blocked by incubation with brefeldin A, ROMK currents decreased with a half-time of ~6 h. Coexpression with the Lys44-->Ala dominant-negative mutant dynamin, but not wild-type dynamin, reduced the rate of reduction of ROMK in the presence of brefeldin A. Mutation of Asn371 to Ile in the putative NPXY internalization motif of ROMK1 abolished the effect of the Lys44-->Ala dynamin mutant on endocytosis of the channel. Coimmunoprecipitation study and confocal fluorescent imaging revealed that ROMK channels associated with clathrin coat proteins in Madin-Darby canine kidney cells. These results provide compelling evidence for endocytosis of ROMK channels via clathrin-coated vesicles.

The basolateral expression of mUT-A3 in the mouse kidney.

Facilitative UT-A urea transporters play a central role in the urinary concentrating mechanism. There are three major UT-A isoforms found in the mouse kidney: mUT-A1, mUT-A2, and mUT-A3. The major aim of this study was to identify the location and function of mUT-A3. UT-A proteins were investigated using three novel mouse UT-A-targeted antibodies: ML446, MQ2, and ML194. ML446 detected mUT-A1 and mUT-A3. ML194 detected mUT-A1 and mUT-A2. Importantly, MQ2 was found to be selective for mUT-A3. MQ2 detected a 45- to 65-kDa signal in the mouse kidney inner medulla, which was deglycosylated to a 40-kDa protein band. Immunolocalization studies showed that mUT-A3 was strongly detected in the papillary tip, mainly in the basolateral regions of inner medullary collecting duct (IMCD) cells. Immunoblotting of subcellular fractions of inner medullary protein suggested that in mouse kidney mUT-A3 was present in plasma membranes. Consistent with this, immunoelectron microscopy demonstrated that mUT-A3 was predominantly localized at the basal plasma membrane domains of the IMCD cells in mouse kidney. Heterologous expression of mUT-A3-enhanced green fluorescent protein in Madin-Darby canine kidney cells showed that the protein localized to the basolateral membrane. In conclusion, our study indicates that mUT-A3 is a basolateral membrane transporter expressed in IMCD cells.