Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

PAR-3 is required for epithelial cell polarity in the distal spermatheca of C. elegans

PAR-3 is localized asymmetrically in epithelial cells in a variety of animals from Caenorhabditis elegans to mammals. Although C. elegans PAR-3 is known to act in early blastomeres to polarize the embryo, a role for PAR-3 in epithelial cells of C. elegans has not been established. Using RNA interference to deplete PAR-3 in developing larvae, we discovered a requirement for PAR-3 in spermathecal development. Spermathecal precursor cells are born during larval development and differentiate into an epithelium that forms a tube for the storage of sperm. Eggs must enter the spermatheca to complete ovulation. PAR-3-depleted worms exhibit defects in ovulation. Consistent with this phenotype, PAR-3 is transiently expressed and localized asymmetrically in the developing somatic gonad, including the spermathecal precursor cells of L4 larvae. We found that the defect in ovulation can be partially suppressed by a mutation in IPP-5, an inositol polyphosphate 5-phosphatase, indicating that one effect of PAR-3 depletion is disruption of signaling between oocyte and spermatheca. Microscopy revealed that the distribution of AJM-1, an apical junction marker, and apical microfilaments are severely affected in the distal spermatheca of PAR-3-depleted worms. We propose that PAR-3 activity is required for the proper polarization of spermathecal cells and that defective ovulation results from defective distal spermathecal development.     

Transcellular and paracellular elements of salt chemosensation in toad skin

Dehydrated toads absorb water by pressing a specialized (seat patch) area of the skin to moist surfaces. This behavior, the water absorption response (WR), is preceded by periods of more limited skin contact (seat patch down, SPD) in which the suitability of the rehydration source is evaluated. WR and SPD behaviors were suppressed on 250 mM NaCl and 200 mM KCl solutions. Ten micromolar amiloride partially restored SPD and WR on NaCl solutions. The addition of 5 mM La(3+) also partially restored the initiation of WR and this effect was additive to the effect of amiloride, suggesting transcellular and paracellular pathways exist in parallel. Similarly, 5 mM La(3+) partially restored the initiation of WR on KCl solutions, to levels comparable to those with K(+)gluconate, suggesting a paracellular pathway for detection of K(+). Hyperosmotic (250 mM) NaCl solutions bathing the mucosal surface rapidly and reversibly increased the paracellular conductance of isolated skin and this increase was partially inhibited by 5 mM La(3+). These results suggest that the regulation of tight junctions has a chemosensory role in toad skin.     

Members of a New Group of Chitinase-Like Genes are Expressed Preferentially in Cotton Cells with Secondary Walls

Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the beta -d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.     

Synthesis and activity of 1-aryl-1'-imidazolyl methyl ethers as non-thiol farnesyltransferase inhibitors

A series of imidazole-containing methyl ethers (4-5) have been designed and synthesized as potent and selective farnesyltransferase inhibitors (FTIs) by transposition of the D-ring to the methyl group on the imidazole of the previously reported FTIs 3. Several compounds such as 4h and 5b demonstrate superior enzymatic activity to the current benchmark compound tipifarnib (1) with IC(50) values in the lower subnanomolar range, while maintaining excellent cellular activity comparable to tipifarnib. The compounds are characterized as being simple, easier to make, and possess no chiral center involved.     

Design and synthesis of 4-phenyl piperidine compounds targeting the mu receptor

Small molecule mu agonists based on the 4-phenyl piperidine scaffold were designed and synthesized to further investigate the therapeutic potential of loperamide analogs. The resulting compounds show excellent agonistic activity towards the human mu receptor with interesting SAR trends within the series.     

Design, synthesis, and activity of 4-quinolone and pyridone compounds as nonthiol-containing farnesyltransferase inhibitors

As a part of our efforts to identify potent inhibitors of farnesyltransferase (FTase), modification of the structure of tipifarnib through structure-based design was undertaken by replacing the 2-quinolones with 4-quinolones and pyridones, and subsequent relocation of the D-ring to the N-methyl group on the imidazole ring. This study has yielded a novel series of potent and selective FTase inhibitors. The X-ray structure of tipifarnib (1) in complex with FTase was described.     

Novel pyrazolopyrimidine derivatives as GSK-3 inhibitors

A series of [1-aryl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]arylhydrazones were discovered as novel inhibitors glycogen synthase kinase-3 (GSK-3). Based on initial modeling a detailed SAR was constructed. Modification of the interior binding aryl ring (Ar(1)) determined this to be a tight binding region with little room for modification. As predicted from the model, a large variety of modifications could be incorporated into the hydrazone aryl ring. This work led to GSK-3 inhibitors in the low nano-molar range.     

A fresh look at the wolf-pack theory of companion-animal dog social behavior

A popular perspective on the social behavior of dogs in multiple-dog households sees the dogs' behavior as reflecting the sociobiological laws of the rigidly structured dominance hierarchy that has been described for wolf packs. This view suggests that aggression problems among dogs are natural expressions of conflict that arise whenever dominance status is in contention. One recommended solution has been for the owner to endorse and enforce a particular dominance hierarchy because, on the wolf pack model, aggression is minimized when the structure of the hierarchy is clear, strong, and stable. This article questions the validity of this perspective on 2 principal grounds. First, because it does not seem to occur in the wild, this article suggests the strong dominance hierarchy that has been described for wolves may be a by-product of captivity. If true, it implies that social behavior—even in wolves—may be a product more of environmental circumstances and contingencies than an instinctive directive. Second, because feral dogs do not exhibit the classic wolf-pack structure, the validity of the canid, social dominance hierarchy again comes into question. This article suggests that behavioral learning theory offers another perspective regarding the behavior of dogs and wolves in the wild or in captivity and offers an effective intervention for aggression problems.