IL-17E, a novel proinflammatory ligand for the IL-17 receptor homolog IL-17Rh1

We report identification of interleukin (IL)-17E, a novel member of the IL-17 family of cytokines. IL-17E is a ligand for the recently identified protein termed EVI27/IL-17BR, which we term IL-17 receptor homolog 1 (IL-17Rh1) in light of the multiple reported ligand-receptor relationships. Murine EVI27 was identified through its location at a common site of retroviral integration in BXH2 murine myeloid leukemias. IL-17Rh1 shows highest level expression in kidney with moderate expression in multiple other organs, whereas IL-17E mRNA was detected at very low levels in several peripheral tissues. IL-17E induces activation of NF-kappaB and stimulates production of the proinflammatory chemokine IL-8.     

Increased insulin sensitivity in Gsalpha knockout mice

The stimulatory guanine nucleotide-binding protein (G(s)) is required for hormone-stimulated cAMP generation. Gnas, the gene encoding the G(s) alpha-subunit, is imprinted, and targeted disruption of this gene in mice leads to distinct phenotypes in heterozygotes depending on whether the maternal (m-/+) or paternal (+/p-) allele is mutated. Notably, m-/+ mice become obese, whereas +/p- mice are thinner than normal. In this study we show that despite these opposite changes in energy metabolism, both m-/+ and +/p- mice have greater sensitivity to insulin, with low to normal fasting glucose levels, low fasting insulin levels, improved glucose tolerance, and exaggerated hypoglycemic response to administered insulin. The combination of increased insulin sensitivity with obesity in m-/+ mice is unusual, because obesity is typically associated with insulin resistance. In skeletal muscles isolated from both m-/+ and +/p- mice, the basal rate of 2-deoxyglucose uptake was normal, whereas the rate of 2-deoxyglucose uptake in response to maximal insulin stimulation was significantly increased. The similar changes in muscle sensitivity to insulin in m-/+ and +/p- mice may reflect the fact that muscle G(s)alpha expression is reduced by approximately 50% in both groups of mice. GLUT4 expression is unaffected in muscles from +/p- mice. Increased responsiveness to insulin is therefore the result of altered insulin signaling and/or GLUT4 translocation. This is the first direct demonstration in a genetically altered in vivo model that G(s)-coupled pathways negatively regulate insulin signaling.     

Interaction of 11-cis-retinol dehydrogenase with the chromophore of retinal g protein-coupled receptor opsin

Vertebrate opsins in both photoreceptors and the retinal pigment epithelium (RPE) have fundamental roles in the visual process. The visual pigments in photoreceptors are bound to 11-cis-retinal and are responsible for the initiation of visual excitation. Retinochrome-like opsins in the RPE are bound to all-trans-retinal and play an important role in chromophore metabolism. The retinal G protein-coupled receptor (RGR) of the RPE and Müller cells is an abundant opsin that generates 11-cis-retinal by stereospecific photoisomerization of its bound all-trans-retinal chromophore. We have analyzed a 32-kDa protein (p32) that co-purifies with bovine RGR from RPE microsomes. The co-purified p32 was identified by mass spectrometric analysis as 11-cis-retinol dehydrogenase (cRDH), and enzymatic assays have confirmed the isolation of an active cRDH. The co-purified cRDH showed marked substrate preference to 11-cis-retinal and preferred NADH rather than NADPH as the cofactor in reduction reactions. cRDH did not react with endogenous all-trans-retinal bound to RGR but reacted specifically with 11-cis-retinal that was generated by photoisomerization after irradiation of RGR. The reduction of 11-cis-retinal to 11-cis-retinol by cRDH enhanced the net photoisomerization of all-trans-retinal bound to RGR. These results indicate that cRDH is involved in the processing of 11-cis-retinal after irradiation of RGR opsin and suggest that cRDH has a novel role in the visual cycle.     

Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells

The various molecular forms of gastrin can act as promoters of proliferation and differentiation in different regions of the gastrointestinal tract. We report a novel stimulatory effect of glycine-extended gastrin(17) only on cell/cell dissociation and cell migration in a non-tumorigenic mouse gastric epithelial cell line (IMGE-5). In contrast, both amidated and glycine-extended gastrin(17) stimulated proliferation of IMGE-5 cells via distinct receptors. Glycine-extended gastrin(17)-induced dissociation preceded migration and was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) but did not require mitogen-activated protein (MAP) kinase activation. Furthermore, glycine-extended gastrin(17) induced a PI3-kinase-mediated tyrosine phosphorylation of the adherens junction protein beta-catenin, partial dissociation of the complex between beta-catenin and the transmembrane protein E-cadherin, and delocalization of beta-catenin into the cytoplasm. Long lasting activation of MAP kinases by glycine-extended gastrin(17) was specifically required for the migratory response, in contrast to the involvement of a rapid and transient MAP kinase activation in the proliferative response to both amidated and glycine-extended gastrin(17). Therefore, the time course of MAP kinase activation appears to be a critical determinant of the biological effects mediated by this pathway. Together with the involvement of PI3-kinase in the dissociation of adherens junctions, long term activation of MAP kinases seems responsible for the selectivity of this novel effect of G(17)-Gly on the adhesion and migration of gastric epithelial cells.     

Crystal structure of activated CheY. Comparison with other activated receiver domains

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.     

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R(f) values for the long wavelength-absorbing fluorophores. Glycation with [U-(14)C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A(330)-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.     

Capture-recapture, epidemiology, and list mismatches: several lists

In applying capture-recapture methods for closed populations to epidemiology, e.g., in the estimation of the size of a diabetes population, one comes up against the problem of list errors due to mistyping or misinformation. This problem has been studied for just two lists by Seber, Huakau, and Simmons (2000, Biometrics 56, 1227 1232) using the concept of tag loss borrowed from animal population studies. In this article, we discuss a similar method that can be extended to an arbitrary number of lists. The methods are applied to an example.     

An Ochrobactrum anthropi gene conferring paraquat resistance to the heterologous host Escherichia coli

A new gene, pqrA, conferring paraquat resistance to the heterologous host Escherichia coli, from a chromosomal DNA library of Ochrobactrum anthropi JW2, was cloned and analyzed. Cells of E. coli transformed with a plasmid carrying the pqrA gene showed elevated resistance to paraquat, but not to hydrogen peroxide. The predicted amino acid sequence of the PqrA polypeptide showed 71% identity with mll7495 hypothetical membrane protein in Mesorhizobium loti, 49% identity with PA2269 protein in Pseudomonas aeruginosa, and significant identity with other previously reported drug transport proteins. The hydropathy pattern of the PqrA polypeptide showed a significant homology to those of 12-transmembrane-segment (TMS) family export proteins. Immunoblot analysis demonstrated that the PqrA protein found in the membrane protein fraction of O. anthropi JW2 has a molecular mass of 42 kDa. These results suggest that the PqrA protein is a membrane protein that plays an important role in protecting cells against paraquat toxicity.