We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

The human erythropoietin gene has been isolated from a genomric phage library by using mixed 20-mer and 17-mer oligonucleotide probes. Construction of Oligonucleotide Probes. Purified human urinary Epo'isolated from the urine of patients with aplastic anemia (16) was subjected to tryptic digestion. The resulting fragments were isolated and sequenced by using an Applied Biosystems gas-phase microsequencer (unpublished data). A hexapeptide and a heptapeptide containing the least codon degeneracy were selected for oligodeoxyribonucleotide probe synthesis. The phosphoramidite method was l4sed for oligonucleotide synthesis (19,20). Each' probe mixture contained a pool of 128-oligonucleotide sequences. The probe mixtures were Probe mixture EpV = Val-Asn-Phe-Tyr-Ala-Trp-Lys 3' CAA TTG AAG ATG CGA ACC TT 5'Probe mixture EpQ= Gln-Pro-Trp-Glu-Pro-Leu 3' GTT GGA ACC CTT GGA GA 5'The probe mixtures were labeled at the 5' end with [-32P]ATP, 7500-8000 Ci/mmol (ICN) (1 Ci = 37 GBq), by using T4 polynucleotide kinase (21). Hybridization Procedures. Phage plaques were amplified according to the procedures of Woo (22) except that GeneScreenPlus filters and NZYAM plates [NaCl, 5 g; MgCl2-6H2O, 2 g; NZ-Amine A, 10 g; yeast extract, 5 g; Casamino acids, 2 g; maltose, 2 g; and agar, 15 g (per liter)] were utilized. Phage particles were disrupted and the DNAs were fixed on filters (50,000 plaques per 8.4 x 8.4 cm filter). The air-dried filters were baked at 80'C for 1 hr and then subjected to proteinase K digestion [50 ,ug ofproteinase 'K per ml of buffer solution containing 0.1 M Tris HCl (pH 8.0), 0.15 M NaCl, 10 mM EDTA, and 0.2% NaDodSO4] for 30 min at 550C. Prehybridization with a 1 M NaCl/1% NaDodSO4 solution was carried out at 550C for 4 hr or longer.The hybridization buffer contained 0.025 pmol/ml of each of the 128 probe sequences in 0.9 M NaCl/5 mM EDTA/50 mM sodium phosphate, pH 6.5/0.5% NaDodSO4/100 jg of yeast tRNA per ml. Hybridization was carried out at 480C'for 20 hr by using the EpV probe mixture. This is 2TC below the lowest calculated dissociation temperature (td) (23) for members of the mixture. At the completion of hybridization, the filters were washed three times with 0.9 M NaCl/90 mM sodium citrate, pH 7.0/0. 1% NaDodSO4 at room temperature Abbreviations: Epo, erythropoietin; CHO, Chinese hamster ovary; DHFR, dihydrofolate reductase; kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); SV40, simian virus 40; td, dissociation temperature. 7580The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Members of the fibroblast growth factor (FGF) family of proteins stimulate the proliferation and differentiation of a variety of cell types through receptor-mediated pathways. The three-dimensional structures of two members of this family, bovine acidic FGF and human basic FGF, have been crystallographically determined. These structures contain 12 antiparallel beta strands organized into a folding pattern with approximate threefold internal symmetry. Topologically equivalent folds have been previously observed for soybean trypsin inhibitor and interleukins-1 beta and -1 alpha. The locations of sequences implicated in receptor and heparin binding by FGF are presented. These sites include beta-sheet strand 10, which is adjacent to the site of an extended sequence insertion in several oncogene proteins of the FGF family, and which shows sequence conservation among the FGF family and interleukin-1 beta.

Background Lactation improves glucose metabolism, but its role in preventing type 2 diabetes mellitus (DM) after gestational diabetes mellitus (GDM) remains uncertain. Objective To evaluate lactation and the 2-year incidence of DM after GDM pregnancy. Design Prospective, observational cohort of women with recent GDM. (ClinicalTrials.gov: NCT01967030) Setting Integrated health care system. Participants 1035 women diagnosed with GDM who delivered singletons at 35 weeks' gestation or later and enrolled in the Study of Women, Infant Feeding and Type 2 Diabetes After GDM Pregnancy from 2008 to 2011. Measurements Three in-person research examinations from 6 to 9 weeks after delivery (baseline) and annual follow-up for 2 years that included 2-hour, 75-g oral glucose tolerance testing; anthropometry; and interviews. Multivariable Weibull regression models evaluated independent associations of lactation measures with incident DM adjusted for potential confounders. Results Of 1010 women without diabetes at baseline, 959 (95%) were evaluated up to 2 years later; 113 (11.8%) developed incident DM. There were graded inverse associations for lactation intensity at baseline with incident DM and adjusted hazard ratios of 0.64, 0.54, and 0.46 for mostly formula or mixed/inconsistent, mostly lactation, and exclusive lactation versus exclusive formula feeding, respectively (P trend = 0.016). Time-dependent lactation duration showed graded inverse associations with incident DM and adjusted hazard ratios of 0.55, 0.50, and 0.43 for greater than 2 to 5 months, greater than 5 to 10 months, and greater than 10 months, respectively, versus 0 to 2 months (P trend = 0.007). Weight change slightly attenuated hazard ratios. Limitation Randomized design is not feasible or desirable for clinical studies of lactation. Conclusion Higher lactation intensity and longer duration were independently associated with lower 2-year incidences of DM after GDM pregnancy. Lactation may prevent DM after GDM delivery.