Functional consequences of AXL sequence variants in hypogonadotropic hypogonadism.

CONTEXT: Prior studies showed that Axl /Tyro3 null mice have delayed first estrus and abnormal cyclicity due to developmental defects in GnRH neuron migration and survival. OBJECTIVE: The objective of the study was to test whether the absence of Axl would alter reproductive function in mice and that mutations in AXL are present in patients with Kallmann syndrome (KS) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). DESIGN AND SETTING: The sexual maturation of Axl null mice was examined. The coding region of AXL was sequenced in 104 unrelated, carefully phenotyped KS or nIHH subjects. Frequency of mutations was compared with other causes of GnRH deficiency. Functional assays were performed on the detected mutations. RESULTS: Axl null mice demonstrated delay in first estrus and the interval between vaginal opening and first estrus. Three missense AXL mutations (p.L50F, p.S202C, and p.Q361P) and one intronic variant 6 bp upstream from the start of exon 5 (c.586-6 C>T) were identified in two KS and 2 two nIHH subjects. Comparison of the frequencies of AXL mutations with other putative causes of idiopathic hypogonadotropic hypogonadism confirmed they are rare variants. Testing of the c.586-6 C>T mutation revealed no abnormal splicing. Surface plasmon resonance analysis of the p.L50F, p.S202C, and p.Q361P mutations showed no altered Gas6 ligand binding. In contrast, GT1-7 GnRH neuronal cells expressing p.S202C or p.Q361P demonstrated defective ligand dependent receptor processing and importantly aberrant neuronal migration. In addition, the p.Q361P showed defective ligand independent chemotaxis. CONCLUSIONS: Functional consequences of AXL sequence variants in patients with idiopathic hypogonadotropic hypogonadism support the importance of AXL and the Tyro3, Axl, Mer (TAM) family in reproductive development.

Morbidity effects on productivity and profitability of stocker cattle grazing in the Southern Plains.

Effects of bovine respiratory disease (BRD) on stocker cattle systems are unknown under extensive rangeland environments. Three experiments were conducted to test the hypothesis that BRD-based morbidity is a major factor affecting the productivity and profitability of stocker cattle grazing Southern Plains rangelands. In Exp. 1 (658 male calves; average BW = 231 kg), 17% of the cattle were treated for BRD <8 d, 6% for 8 to 14 d, and 8% for >14 d. Morbid cattle had lower ADG than did healthy cattle (P < 0.10). Cattle requiring 14 d of pharmaceutical therapy gained less than cattle having <14 d therapy (P < 0.01). In Exp. 2, (279 steers and bulls; average BW = 216 kg), the ADG by steers (0.74 kg x animal(-1) x d(-1)) was greater (P < 0.05) than by bulls castrated after arrival (0.64 kg x animal(-1) x d(-1)). Castration after arrival led to a 13.5% loss in daily gain and a 10.3% loss in season-long gain. More (P < 0.05) bulls castrated after arrival (60%) were morbid compared with steers (28%). In Exp. 3, 633 heifers (average BW = 251 kg) were used to test the effects of morbidity on weight gain and reproduction. Heifers with lower initial weights exhibited increased (P < 0.05) morbidity. Heifers requiring two or more antibiotic treatments gained 0.03 kg/d less (P < 0.10) than did healthy heifers and had lower (P < 0.05) conception rates (66 vs. 81%). Conception rate in twice-treated heifers was 19% less than healthy heifers. Morbid heifers conceived 0.6 mo later (P < 0.05) than healthy heifers. Under the conditions of Exp. 1 and Exp. 2, morbidity decreased net returns 9.7 to 21.3% per animal. Adjusted gross returns per animal in Exp. 3 for replacement heifers were 3 to 7.8% less for morbid heifers.

Critical nature of a specific uridine O2-carbonyl for cleavage by the hammerhead ribozyme.

Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.

Importance of specific adenosine N3-nitrogens for efficient cleavage by a hammerhead ribozyme.

Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N3-nitrogens have been excised and replaced with carbon. The modified complexes were chemically synthesized with the substitution of a single 3-deazzaadenosine (c3A) base analogue for residues A6, A9, A13, A14, or A15.1. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/K(M), for the c3A6, c3A9, and c3A14 complexes were only marginally reduced (< or = 5-fold) relative to the native complex. By comparison, the cleavage efficiencies for the c3A13 and c315.1 complexes were reduced by 9-fold and 55-fold, respectively. these reductions in cleavage efficiency are primarily a result of lower kcat values. Profiles of pH and cleavage rate suggest that the chemical cleavage step is the rate-limiting reaction for these complexes. These results suggest that the N3-nitrogen of the A13 residue and particularly the A15.1 residue in the hammerhead ribozyme/substrate complex are critical for transition state stabilization and efficient cleavage activity. We have additionally compared the locations of these critical functional groups, as well as those identified from other studies, with recent crystallographic analyses. In some cases, the critical functional groups are clustered around proposed metal binding sites and may reflect functional groups critical for binding the metal cofactor. In other cases, clusters of functional groups may form a network of hydrogen bonds necessary for transition state stabilization.