Site-Directed Mutations and the Polymorphic Variant Ala160Thr in the Human Thromboxane Receptor Uncover a Structural Role for Transmembrane Helix 4

The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala1604.53 is highly conserved as is Gly1644.57. Here we target Ala1604.53 and Gly1644.57 in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala1604.53 and Gly1644.57 in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions

Hypothyroidism of hypothalamic origin in pyridoxine-deficient rats

Pyridoxine-deficient young rats (3 weeks old) had significantly reduced levels of pituitary TSH, serum thyroxine (T4) and tri iodothyn nine (T,,) Compared with pyridoxine-supplemented rats. The status of the pituitary-thyroid axis of normal, pyridoxine-supplemented and pyridoxine-deficient rats was evaluated by studying the binding parameters of [3H](3-nicthylhistidine2) TRH in the pituitary of these rats. The effects of TRH and 1'4 injections on pituitary TSH and serum TSH, T4 and T3 of these two groups were also compared. The maximal binding of TRH receptors in the pituitary of pyridoxine-deficient rats was significantly higher than that of pyridoxine-supplemented control and normal rats, but there was no change in the binding affinity. Treatment with TRH stimulated TSH synthesis and release. It also increased serum T4 and T3 in both pyridoxine-supplemented and pyridoxine-deficient rats. Treatment with T4 decreased serum and pituitary TSH in both pyridoxine-supplemented and pyridoxine-deficient rats, compared with saline-treated rats. The increased pituitary TRH receptor content, response to TRH administration and the fact that regulation at the level of the pituitary is not affected in the pyridoxinedeficient rat indicates a hypothalamic origin for the hypothyroidism of the pyridoxine-deficient rat

Serial serum brain-type natriuretic peptide (BNP) identifies compromised blood flow in infants with hemodynamically significant patent ductus arteriosus

Background: The physiological correlates of elevated serum brain-type natriuretic peptide (BNP) in hemodynamically significant patent ductus arteriosus (HSPDA) are unclear. Objective: To determine if serial BNP measured at 48-72 hours of age, before and after non-steroidal anti-inflammatory drugs (NSAID) treatment of HSPDA reflect compromised blood flow indices, in infants < 31 weeks of gestational age (GA). Design/methods: In a prospective blinded study, 70 infants < 31 weeks GA, admitted to Winnipeg NICUs from August 2010 to September 2011, had serum BNPs and echocardiograms at 48-72 hours of age, before and after medical treatment of HSPDA. All BNP and logarithm of BNP (logBNP) were correlated by linear regression with contemporaneous blood flow indices for: 1) systemic hemodynamic indices (corrected left and right ventricular outputs [LVO, RVO], RVO/LVO ratio, superior vena cava flow [SVCF], SVCF/LVO); 2) regional blood flow indices (middle cerebral artery flow [MCAF], MCAF/LVO ratio, middle cerebral artery resistive index [MCARI], middle cerebral artery pulsatility index [MCAPI]; celiac artery flow [CAF], CAF/LVO ratio, celiac artery resistive index [CARI], and celiac artery pulsatility index [CAPI]). Results: Twenty-six of 70 infants developed HSPDA at 6 ± 2 days. Both BNP and logBNP had similar correlations with all indices, but logBNP showed better goodness of fit. The best correlation was at 48-72 hours of life. Analyzing systemic hemodynamics, logBNP best correlated with SVCF (β -0.49, R2 0.24, p < 0.0001), SVCF/LVO (β -0.55, R2 0.31, p < 0.0001), RVO/LVO (β -0.59, R2 0.35, p < 0.0001), LVO (β 0.4, R2 0.16, p < 0.0001), and RVO (β -0.35, R2 0.12, p < 0.0001). For regional blood flow, logBNP best correlated with MCARI (β 0.6, R2 0.35, p < 0.0001), MCAPI (β 0.5, R2 0.29, p < 0.001), and MCAF (β -0.34, R2 0.12, p < 0.0001). Conclusions: BNP correlates with blood flow indices in preterm infants with HSPDA mainly at 48-72 hours reflecting the value of pre-symptomatic physiologic prediction by BNP

Thyroid function in pyridoxine-deficient young rats

In pyridopaminedoxine-deficient young rats hypothalamic serum TSH concentration was detected. Highly signifiserotonin was decreased with no changes in the cant decreases in the content of pituitary TSH and in and noradrenaline content. Serum the number of pituitary thyrotroph secretory granules and tri-iodothyronine concentrations were were found. These results suo mmuuchch lower in the deficient rats as compared to thyroidism of suggest that the hypocontrols. No significant of hypothalamicp yorirdigoxinin.e -deficient young rats might bbee difference between deficient and control groups in th

Testicular Function in Biotin-Deficient Adult Rats

We have studied testicular function in the biotin- deficient rat biochemically and morphologically. Serum testosterone and luteinizing hormone (LH) levels were decreased significantly in the deficient rats. Administration of biotin or gonadotropins to the deficient rats reversed this decrease in serum testosterone. There was no difference in the serum cholesterol level between the control and biotin-deficient rats. A significant degree of sloughing of seminiferous tubule germinal epithelium was noticed in the biotin-deficient rat testes. Biotin treatment of biotindeficient rats reversed this condition whereas testosterone treatment was without any effect. The development and maintenance of morphological and functional integrity of the seminiferous tubules appears to require a biotin-mediated step in addition to testosterone

Pre-symptomatic prediction of morbitidies in preterm infants with patent ductus arteriosus by targeted neonatal echocardiography and brain-type natriuretic peptide

Objectives: Our objective was to compare patent ductus arteriosus (PDA) diameter, PDA score and brain-type natriuretic peptide (BNP) measurements at 48-72 hours of life, for prediction of neonatal morbidities. We hypothesized that use of a PDA score with BNP, may improve pre-symptomatic prediction of PDAs associated with adverse outcomes. Method: Infants < 31 weeks GA were prospectively studied by targeted neonatal echocardiogram (TNE) at 48-72 hours age, composite PDA score and serum BNP assay; the clinical team remained blinded. PDA was independently diagnosed by echocardiography at time of clinical suspicion (6 ± 2 days), and treated at discretion of the clinical team. Primary outcome was survival with one or more of adverse outcomes (intraventricular hemorrhage [IVH], bronchopulmonary dysplasia [BPD], retinopathy of prematurity [ROP], necrotizing enterocolitis [NEC]). Results: A PDA was present in 56 of 70 infants studied at 48-72 hours; 30 were eventually diagnosed with PDA but never required treatment, 19 required medical treatment, 7 surgical ligation. After adjustment for gestation, PDA diameter did not predict any adverse outcome, PDA score was associated with increased risk of any adverse outcome and high BNP was associated with IVH, BPD, or survival with any adverse outcome. Conclusions: Comprehensive PDA evaluation at 48-72 hours of age may predict the subsequent occurrence of adverse outcomes and may be useful to define the PDA treatment threshold