The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

INTRODUCTION: The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. OBJECTIVES/METHODS: We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). RESULTS: Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. CONCLUSIONS: Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.

Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults.

Many areas of the Earth's crust deform by distributed extensional faulting and complex fault interactions are often observed. Geodetic data generally indicate a simpler picture of continuum deformation over decades but relating this behaviour to earthquake occurrence over centuries, given numerous potentially active faults, remains a global problem in hazard assessment. We address this challenge for an array of seismogenic faults in the central Italian Apennines, where crustal extension and devastating earthquakes occur in response to regional surface uplift. We constrain fault slip-rates since ~18 ka using variations in cosmogenic 36Cl measured on bedrock scarps, mapped using LiDAR and ground penetrating radar, and compare these rates to those inferred from geodesy. The 36Cl data reveal that individual faults typically accumulate meters of displacement relatively rapidly over several thousand years, separated by similar length time intervals when slip-rates are much lower, and activity shifts between faults across strike. Our rates agree with continuum deformation rates when averaged over long spatial or temporal scales (104 yr; 102 km) but over shorter timescales most of the deformation may be accommodated by <30% of the across-strike fault array. We attribute the shifts in activity to temporal variations in the mechanical work of faulting.

Novel Lethal Form of Congenital Hypopituitarism Associated With the First Recessive LHX4 Mutation.

BACKGROUND: LHX4 encodes a member of the LIM-homeodomain family of transcription factors that is required for normal development of the pituitary gland. To date, only incompletely penetrant heterozygous mutations in LHX4 have been described in patients with variable combined pituitary hormone deficiencies. OBJECTIVE/HYPOTHESIS: To report a unique family with a novel recessive variant in LHX4 associated with a lethal form of congenital hypopituitarism that was identified through screening a total of 97 patients. METHOD: We screened 97 unrelated patients with combined pituitary hormone deficiency, including 65% with an ectopic posterior pituitary, for variants in the LHX4 gene using Sanger sequencing. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) were consulted upon identification of variants. RESULTS: We identified the first novel homozygous missense variant (c.377C>T, p.T126M) in two deceased male patients of Pakistani origin with severe panhypopituitarism associated with anterior pituitary aplasia and posterior pituitary ectopia. Both were born small for gestational age with a small phallus, undescended testes, and mid-facial hypoplasia. The parents' first-born child was a female with mid-facial hypoplasia (DNA was unavailable). Despite rapid commencement of hydrocortisone and T4 in the brothers, all three children died within the first week of life. The LHX4(p.T126M) variant is located within the LIM2 domain, in a highly conserved location. The absence of homozygosity for the variant in over 65 000 controls suggests that it is likely to be responsible for the phenotype. CONCLUSION: We report, for the first time to our knowledge, a novel homozygous mutation in LHX4 associated with a lethal phenotype, implying that recessive mutations in LHX4 may be incompatible with life.

Effect of baroreceptor denervation on the inhibition of renin release by vasopressin.

Previous studies have suggested that the inhibition of renin secretion by acute administration of vasopressin in conscious dogs results from a reflex reduction in renal nerve activity. In the present investigation, this hypothesis was tested by studying the effect of total baroreceptor denervation or selective low pressure baroreceptor denervation on the suppression of PRA by vasopressin in conscious, chronically prepared dogs. In eight sham-operated dogs, a 45-min infusion of vasopressin (2.0 ng/kg.min, iv) decreased PRA from 10.5 +/- 1.9 to 5.9 +/- 1.0 ng/ml.3 h (P less than 0.01). Mean arterial pressure did not change (110 +/- 10 to 107 +/- 7 mm Hg), but heart rate decreased from 84 +/- 9 to 69 +/- 8 beats/min (P less than 0.05). In contrast, vasopressin infusion failed to significantly decrease PRA in seven sinoaortic/cardiac denervated dogs (9.5 +/- 1.7 to 7.4 +/- 2.0 ng/ml.3 h), although decreases did occur in three of the dogs. Mean arterial pressure increased from 104 +/- 5 to 125 +/- 6 mm Hg (P less than 0.01), but heart rate did not change (112 +/- 4 to 107 +/- 5 beats/min). When renal perfusion pressure was maintained at the preinfusion level in three sinoaortic/cardiac denervated dogs, vasopressin infusion failed to decrease PRA (2.3 +/- 0.6 to 2.4 +/- 0.6 ng/ml.3 h). In six cardiac denervated dogs, vasopressin infusion decreased PRA from 5.3 to 0.9 to 3.1 +/- 0.7 ng/ml.3 h (P less than 0.01). Results obtained with two lower doses of vasopressin (0.5 and 1.0 ng/kg.min) were generally similar to the responses observed during infusion at 2.0 ng/kg.min. Angiotensin II (5.0 ng/kg.min) suppressed PRA in all groups of dogs. These experiments demonstrate that the inhibition of renin secretion by acute administration of vasopressin in conscious dogs is prevented by total baroreceptor denervation, but not by denervation of the low pressure baroreceptors alone. These results suggest that the suppression of renin release by vasopressin is a reflex response resulting from activation of the high pressure baroreceptors.

Effect of subunit III removal on control of cytochrome c oxidase activity by pH.

Studies were undertaken to assess the postulated involvement of subunit III in the proton-linked functions of cytochrome c oxidase. The effect of pH on the steady-state kinetic [corrected] parameters of subunit III containing and subunit III depleted cytochrome oxidase was determined by using beef heart and rat liver enzymes reconstituted into phospholipid vesicles. The TNmax and Km values for the III-containing enzyme increase with decreasing pH in a manner quantitatively similar to that reported by Thornstrom et al. [(1984) Chem. Scr. 24, 230-235], giving three apparent pKa values of less than 5.0, 6.2, and 7.8. The maximal activities of the subunit III depleted enzymes (beef heart and rat liver) show a similar dependence on pH, but the Km values are consistently higher than those of the III-containing enzyme, an effect that is accentuated at low pH. The pH dependence of TNmax/Km for both forms of the enzyme (+/- subunit III) indicates that protonation of a group with an apparent pKa of 5.7 lowers the affinity for substrate (cytochrome c) independently of a continued increase in maximal velocity. N,N'-Dicyclohexylcarbodiimide (DCCD) decreases the pH responsiveness of the electron-transfer activity to the same extent in both III-containing and III-depleted enzymes, indicating that this effect is mediated by a peptide other than subunit III. Control of intramolecular electron transfer by a transmembrane pH gradient (or alkaline intravesicular pH) is shown to occur in cytochrome oxidase vesicles with cytochrome c as the electron donor, in agreement with results of Moroney et al. [(1984) Biochemistry 23, 4991-4997] using hexaammineruthenium(II) as the reductant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of renal denervation on the suppression of renin secretion by vasopressin in conscious dogs.

Previous studies have shown that the inhibition of renin secretion by vasopressin (AVP) in conscious dogs is related to vasoconstrictor activity and may be a reflex response mediated by the renal nerves. The aim of the present experiments was to determine whether the suppression of plasma renin activity (PRA) by AVP is blocked by renal denervation. AVP and, for comparison, angiotensin II (ANG II) were infused intravenously for 45 min in seven conscious dogs before and after bilateral renal denervation. Before denervation, AVP infusion at 0.2 and 1.0 ng X kg-1 X min-1 suppressed PRA from 7.4 +/- 1.1 to 4.7 +/- 1.0 (P less than 0.01) and from 7.9 +/- 1.8 to 3.8 +/- 0.8 ng X ml-1 X 3 h-1 (P less than 0.01), respectively. ANG II infusion at 5.0 and 10.0 ng X kg-1 X min-1 decreased PRA from 7.5 +/- 2.3 to 2.5 +/- 0.7 (P less than 0.01) and from 6.0 +/- 1.1 to 1.8 +/- 0.4 ng X ml-1 X 3 h-1 (P less than 0.01), respectively. One to three weeks following renal denervation, PRA had decreased from 6.7 +/- 1.3 to 2.9 +/- 0.5 ng X ml-1 X 3 h-1 (P less than 0.01), and renal norepinephrine was undetectable. After denervation, neither AVP infusion at 0.2 (3.0 +/- 0.5 to 2.4 +/- 0.4 ng X ml-1 X 3 h-1) nor 1.0 ng X kg-1 X min-1 (3.1 +/- 0.8 to 2.8 +/- 1.0 ng X ml-1 X 3 h-1) suppressed PRA.(ABSTRACT TRUNCATED AT 250 WORDS)

Eight compact analysis systems evaluated for measuring total cholesterol.

We examined the analytical performance of eight compact systems for measuring total cholesterol: AccuMeter, Cobas Ready, Discovery f2, DT60, L-D-X, Reflotron, QCA, and Vision. We determined average bias at two decision levels, the mean absolute bias, and the percentage of results differing from the comparison method results by > 8.9% allowable total error limit for multiple reagent lots. Average bias was < 3% for all lots tested for AccuMeter, Discovery f2, and DT60, but > 3% for one or more lots or sample types tested with the other systems. Of results from each reagent lot, > 95% were within the 8.9% total error specifications with Discovery f2, DT60, and QCA, whereas the performance of L-D-X, Vision, and Reflotron depended on reagent lot and (or) sample type. Of all results from each lot tested with AccuMeter and Cobas Ready, > 5% exceeded the total allowable error limit. We determined imprecision for five systems: Cobas Ready, Discovery f2, and QCA had CVs < 3%, whereas CVs for AccuMeter and L-D-X were > 3% but < 5%.

Effect of vasopressin blockade on blood pressure during water deprivation in intact and baroreceptor-denervated conscious dogs.

Previous studies have provided evidence that vasopressin plays an important role in blood pressure regulation during water deprivation. However, these investigations have been complicated by reflex compensatory increases in cardiac output and renin secretion. The aim of the present study was to investigate the effect of blockade of the vasoconstrictor action of vasopressin in conscious water-deprived dogs in which the low- and/or high-pressure baroreceptors were denervated to minimize reflex responses. Vasopressin blockade in sham-operated dogs (n = 7) did not change arterial pressure. Heart rate rose from 78 +/- 9 to 119 +/- 13 beats/min (P less than 0.01), and plasma renin activity increased from 10.9 +/- 2.1 to 21.6 +/- 4.6 ng.ml-1.3 h-1 (P less than 0.01). In carotid sinus-denervated dogs (n = 6), vasopressin blockade again failed to decrease arterial pressure. Heart rate increased from 105 +/- 10 to 132 +/- 10 beats/min (P less than 0.01), and plasma renin activity rose from 6.8 +/- 1.7 to 15.5 +/- 2.4 ng.ml-1.3 h-1 (P less than 0.01). The antagonist also failed to change blood pressure in cardiac-denervated dogs (n = 5). Heart rate increased from 111 +/- 9 to 119 +/- 1 beats/min (P less than 0.01), but plasma renin activity did not increase significantly. In marked contrast, vasopressin blockade in sinoaortic/cardiac-denervated dogs (n = 7) promptly decreased arterial pressure from 115 +/- 8 to 94 +/- 7 mmHg (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of small C-ANF receptor ligands on plasma levels of atrial natriuretic factor, blood pressure, and renal function in the rat.

In this article, after a very brief review on ANF receptors, we report our study on the effects of small C-ANF receptor ligands in the rat. Two small ligands were synthesized: 2-naphthoxyacetyl-isonipecotyl-rANF11-15-NH2 (5 aa), containing 5 amino acids; and Ala7-rANF8-17-NH2 (10 aa), containing 10 amino acids from the ring structure of ANF1-28. After control periods, 5 aa or 10 aa were infused i.v. at a dose of 10 micrograms.min-1.kg-1 body weight for 70 min in anesthetized rats, followed by a 60-min recovery period. The 5 aa and 10 aa peptides significantly and reversibly increased plasma levels of endogenous immunoreactive ANF by 106 +/- 29 and 52 +/- 24 pg/mL, respectively. Infusion of the 5 aa peptide significantly decreased mean arterial blood pressure from 113 +/- 1 to 100 +/- 3 mmHg (1 mmHg = 133.32 Pa) and increased glomerular filtration rate from 1.6 +/- 0.2 to 2.3 +/- 0.2 mL/min, sodium excretion from 0.6 +/- 0.3 to 3.4 +/- 0.4 mumol/min, and potassium excretion from 0.5 +/- 0.2 to 1.2 +/- 0.2 mumol/min. Similar results were obtained with the 10 aa peptide. The effects of both peptides on blood pressure and sodium excretion persisted throughout the recovery period. The results confirm and extend previous observations showing that C-ANF receptors mediate the removal of ANF from the circulation. The shortening of the minimal peptide length necessary to bind to C-ANF receptors markedly enhances the possibility of developing orally active C-ANF receptor ligands for the treatment of cardiovascular and renal diseases.

Isolation and functional expression of the human atrial natriuretic peptide clearance receptor cDNA.

The amino acid sequence of the human atrial natriuretic peptide clearance receptor (ANP C-receptor) was deduced from the nucleotide sequence of cDNA clones obtained from human placental and kidney cDNA libraries. The human sequence is highly homologous to the bovine C-receptor sequence already described, and the corresponding mRNA is expressed in human placenta, adult and fetal kidney and fetal heart. Upon transfection of this cDNA into mammalian cells, recombinant expression experiments revealed that the human ANP C-receptor has a high affinity for ANP (6 x 10(-9) M), similar to that observed for the receptor in other species. These data indicate that the human ANP C-receptor, previously characterized in other mammalian species, is highly conserved structurally and is expressed in various human tissues.

In vitro interference of the red cell substitute pyridoxalated hemoglobin-polyoxyethylene with blood compatibility, coagulation, and clinical chemistry testing.

OBJECTIVES: Pyridoxalated hemoglobin-polyoxyethylene (PHP) is a prototypical red cell substitute approved for phase I studies. Peripheral blood smears of human blood mixed with PHP in 1 to 4 g/dL concentrations showed dose-dependent red cell aggregation and rouleaux. Whether this aggregation limits interpretation of blood compatibility testing and whether the intense coloration of serum or plasma containing PHP affects routine coagulation and clinical chemistry measurements was tested. DESIGN: In vitro studies. SETTING: University hospital laboratory. PARTICIPANTS: Four healthy volunteers, blood types A, B, AB, and O. All were Rh+. MEASUREMENTS AND MAIN RESULTS: ABO typing, Rh typing, and antibody screening and coagulation studies were performed on blood: PHP admixtures having final concentrations of 1, 2, and 4 g/dL. For clinical chemistry interference studies, known concentrations of analytes were added to a serum matrix containing PHP. ABO (forward) and Rh typing showed no interference in the three concentrations tested. Reverse ABO typing and antibody screening showed rouleaux at 4 g/dL, which corrected with routine saline replacement. Partial thromboplastin time (PTT), prothrombin time (PT), and fibrinogen showed no clinically significant differences from the controls. Results for electrolytes, renal function analytes, and markers of cardiac injury were acceptable by standard laboratory methods. However, results of liver function tests were unacceptable in PHP-containing specimens. CONCLUSIONS: PHP-induced aggregation was observed with high PHP concentration; however, compatibility testing was not affected because agglutination was corrected by saline replacement, which is standard practice. Although routine blood banking, coagulation, and most clinical chemistry analytes can be measured reliably, alternative methods and strategies are needed for assessing liver function in the presence of PHP.