Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Neuropeptide Y restores non-receptor-mediated vasoconstrictive action in superior mesenteric arteries in portal hypertension

BACKGROUND & AIMS Vascular hyporeactivity to vasoconstrictors contributes to splanchnic arterial vasodilatation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY), a sympathetic cotransmitter, has been shown to improve adrenergic vascular contractility in portal hypertensive rats and markedly attenuate hyperdynamic circulation. To further characterize the NPY-effects in portal hypertension, we investigated its role for non-receptor-mediated vasoconstriction in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham-operated rats. METHODS Ex vivo SMA perfusion of PVL and sham rats was used to analyse the effects of NPY on pressure response to non-receptor-mediated vasoconstriction. Dose-response curves to KCl (30-300 mM) were used to bypass G protein-coupled receptor mechanisms. Potential involvement of the cyclooxygenase-pathway was tested by non-selective cyclooxygenase-inhibition using indomethacin. RESULTS KCl-induced vascular contractility but not vascular sensitivity was significantly attenuated in PVL rats as compared with sham rats. Administration of NPY resulted in an augmentation of KCl-evoked vascular sensitivity being not different between study groups. However, KCl-induced vascular contractility was markedly more enhanced in PVL rats, thus, vascular response was no more significantly different between PVL and sham rats after addition of NPY. Administration of indomethacin abolished the NPY-induced enhancement of vasoconstriction. CONCLUSIONS Receptor-independent vascular contractility is impaired in mesenteric arteries in portal hypertension. NPY improves non-receptor mediated mesenteric vasoconstriction more effective in portal hypertension than in healthy conditions correcting splanchnic vascular hyporesponsiveness. This beneficial vasoactive action of NPY adds to its well known more pronounced effects on adrenergic vasoconstriction in portal hypertension making it a promising therapeutic agent in portal hypertension.

Thermal release rate studies of nuclear reaction products from polycrystalline metal matrices

The thermal release rate of nuclear reaction products was investigated in offline annealing experiments. This work was motivated by the search for a high melting catcher material for recoiling products from heavy ion induced nuclear fusion reactions. Polycrystalline refractory metal foils of Ni, Y, Zr, Nb, Mo, Hf, W, and Re were investigated as catcher metals. Diffusion data for various tracer/host combinations were deduced from the measured release rates. This work focuses on the diffusion and the release rate of volatile p-elements from row 5 and 6 of the periodic table as lighter homologues of the superheavy elements with Z ≥ 113 to be studied in future experiments. A massive radiation damage enhancement of the diffusion velocity was observed. Diffusion trends have been established along the groups and rows of the periodic table based on the dependence of diffusion velocity on atomic sizes.

Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.

Lowered microvascular vessel wall oxygen consumption augments tissue pO2 during PgE1-induced vasodilation

Continuous infusion of intravenous prostaglandin E1 (PgE1, 2.5 mug/kg/min) was used to determine how vasodilation affects oxygen consumption of the microvascular wall and tissue pO(2) in the hamster window chamber model. While systemic measurements (mean arterial pressure and heart rate) and central blood gas measurements were not affected, PgE1 treatment caused arteriolar (64.6 +/- 25.1 microm) and venular diameter (71.9 +/- 29.5 microm) to rise to 1.15 +/- 0.21 and 1.06 +/- 0.19, respectively, relative to baseline. Arteriolar (3.2 x 10(-2) +/- 4.3 x 10(-2) nl/s) and venular flow (7.8 x 10(-3) +/- 1.1 x 10(-2)/s) increased to 1.65 +/- 0.93 and 1.32 +/- 0.72 relative to baseline. Interstitial tissue pO(2) was increased significantly from baseline (21 +/- 8 to 28 +/- 7 mmHg; P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the microvascular wall decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). The arteriolar vessel wall gradient, a measure of oxygen consumption by the vascular wall, decreased from 20 +/- 6 to 16 +/- 3 mmHg (P < 0.001). This reduction reflects a 20% decrease in oxygen consumption by the vessel wall and up to 50% when cylindrical geometry is considered. The venular vessel wall gradient decreased from 12 +/- 4 to 9 +/- 4 mmHg (P < 0.001). Thus PgE1-mediated vasodilation has a positive microvascular effect: enhancement of tissue perfusion by increasing flow and then augmentation of tissue oxygenation by reducing oxygen consumption by the microvascular wall.

Sex determination and temperature-induced sex differentiation in three natural populations of Nile tilapia (Oreochromis niloticus) adapted to extreme temperature conditions

As a species of major interest for aquaculture, the sex determination system (SDS) of Nile tilapia, Oreochromis niloticus, has been widely investigated. In this species, sex determination is considered to be governed by the interactions between a complex system of genetic sex determination factors (GSD) and the influence of temperature (TSD) during a critical period. Previous studies were exclusively carried out on domestic stocks with the genetic and maintenance limitations associated. Given the wide distribution and adaptation potential of the Nile tilapia, we investigated under controlled conditions the sex determination system of natural populations adapted to three extreme thermal regimes: stable extreme environments in Ethiopia, either cold temperatures in a highland lake (Lake Koka), or warm temperatures in hydrothermal springs (Lake Metahara), and an environment with large seasonal variations in Ghana (Kpandu, Lake Volta). The sex ratio analysis was conducted on progenies reared under constant basal (27 degrees C) or high (36 degrees C) temperatures during the 30 days following yolk-sac resorption. Sex ratios of the progenies reared at standard temperature suggest that the three populations share a similar complex GSD system based on a predominant male heterogametic factor with additional influences of polymorphism at this locus and/or action of minor factors. The three populations presented a clear thermosensitivity of sex differentiation, with large variations in the intensity of response depending on the parents. This confirms the presence of genotype-environment interactions in TSD of Nile tilapia. Furthermore the existence of naturally sex-reversed individuals is strongly suggested in two populations (Kpandu and Koka). However, it was not possible here to infer if the sex-inversion resulted from minor genetic factors and/or environmental influences. The present study demonstrated for the first time the conservation of a complex SDS combining polymorphic GSD and TSD components in natural populations of Nile tilapia. We discuss the evolutionary implications of our findings and highlight the importance of field investigations of sex determination. (c) 2007 Elsevier B.V. All rights reserved.

Thermal right-handed neutrino production rate in the relativistic regime

The production rate of right-handed neutrinos from a Standard Model plasma at a temperature above a hundred GeV is evaluated up to NLO in Standard Model couplings. The results apply in the so-called relativistic regime, referring parametrically to a mass M ~ πT, generalizing thereby previous NLO results which only apply in the non-relativistic regime M ≫ πT. The non-relativistic expansion is observed to converge for M ≳ 15T, but the smallness of any loop corrections allows it to be used in practice already for M ≳ 4T. In the latter regime any non-covariant dependence of the differential rate on the spatial momentum is shown to be mild. The loop expansion breaks down in the ultrarelativistic regime M ≪ πT, but after a simple mass resummation it nevertheless extrapolates reasonably well towards a result obtained previously through complete LPM resummation, apparently confirming a strong enhancement of the rate at high temperatures (which facilitates chemical equilibration). When combined with other ingredients the results may help to improve upon the accuracy of leptogenesis computations operating above the electroweak scale.

Enhancement of Drug-Specific Lymphocyte Proliferation Using CD25-Depleted CD3(+) Effector Cells

Background: The lymphocyte transformation test (LTT) is used for in vitro diagnosis of drug hypersensitivity reactions. While its specificity is over 90%, sensitivity is limited and depends on the type of reaction, drug and possibly time interval between the event and analysis. Removal of regulatory T cells (Treg/CD25(hi)) from in vitro stimulated cell cultures was previously reported to be a promising method to increase the sensitivity of proliferation tests. Objective: The aim of this investigation is to evaluate the effect of removal of regulatory T cells on the sensitivity of the LTT. Methods: Patients with well-documented drug hypersensitivity were recruited. Peripheral blood mononuclear cells, isolated CD3(+) and CD3(+) T cells depleted of the CD25(hi) fraction were used as effector cells in the LTT. Irrelevant drugs were also included to determine specificity. (3)H-thymidine incorporation was utilized as the detection system and results were expressed as a stimulation index (SI). Results: SIs of 7/11 LTTs were reduced after a mean time interval of 10.5 months (LTT 1 vs. LTT 2). Removal of the CD25(hi) fraction, which was FOXP3(+) and had a suppressive effect on drug-induced proliferation, resulted in an increased response to the relevant drugs. Sensitivity was increased from 25 to 82.35% with dramatically enhanced SI (2.05 to 6.02). Specificity was not affected. Conclusion: Removal of Treg/CD25(hi) cells can increase the frequency and strengths of drug-specific proliferation without affecting specificity. This approach might be useful in certain drug hypersensitivity reactions with borderline responses or long time interval since the hypersensitivity reaction. © 2014 S. Karger AG, Basel.

The effect of desmopressin on platelet function: a selective enhancement of procoagulant COAT platelets in patients with primary platelet function defects.

1-deamino-8-d-arginine vasopressin (desmopressin [DDAVP]) is clinically efficacious in patients with mild platelet function disorders but it is not known which mechanisms mediate this effect. Our aim was to evaluate the impact of in vivo DDAVP administration in these patients. We assessed von Willebrand factor (VWF), factor VIII, platelet activation and aggregation, platelet-dependent thrombin generation, and platelet intracellular Na(+)/Ca(2+) fluxes, before and 2 and 4 hours after DDAVP (0.3 µg/kg). We found (1) no significant changes for P-selectin expression, PAC-1 binding, δ-granule content and secretion, and platelet-aggregation; (2) significant decreases of secretion of α-granules and GPIIb-IIIa activation induced by adenosine 5'-diphosphate, convulxin, and thrombin; (3) significant increases of procoagulant platelets induced by convulxin/thrombin and platelet-dependent thrombin generation; and (4) significant increases of intracellular Na(+)/Ca(2+) concentrations. We show that in vivo DDAVP selectively and markedly enhances the ability to form procoagulant platelets and increases platelet-dependent thrombin generation by enhancing Na(+)/Ca(2+) mobilization. This report indicates that the beneficial hemostatic effect of DDAVP is not limited to an increase in large VWF multimers. An enhancement of platelet procoagulant activity appears to be an additional and (at least in platelet disorders) -possibly clinically relevant mechanism of DDAVP's action.

5-ALA complete resections go beyond MR contrast enhancement: shift corrected volumetric analysis of the extent of resection in surgery for glioblastoma

BACKGROUND The technique of 5-aminolevulinic acid (5-ALA) tumor fluorescence is increasingly used to improve visualization of tumor tissue and thereby to increase the rate of patients with gross total resections. In this study, we measured the resection volumes in patients who underwent 5-ALA-guided surgery for non-eloquent glioblastoma and compared them with the preoperative tumor volume. METHODS We selected 13 patients who had received a complete resection according to intraoperative 5-ALA induced fluorescence and CRET according to post-operative T1 contrast-enhanced MRI. The volumes of pre-operative contrast enhancing tissue, post-operative resection cavity and resected tissue were determined through shift-corrected volumetric analysis. RESULTS The mean resection cavity (29 cm(3)) was marginally smaller than the pre-operative contrast-enhancing tumor (39 cm(3), p = 0.32). However, the mean overall resection volume (84 cm(3)) was significantly larger than the pre-operative contrast-enhancing tumor (39 cm(3), p = 0.0087). This yields a mean volume of resected 5-ALA positive, but radiological non-enhancing tissue of 45 cm(3). The mean calculated rim of resected tissue surpassed pre-operative tumor diameter by 6 mm (range 0-10 mm). CONCLUSIONS Results of the current study imply that (i) the resection cavity underestimates the volume of resected tissue and (ii) 5-ALA complete resections go significantly beyond the volume of pre-operative contrast-enhancing tumor bulk on MRI, indicating that 5-ALA also stains MRI non-enhancing tumor tissue. Use of 5-ALA may thus enable extension of coalescent tumor resection beyond radiologically evident tumor. The impact of this more extended resection method on time to progression and overall survival has not been determined, and potentially puts adjacent and functionally intact tissue at risk.

Thermal neutron capture effects in radioactive and stable nuclide systems

Neutron capture effects in meteorites and lunar surface samples have been successfully used in the past to study exposure histories and shielding conditions. In recent years, however, it turned out that neutron capture effects produce a nuisance for some of the short-lived radionuclide systems. The most prominent example is the 182Hf-182W system in iron meteorites, for which neutron capture effects lower the 182W/184W ratio, thereby producing too old apparent ages. Here, we present a thorough study of neutron capture effects in iron meteorites, ordinary chondrites, and carbonaceous chondrites, whereas the focus is on iron meteorites. We study in detail the effects responsible for neutron production, neutron transport, and neutron slowing down and find that neutron capture in all studied meteorite types is not, as usually expected, exclusively via thermal neutrons. In contrast, most of the neutron capture in iron meteorites is in the epithermal energy range and there is a significant contribution from epithermal neutron capture even in stony meteorites. Using sophisticated particle spectra and evaluated cross section data files for neutron capture reactions we calculate the neutron capture effects for Sm, Gd, Cd, Pd, Pt, and Os isotopes, which all can serve as neutron-dose proxies, either in stony or in iron meteorites. In addition, we model neutron capture effects in W and Ag isotopes. For W isotopes, the GCR-induced shifts perfectly correlate with Os and Pt isotope shifts, which therefore can be used as neutron-dose proxies and permit a reliable correction. We also found that GCR-induced effects for the 107Pd-107Ag system can be significant and need to be corrected, a result that is in contrast to earlier studies.

Distortion-free enhancement of terahertz signals measured by electro-optic sampling. II. Experiment

Three methods for distortion-free enhancement of electro-optic sampling measurements of terahertz signals are tested. In the first part of this two-paper series [J. Opt. Soc. Am B 31, 904–910 (2014)], the theoretical framework for describing the signal enhancement was presented and discussed. As the applied optical bias is decreased, individual signal traces become enhanced but distorted. Here we experimentally show that nonlinear signal components that distort the terahertz electric field measurement can be removed by subtracting traces recorded with opposite optical bias values. In all three methods tested, we observe up to an order of magnitude increase in distortion-free signal enhancement, in agreement with the theory, making possible measurements of small terahertz-induced transient birefringence signals with increased signal-to-noise ratio.

Daily and seasonal thermal stresses in tilings: a field survey combined with numeric modeling

This study investigates thermally induced tensile stresses in ceramic tilings. Daily and seasonal thermal cycles, as well as, rare but extreme events, such as a hail-storm striking a heated terrace tiling, were studied in the field and by numerical modeling investigations. The field surveys delivered temperature– time diagrams and temperature profiles across tiling systems. These data were taken as input parameters for modeling the stress distribution in the tiling system in order to detect potential sites for material failure. Dependent on the thermal scenario (e.g., slow heating of the entire structure during morning and afternoon, or a rapid cooling of the tiles by a rain storm) the modeling indicates specific locations with high tensile stresses. Typically regions along the rim of the tiling field showed stresses, which can become critical with respect to the adhesion strength. Over the years, ongoing cycles of thermal expansion–contraction result in material fatigue promoting the propagation of cracks. However, the installation of flexible waterproofing membranes (applied between substrate and tile adhesive) represents an efficient technical innovation to reduce such crack propagation as confirmed by both numerical modeling results and microstructural studies on real systems.

Acupuncture-induced changes of pressure pain threshold are mediated by segmental inhibition-a randomized controlled trial

Our aim was to distinguish between spinal and supraspinal mechanisms in the intact nervous system by comparing homosegmental and heterosegmental effects of electroacupuncture (EA) and manual acupuncture (MA) on sensory perception in healthy volunteers by means of quantitative sensory testing. Seventy-two healthy volunteers were randomly assigned to receive either MA or EA at SP 6, SP 9, GB 39, and ST 36 at the left leg or relaxed for 30 minutes (control group [CG]). Blinded examiners assessed 13 sensory modalities (thermal and mechanical detection and pain thresholds) at the upper arms and lower legs before and after intervention by means of a standardized quantitative sensory testing battery. Change scores of all 13 sensory thresholds were compared between groups. The main outcome measure was the change score of the pressure pain threshold (PPT). There were no baseline differences between groups. Pressure pain threshold change scores at the lower left leg, in the same segment as the needling site, differed significantly (P = 0.008) between the EA (median: 103.01 kPa) and CG groups (median: 0.00 kPa) but not between the MA (median: 0.00 kPa) and CG groups. No further significant change score differences were found between one of the acupuncture groups and the CG. The PPT can be changed by EA. The PPT increase was confined to the segment of needling, which indicates that it is mainly mediated by segmental inhibition in the spinal cord. This underscores the importance of segmental needling and electrical stimulation in clinical practice.