Fast and sensitive detection of indels induced by precise gene targeting

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

The biological activity of catfish pancreatic somatostatin

Catfish pancreatic somatostatin, which contains eight additional amino acids on the amino terminus of a tetradecapeptide with considerable homology to tetradecapeptide somatostatin (SRIF), is a naturally occurring homology of the hypothalamic peptide. The purpose of these studies was to determibe the biological activity of this somatostatin homolog. Inhibition of ¹²⁵I-labelled tyr¹-SRIF binding to bovine pituitart plasma membranes by catfish pancreatic somatostatin was approximately 33% that of SRIF. Pancreatic somatostatin has full biological activity measured by inhibition of growth hormone release from isolated rat pituitary cells, but 0.01–0.1% the potency of SRIF. Pancreatic somatostatin at 100 ng/ml produced a 50–60% inhibition of insulin and glucagon secretion from perfused rat pancreas, while SRIF produced comparable inhibition at 10 ng/ml. This report demonstrates that a larger molecular form and natural homolog of SRIF, isolated from fish pancreas, has the same (but reduced) biological activities in rat assay systems as somatostatin originally isolated from sheep hypothalamus.     

Saccharomyces cerevisiae leukotriene A4 hydrolase: formation of leukotriene B4 and identification of catalytic residues

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.     

Dietary vitamin A intake below the recommended daily intake during pregnancy and the risk of congenital diaphragmatic hernia in the offspring

BACKGROUND

Vitamin A has been related to the etiology of congenital diaphragmatic hernia (CDH). We performed a case‐control study to investigate whether maternal dietary vitamin A intake is related to CDH in the offspring.

METHODS

Thirty‐one pregnancies diagnosed with CDH and 46 control pregnancies were included during the study. After CDH diagnosis and inclusion of controls by risk set sampling, maternal vitamin A intake was investigated with a food frequency questionnaire. Serum retinol and retinol‐binding protein were determined. Univariable and multivariable logistic regression models were used to calculate risk estimates with adjustment for potential confounders.

RESULTS

We found no significant differences in the overall nutrient and vitamin A intake between case and control mothers. After stratification in body mass index (BMI) categories, case mothers with normal weight showed a lower energy adjusted vitamin A intake (685 vs. 843 μg retinol activity equivalents [RAEs] / day; p = 0.04) and a slightly lower serum retinol (1.58 vs. 1.67 μmol/L; p = 0.08) than control mothers. Vitamin A intake <800 μg retinol activity equivalents (recommended daily intake) in normal weight mothers was associated with a significantly increased CDH risk (odds ratio [OR], 7.2; 95% confidence interval [CI], 1.5–34.4; p = 0.01). Associations were not significantly different in underweight and overweight mothers.

CONCLUSIONS

In normal‐weight mothers, dietary vitamin A intake during pregnancy below the recommended daily intake is significantly associated with an increased risk of a child with CDH. This finding supports the retinoid hypothesis in human CDH, but warrants further investigation in larger study populations. Birth Defects Research (Part A), 2013. © 2013 Wiley Periodicals, Inc.     

Interleukin-6 Induced Suppression of Bovine Parathyroid Hormone Secretion

Calcium disturbances in the critically ill coincide with elevations of proinflammatory cytokines. The effects of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) on parathyroid hormone (PTH) secretion were investigated. IL-6 and TNF- had no acute effect on PTH secretion in extracellular Ca²⁺ concentrations of 0.5, 1.25 and 3.0 mM. In contrast to TNF-, cultures for 24 h in the presence of l0 ng/mL of IL-6 showed decreased PTH secretion by 51% and 29% in 0.5 mM and 1.25 mMCa²⁺ respectively. Neither IL-6 nor TNF-, affected cytoplasmic Ca²⁺ of the cells. We conclude that PTH secretion in vitro can be suppressed by IL-6 at clinically relevant concentrations. This suppression may aggravate hypocalcemia of the critically ill and attenuate the conventionally strong stimulation of the PTH release by reduction in serum calcium.     

Local vasodilatation with metacholine, but not with nitroprusside, increases forearm glucose uptake

Insulin is known to increase blood flow in parallel to glucose uptake in skeletal muscle. However, it is not known if an increase in blood flow by itself is associated with an increase in glucose uptake in the absence of hyperinsulinemia. To investigate further this matter, the effect of increased blood flow on forearm glucose uptake was studied in the fasting state during intra-arterial infusions of two different vasodilators, metacholine and nitroprusside, in 19 hypertensive subjects. Both metacholine (4 microg/min) and nitroprusside (10 microg/min) increased resting forearm blood flow, measured by venous occlusion plethysmography, to a similar degree (180 % and 170 %, respectively, p<0.0001 for both). However, metacholine infusion increased the forearm glucose uptake from 2.0+/-0.9 (S.D.) during rest to 5.5+/-3.0 umol/min/100 ml tissue (p<0.0001), while no significant change in glucose uptake was seen during nitroprusside infusion (2.3+/-1.4 micromol/min/100 ml tissue). In conclusion, vasodilatation induced by metacholine, but not by nitroprusside, increased glucose uptake in the forearm of hypertensive patients. Thus, an increase in forearm blood flow does not necessarily improve glucose uptake in the forearm during the fasting state.