Differential Pre-mRNA Splicing Regulates Nnat Isoforms in the Hypothalamus after Gastric Bypass Surgery in Mice

Background

Neuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat.

Methods

Expression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA.

Results

Hypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R² = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R² = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine.

Conclusions

Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss.     

A second-generation linkage map of the sheep genome

A genetic map of Ovis aries (haploid n = 27) was developed with 519 markers (504 microsatellites) spanning ∼3063 cM in 26 autosomal linkage groups and 127 cM (female specific) of the X Chromosome (Chr). Genotypic data were merged from the IMF flock (Crawford et al., Genetics 140, 703, 1995) and the USDA mapping flock. Seventy-three percent (370/504) of the microsatellite markers on the map are common to the USDA-ARS MARC cattle linkage map, with 27 of the common markers derived from sheep. The number of common markers per homologous linkage group ranges from 5 to 22 and spans a total of 2866 cM (sex average) in sheep and 2817 cM in cattle. Marker order within a linkage group was consistent between the two species with limited exceptions. The reported translocation between the telomeric end of bovine Chr 9 (BTA 9) and BTA 14 to form ovine Chr 9 is represented by a 15-cM region containing 5 common markers. The significant genomic conservation of marker order will allow use of linkage maps in both species to facilitate the search for quantitative trait loci (QTLs) in cattle and sheep. Received: 20 September 1992 / Accepted: 18 November 1997     

Roadside Revegetation in Glacier National Park, U.S.A.: Effects of Herbicide and Seeding Treatments

From 1992 to 1995 we experimentally evaluated the effectiveness of several revegetation treatments along a segment of Going-to-the-Sun Highway in Glacier National Park, U.S.A. This segment, reconstructed during the spring and summer of 1992, is bordered by fescue prairie vegetation and is known to be susceptible to invasion by several alien species, including Centaurea maculosa (spotted knapweed) and Phleum pratense (common timothy). We used a split plot study design to evaluate the effectiveness of herbicide and seeding treatments on assisting recovery of native flora and limiting the establishment of alien species. The herbicide treatment consisted of a yearly herbicide spray application of clopyralid (3,6-dichloropicolinic acid). Five seeding treatments were evaluated, three of which included an indigenous graminoid-forb seed mix. Percent canopy coverages of four species groups—alien graminoids, native graminoids, alien forbs, and native forbs—were determined in July 1995. In addition, community-level patterns in sprayed plots and unsprayed plots were compared with a reference site of native fescue prairie. Herbicide treatments decreased mean canopy coverage of alien forbs (treated = 4.2%, untreated = 23.4%) and increased mean canopy coverage of native graminoids slightly (treated = 6.3%, untreated = 4.0%). But herbicide treatments reduced mean coverage of native forbs (treated = 3.9%, untreated = 8.9%) and likely increased coverage of alien graminoids. Treatments that included a fall 1992 seed mix increased native graminoid coverages 2.8–4.6 times, although coverages were still lower than those attained by alien graminoids. Native and alien forb coverage appeared unaffected by seeding treatments. Species composition was less diverse in sprayed plots and more dominated by alien grasses than in unsprayed plots and the reference site. Areas for additional study are suggested, including seed bank assays to determine treatment effects on recruitment of alien versus native species and the use of native graminoids to create low-diversity communities with high canopy coverages to resist establishment of alien species.     

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells^3-5. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells⁶, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies¹. Originally published by Naal et al.¹, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease^7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function². In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε₂₈₀ = 4,200 L/M/cm)¹². This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Differential Effector Engagement by Oncogenic KRAS

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Diversity and composition of macroinvertebrate communities in a rare inland salt marsh

Inland salt marshes are rare habitats in the Great Lakes region of North America, formed on salt deposits from the Silurian period. These patchy habitats are abiotically stressful for the freshwater invertebrates that live there, and provide an opportunity to study the relationship between stress and diversity. We used morphological and COI metabarcoding data to assess changes in diversity and composition across both space (a transect from the salt seep to an adjacent freshwater area) and time (three sampling seasons). Richness was significantly lower at the seep site with both datatypes, while metabarcoding data additionally showed reduced richness at the freshwater transect end, consistent with a pattern where intermediate levels of stress show higher diversity. We found complementary, rather than redundant, patterns of community composition using the two datatypes: not all taxa were equally sequenced with the metabarcoding protocol. We identified taxa that are abundant at the salt seep of the marsh, including biting midges (Culicoides) and ostracods (Heterocypris). We conclude that (as found in other studies) molecular and morphological work should be used in tandem to identify the biodiversity in this rare habitat. Additionally, salinity may be a driver of community membership in this system, though further ecological research is needed to rule out alternate hypotheses.