Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3.

BACKGROUND: The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) is a highly destructive pest of pine forests in western North America. During flight to a new host tree and initiation of feeding, mountain pine beetles release aggregation pheromones. The biosynthetic pathways of these pheromones are sex-specific and localized in the midgut and fat body, but the enzymes involved have not all been identified or characterized. RESULTS: We used a comparative RNA-Seq analysis between fed and unfed male and female MPB midguts and fat bodies to identify candidate genes involved in pheromone biosynthesis. The 13,407 potentially unique transcripts showed clear separation based on feeding state and gender. Gene co-expression network construction and examination using petal identified gene groups that were tightly connected. This, as well as other co-expression and gene ontology analyses, identified all four known pheromone biosynthetic genes, confirmed the tentative identification of four others from a previous study, and suggested nine novel candidates. One cytochrome P450 monooxygenase, CYP6DE3, identified as a possible exo-brevicomin-biosynthetic enzyme in this study, was functionally characterized and likely is involved in resin detoxification rather than pheromone biosynthesis. CONCLUSIONS: Our analysis supported previously characterized pheromone-biosynthetic genes involved in exo-brevicomin and frontalin biosynthesis and identified a number of candidate cytochrome P450 monooxygenases and a putative cyclase for further studies. Functional analyses of CYP6DE3 suggest its role in resin detoxification and underscore the limitation of using high-throughput data to tentatively identify candidate genes. Further functional analyses of candidate genes found in this study should lead to the full characterization of MPB pheromone biosynthetic pathways and the identification of molecular targets for possible pest management strategies.

Muscle growth in young horses: Effects of age, cytokines, and growth factors.

Success as equine athletes requires proper muscle growth in young horses. Muscle hypertrophy occurs through protein synthesis and the contribution of muscle satellite cells, which can be stimulated or inhibited by cytokines and growth factors present during exercise and growth. The hypotheses of this study were that 1) the LM area in young horses would increase over 1 yr, and 2) specific cytokines and growth factors (IL-1ß, IL-6, tumor necrosis factor [TNF]-α, IGF-I, and fibroblast growth factor [FGF]-2) would alter proliferation and differentiation of satellite cells isolated from young horses. Fourteen horses were divided into 3 age groups: weanlings ( = 5), yearlings to 2 yr olds ( = 4), and 3 to 4 yr olds ( = 5). The area, height, and subcutaneous fat depth of the LM were measured using ultrasonography, and BW and BCS were taken in October (Fall1), April (Spring), and October of the following year (Fall2). Satellite cells obtained from 10-d-old foals ( = 4) were cultured in the presence of IL-6, IL-1ß, TNF-α, IGF-I, or FGF-2 before evaluation of proliferation and differentiation. Data were analyzed using PROC MIXED in SAS. Body weight increased from Fall1 to Spring in weanlings ( < 0.001) and increased in all horses from Spring to Fall2 ( ≤ 0.02). Area and height of the LM increased over time ( < 0.001) and with increasing age group of horse ( ≤ 0.03), although there was no interaction of time and age ( > 0.61). There was a significant increase in LM area in all animals from Spring to Fall2 ( < 0.001) but not from Fall1 to Spring. Interleukin-6 and TNF-α decreased satellite cell proliferation by 14.9 and 11.5%, respectively ( ≤ 0.01). Interleukin-6 increased fusion 6.2%, whereas TNF-α decreased fusion 8.7% compared with control cells ( ≤ 0.001). Interleukin-1ß had no effect on proliferation ( = 0.32) but tended to decrease fusion ( = 0.06). Satellite cell proliferation was increased 28.8 and 73.0% by IGF-I and FGF-2, respectively ( < 0.0001). Differentiation was decreased 13.1% in the presence of FGF-2 but increased 3.5% in the presence of IGF-I ( ≤ 0.01). In summary, the LM area increases over the course of a year in young horses with the most growth occurring in summer. By stimulating or inhibiting proliferation and differentiation of satellite cells, IL-6, TNF-α, IL-1ß, IGF-I, and FGF-2 may alter muscle growth in young horses, thereby impacting athletic potential.

Comparison of endoscopic, necropsy and histology scoring of equine gastric ulcers.

Equine gastric ulcer syndrome (EGUS) represents a major health problem in performance horses. Much debate exists regarding endoscopic gastric ulcer scoring systems and their ability accurately to predict severity or depth of gastric ulcers. The purpose of this study was to evaluate the ability of an endoscopist to count gastric ulcers and predict gastric ulcer severity or depth using 2 endoscopic scoring systems and compare them to the same gastric ulcers see on necropsy and histopathology. Endoscopic examination of the stomach was performed under general anaesthesia on 23 mixed breed yearling horses, after feed was withheld for 24 h. Gastric ulcers were scored using 2 systems, number/severity-scoring (N/S) and practitioner simplified (PS) systems. After endoscopy, the horses were subjected to euthanasia and the stomach mucosa examined blindly and scored again at necropsy using above scoring systems. Representative gastric ulcers were then placed in 10% formalin and processed routinely for histopathology. The gastric ulcers were scored using a histopathology system (HSS) based on ulcer depth. Number scores in the N/S scoring system and PS on endoscopic and necropsy examinations were compared using Friedman 2 way analysis of variance. Where significant differences between variables were found a post hoc analysis was conducted using a Tukey's Studentised range (HSD) test. Severity scores using the N/S (ENGS) and PS scores recorded for the stomach via endoscopy and scores from HSS were evaluated for significant association using a Mantel-Haenszel Chi-square and Pearson moment correlation coefficient analysis. Significance was P < 0.05. All horses had gastric ulcers in the nonglandular mucosa via endoscopic examination and at necropsy examination. Mean nonglandular ulcer number (ENGN) score was significantly (P = 0.0024) lower on endoscopic examination compared to the score at necropsy (NNGN); whereas PS scores were not significantly different on endoscopy when compared to necropsy examination. A significant but weak association was found between ENGS and HSS (3.89, P = 0.048; r = 0.453, P = 0.045) and no correlation was found between PS and HSS (1.2, P = 0.272; r = 0.117; P = 0.622). Only 1/23 horses had glandular ulcers observed via endoscopic examination whereas, 6/23 horses had glandular ulcers at necropsy and on histopathology. The prevalence of EGUS is high in stalled yearling horses. The endoscopist may underestimate the number of gastric ulcers and may not be able accurately to predict the severity or depth of those ulcers present in the nonglandular equine stomach. Furthermore, the endoscopist may miss glandular gastric ulcers.

Measurement of total body water content in horses, using deuterium oxide dilution.

OBJECTIVE: To measure total body water (TBW) content in horses, using deuterium oxide (D2O) dilution. ANIMALS: Six 8- to 10-year-old healthy untrained mixed-breed horses, weighing (mean +/-SD) 503.4 +/- 64.0 kg. PROCEDURE: After a 12-hour nonfeeding period, 6 horses were given D2O (0.14 g/kg of body weight) via nasogastric tube. Blood samples were collected from a preplaced indwelling jugular vein catheter prior to and 1 to 8, 10, 12, 14, and 24 hours after administration of D2O. Blood samples were centrifuged immediately, and plasma was collected and stored at -70 C until analysis. The D2O content in plasma was measured by zinc reduction to deuterium gas. The resulting gas was measured, using an isotope ratio mass spectrometer. RESULTS: Deuterium oxide was rapidly absorbed from the gastrointestinal tract of all horses, and reached peak (mean +/- SD) plasma concentration (1,454.4 +/- 163 delta D/ml or parts/thousand) 1 hour after administration. Plasma concentration decreased slowly during the next 2 to 3 hours, then remained statistically constant from 2 to 5 hours (early plateau phase) and 3 to 7 hours (late plateau phase) after administration. Mean +/- SEM TBW content was 623.0 +/- 2.2 ml/kg (62.3% of body weight) for the early plateau phase and 630.3 +/- 2.2 ml/kg (63.0% of body weight) for the late plateau phase. CONCLUSION: Deuterium oxide dilution appears to be of value for measurement of TBW content in horses, and has a 4-hour plateau effect. Equilibration of D2O with large intestinal water may be the reason for the prolonged equilibrium time and plateau effect seen in these horses. CLINICAL RELEVANCE: Deuterium oxide appears safe and efficacious for determining TBW content in horses and may be helpful for determining changes in TBW content during exercise and disease.

Effects of orally administered enteric-coated omeprazole on gastric acid secretion in horses.

OBJECTIVES: To determine the effects of orally administered omeprazole, as enteric-coated capsules, on baseline and stimulated gastric acid secretion in horses. ANIMALS: 5 healthy 8-year-old mixed-breed horses fitted with gastric cannulas. PROCEDURE: Enteric-coated granules of omeprazole were mixed with corn syrup and administered orally once daily for 5 consecutive days. On days 1 and 5 beginning 5 hours after omeprazole administration, 4 gastric fluid samples were collected, each for 15 minutes, via the gastric cannula (baseline samples). Pentagastrin was administered IV as a constant infusion for the subsequent 2 hours, and 15-minute gastric fluid samples were again collected (stimulated samples). Fluid volume, acidity (mmol H-/L), and pH and gastric acid production (mmol H+) were determined for all baseline samples and for stimulated samples collected during the second hour of pentagastrin infusion. Control experiments were done in a similar manner after giving corn syrup alone to the same horses. RESULTS: Compared with values obtained during control experiments, baseline and stimulated gastric fluid acidity and gastric acid production significantly decreased, and the mean pH of gastric fluid samples significantly increased, after horses were given 5 daily doses of omeprazole. CONCLUSIONS AND CLINICAL RELEVANCE: Enteric-coated omeprazole (1.0 mg/kg of body weight; PO) administered once daily for 5 days significantly inhibited unstimulated and pentagastrin-stimulated gastric acid secretion in horses. This commercially available formulation of omeprazole may be efficacious in the treatment of gastroduodenal ulcers in horses.

Evaluation of diet as a cause of gastric ulcers in horses.

OBJECTIVE: To measure pH, volatile fatty acid (VFA) concentrations, and lactate concentrations in stomach contents and determine number and severity of gastric lesions in horses fed bromegrass hay and alfalfa hay-grain diets. ANIMALS: Six 7-year-old horses. PROCEDURE: A gastric cannula was inserted in each horse. Horses were fed each diet, using a randomized crossover design. Stomach contents were collected immediately after feeding and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 hours after feeding on day 14. The pH and VFA and lactate concentrations were measured in gastric juice Number and severity of gastric lesions were scored during endoscopic examinations. RESULTS: The alfalfa hay-grain diet caused significantly higher pH in gastric juice during the first 5 hours after feeding, compared with that for bromegrass hay. Concentrations of acetic, propionic, and isovaleric acid were significantly higher in gastric juice, and number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. Valeric acid, butyric acid, and propionic acid concentrations and pH were useful in predicting severity of nonglandular squamous gastric lesions in horses fed alfalfa hay-grain, whereas valeric acid concentrations and butyric acid were useful in predicting severity of those lesions in horses fed bromegrass hay. CONCLUSIONS AND CLINICAL RELEVANCE: An alfalfa hay-grain diet induced significantly higher pH and VFA concentrations in gastric juice than did bromegrass hay. However, number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. An alfalfa hay-grain diet may buffer stomach acid in horses.

Age effects on blood gas, spirometry, airway reactivity, and bronchoalveolar lavage fluid cytology in clinically healthy horses.

BACKGROUND: Despite the increasing number of geriatric horses attended by veterinarians, there is a lack of understanding of aging-related changes on the respiratory system of horses. OBJECTIVE: To identify aging-related changes on the respiratory function and bronchoalveolar lavage fluid (BALF) cytology of horses. ANIMALS: Fifteen healthy young adult (2-11 years) and 16 healthy aged (≥20 years) horses. METHODS: The respiratory system was examined by measurement of arterial blood gases (ABG), use of respiratory inductive plethysmography (RIP) for assessment of breathing pattern and ventilatory parameters, histamine bronchoprovocation, and BALF cytology. RESULTS: No significant differences were detected with regard to values obtained by ABG or bronchoprovocation of young adult and aged healthy horses. In aged horses, there were significant differences in mean ± SD of the following parameters when compared to young horses: prolonged expiratory time (Te) measured by RIP (3.9 ± 1.5 s versus 3.0 ± 0.6 s), decreased percentage of alveolar macrophages (40.6 ± 11.3% versus 53.5 ± 9.6%), and increased percentage of lymphocytes (53.4 ± 9.5% versus 43.9 ± 11.0%). No correlations between airway reactivity and ventilatory parameters, ABG, or BALF cytology were found in this asymptomatic population. CONCLUSIONS: These results suggest that aging does not cause changes in the results obtained by ABG, most RIP-derived variables, and bronchoprovocation in the horse. A decreased percentage of macrophage and an increased percentage of lymphocytes in the BALF cytology may be expected in the asymptomatic geriatric horse and may be a result of aging.

Reform of the Quebec healthcare system.

This article briefly describes issues surrounding the reform process in Quebec and discusses its major thrusts--regionalization, definition of healthcare objectives, strengthening frontline services and greater use of outpatient services--and their effect on institutions which must above all come to terms with the reduction of the provincial deficit. Fewer human resources, longer waiting periods, institutional autonomy and the creation of integrated networks are some of the issues of current concern to the Quebec healthcare community.

Temporal and spatial regulation of 1-aminocyclopropane-1-carboxylate oxidase in the pollination-induced senescence of orchid flowers.

Pollination of many flowers initiates a sequence of precisely regulated developmental events that include senescence of the perianth and development of the ovary. The plant hormone ethylene is known to play a key role in regulating the biochemical and anatomical changes that constitute the postpollination syndrome. For this reason, we have studied the pollination syndrome in Phalaenopsis orchids by examining the spatial and temporal location of ethylene biosynthesis within the orchid flower, and how this biosynthesis is regulated by factors that influence expression of genes that encode key enzymes in the ethylene biosynthetic pathway. In particular, we examined the role in the postpollination syndrome of the expression of the gene for 1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyzes the conversion of ACC to ethylene. In vivo incubation of tissues with the ethylene precursor ACC demonstrated that ACC oxidase activity increases after pollination in the stigma, contrary to the observation that activity is constitutive in petunia and carnation gynoecia. RNA blot hybridization of floral tissues indicates that the increase in ACC oxidase activity is due to de novo synthesis of mRNA and presumably protein, which is induced after pollination. Furthermore, the pattern of induction is consistent with a model of coordinate regulation of gene expression in which the pollination signal travels to other organs of the flower to induce their ethylene production. We have also used in situ hybridization to define further the temporal and spatial expression of ACC oxidase within the floral organs, showing that expression, and,by inference, the capability to oxidize ACC to ethylene, is induced in all living cells of the tissues examined after pollination. These findings contrast with work in petunia that suggests that ACC oxidase is localized to the stigmatic surface.

Identification of a meristem L1 layer-specific gene in Arabidopsis that is expressed during embryonic pattern formation and defines a new class of homeobox genes.

Homeobox genes are master regulatory genes that specify the body plan and control development of many eukaryotic organisms, including plants. We isolated and characterized a cDNA designated ATML1 (for Arabidopsis thaliana meristem L1 layer) that encodes a novel homeodomain protein. The ATML1 protein shares high sequence homology inside and outside of the homeodomain with both the Phalaenopsis O39 and the Arabidopsis GLABRA2 (GL2) homeodomain proteins, which together define a new class of plant homeodomain-containing proteins, designated HD-GL2. The ATML1 gene was first expressed in the apical cell after the first asymmetric division of the zygote and continued to be expressed in all proembryo cells until the eight-cell stage. In the 16-cell proembryo, the ATML1 gene showed a distinct pattern of expression, with its mRNA becoming restricted to the protoderm. In the torpedo stage of embryo development, ATML1 mRNA disappeared altogether but reappeared later only in the L1 layer of the shoot apical meristem in the mature embryo. After germination, this L1 layer-specific pattern of expression was maintained in the vegetative shoot apical meristem, inflorescence, and floral meristems, as well as in the young floral organ primordia. Finally, ATML1 mRNA accumulated in the protoderm of the ovule primordia and integuments and gradually became restricted in its expression to the endothelium surrounding the embryo sac. We propose that ATML1 may be involved in setting up morphogenetic boundaries of positional information necessary for controlling cell specification and pattern formation. In addition, ATML1 provides an early molecular marker for the establishment of both apical-basal and radial patterns during plant embryogenesis.

Effect of nervous excitation on acid secretion in horses.

Nervous excitation was induced by various means in horses provided with a gastric cannula. Insulin hypoglycaemia profoundly inhibited the basal acid output and volume secreted from the stomach. No clear effect on acid secretion was noted after administration of bethanechol, as the acid output was covered by the copious secretion of saliva. Atropine almost abolished the basal acid output. Sensoric stimulation by teasing caused a slight but not significant increase in the total acid output. These data suggest that cholinergic excitation might play a role in the stimulation of both volume and acid secretion in the horse. The inhibitory effect seen on these two parameters after insulin hypoglycaemia may hypothetically be ascribed to inhibitory impulses carried in peptide neurones of the vagal nerves or to inhibitory impulses in adrenergic nerves acting directly or indirectly on the parietal cells.

Characterization of a cytokinesis defective (cyd1) mutant of Arabidopsis.

Although several mutations and genes affecting plant cytokinesis have been identified, mutant screens are not yet saturated and knowledge about gene function is still limited. A novel Arabidopsis mutation, cytokinesis defective1 (cyd1), was identified by partial or missing cell walls in stomata. Stomata with incomplete or no cytokinesis still differentiate and some contain swellings of the outer wall not found in the wild type. The incomplete walls are correctly placed opposite stomatal wall thickenings suggesting that the mutation interferes with the execution of cytokinesis rather than with the placement of the division site. Cytokinesis defects are also detectable in other cell types throughout the plant, defects which include cell wall protrusions, two or more nuclei in one cell, and reduced cell number. The extent of cytokinetic partitioning correlates with nuclear number in abnormal stomata. Many cyd1 epidermal cells, stomata and pollen are larger, and trichomes have more branches. cyd1 is partially lethal with poor seed set and some defective ovules, but many plants are fertile despite abnormalities in vegetative and reproductive development such as missing, reduced, fused or misshapen leaves and floral organs. cyd1 appears to be the only cytokinesis mutant described where defects are known to occur in both mature vegetative and reproductive organs. Thus, the CYD1 gene product appears to be necessary for the execution of cytokinesis throughout the shoot. The examination of stomata by microscopy may be a useful screen for the directed isolation of additional cytokinesis mutations that are not embryo or seedling lethal

Ovule development: identification of stage-specific and tissue-specific cDNAs.

A differential screening approach was used to identify seven ovule-specific cDNAs representing genes that are expressed in a stage-specific manner during ovule development. The Phalaenopsis orchid takes 80 days to complete the sequence of ovule developmental events, making it a good system to isolate stage-specific ovule genes. We constructed cDNA libraries from orchid ovule tissue during archesporial cell differentiation, megasporocyte formation, and the transition to meiosis, as well as during the final mitotic divisions of female gametophyte development. RNA gel blot hybridization analysis revealed that four clones were stage specific and expressed solely in ovule tissue, whereas one clone was specific to pollen tubes. Two other clones were not ovule specific. Sequence analysis and in situ hybridization revealed the identities and domain of expression of several of the cDNAs. O39 encodes a putative homeobox transcription factor that is expressed early in the differentiation of the ovule primordium; O40 encodes a cytochrome P450 monooxygenase (CYP78A2) that is pollen tube specific. O108 encodes a protein of unknown function that is expressed exclusively in the outer layer of the outer integument and in the female gametophyte of mature ovules. O126 encodes a glycine-rich protein that is expressed in mature ovules, and O141 encodes a cysteine proteinase that is expressed in the outer integument of ovules during seed formation. Sequences homologous to these ovule clones can now be isolated from other organisms, and this should facilitate their functional characterization.

Effects of intramuscular omeprazole on gastric acid secretion in horses over a twenty-four hour period.

The effect of intramuscular (i.m.) omeprazole (0.25 or 1.0 mg/kg bwt; LD and HD), respectively, on volume, total acid output (TAO) and pH of the gastric juice was studied during 24 h in 5 horses with a chronically implanted gastric cannula. Whether secretion in controls was basal or stimulated with pentagastrin (8 micrograms/kg bwt/h), volume (NS) and TAO (P < 0.01, NS) gradually decreased and pH increased (P < 0.05, NS). Omeprazole significantly reduced the average basal TAO by 49 +/- 6% (LD) and 88 +/- 3% (HD) and the stimulated TAO by 64 +/- 2% and 97 +/- 1%. Basal pH in controls was 2.1-4.2 and after omeprazole treatment, pH 2.8-4.1 (LD) and 2.4-6.6 (HD). After stimulation, the corresponding pH values were 2.6-3.3, 3.9-4.9 and 5.4-7.2. The biological availability of omeprazole was 70-80%. Due to the simplicity of the administration technique and the higher biological availability, intramuscular administration may offer a practical and less expensive way of treating gastric ulcers in horses.