Noninvasive Assessment of Pulse-Wave Velocity and Flow-Mediated Vasodilation in Anesthetized G?ttingen Minipigs

Few methods for noninvasive assessment of arterial stiffness and endothelial dysfunction in porcine models are available. The aim of this study was to evaluate methods for assessment of arterial stiffness and endothelial dysfunction in anesthetized Göttingen minipigs. Pulse-wave velocity (PWV) was assessed in male Göttingen minipigs (n = 8; age approximately 60 wk) by using applanation tonometry of the carotid and femoral arteries. In addition, flow-mediated vasodilation (FMD) was assessed by using vascular ultrasonography of the brachial artery to evaluate endothelial dysfunction. To evaluate the reproducibility of the methods, minipigs were anesthetized by intravenous infusion of ketamine and midazolam and examined every other day for a total of 3 trials. Neither examination day nor systolic, diastolic, or mean arterial blood pressure statistically influenced PWV or FMD. The median interexamination coefficient of variation was 17% for PWV and 59% for FMD. Measured values of PWV corresponded largely to those in clinically healthy humans, but FMD values were lower than expected for lean, young animals. Although the ketamine–midazolam anesthesia we used has been associated with minor hemodynamic effects in vivo, in vitro studies suggest that both drugs are vasodilatory. Therefore anesthesia might have influenced the endothelial response, contributing to the modest FMD response and the concurrent high coefficients of variation that we noted. We conclude that PWV—but not FMD—showed acceptable interexamination variation for its potential application in porcine models.Abbreviations: FMD, flow-mediated vasodilation; FVI, integrated flow velocity; GTN, glyceryl trinitrate; PWV, pulse-wave velocity; T, transit timeCardiovascular disease has become a global challenge in public health,⁴² and the development and characterization of comparative animal models are of increasing importance. Several animal models of atherosclerosis, including porcine, have been described.^11,12,34 Due to similarities to humans in the anatomy of the cardiovascular system and metabolic physiology, pigs represent a generally useful model in regard to preclinical evaluation and pharmacology.³⁶ Assessment of changes related to atherosclerosis in vivo would be valuable in for example longitudinal assessment of drug effect, but few noninvasive methods for evaluating structural and functional changes in the arteries of pigs are available. In humans, increased arterial stiffness, which occurs with advanced age, also is caused by the pathophysiologic changes associated with atherosclerosis,²⁶ and noninvasive methods for assessing arterial stiffness have been established. The evaluation of pulse-wave velocity (PWV) by using pressure transducers, such as applanation tonometry, is a method recognized as an independent predictor for cardiovascular events in epidemiologic studies.²¹ The method evaluates the velocity with which the pulse wave is propagated through the arterial tree, with arterial stiffness causing increased velocity.^{1,22,29,37,39} Flow-mediated vasodilation (FMD), assessed by vascular ultrasonography, represents a noninvasive evaluation of endothelial-dependent vasodilation. A decrease in vasodilation as a response to increased shear stress has been recognized as a marker of endothelial dysfunction, which precedes the development of atherosclerosis.^5,7 Recent studies have shown that the FMD method is applicable in large animals (that is, dogs and horses) and that a decreased FMD response occurs in dogs with valvular heart disease.^10,15,23,28The aim of this study was to evaluate the reproducibility of methods for assessing arterial stiffness (PWV) and endothelial function (FMD response) in anesthetized Göttingen minipigs, including the influences of arterial blood pressure, heart rate, and room and body temperatures on these methods.     

High Fat,Low Carbohydrate Diet Limit Fear and Aggression in G?ttingen Minipigs

High fat, low carbohydrate diets have become popular, as short-term studies show that such diets are effective for reducing body weight, and lowering the risk of diabetes and cardiovascular disease. There is growing evidence from both humans and other animals that diet affects behaviour and intake of fat has been linked, positively and negatively, with traits such as exploration, social interaction, anxiety and fear. Animal models with high translational value can help provide relevant and important information in elucidating potential effects of high fat, low carbohydrate diets on human behaviour. Twenty four young, male Göttingen minipigs were fed either a high fat/cholesterol, low carbohydrate diet or a low fat, high carbohydrate/sucrose diet in contrast to a standard low fat, high carbohydrate minipig diet. Spontaneous behaviour was observed through video recordings of home pens and test-related behaviours were recorded during tests involving animal-human contact and reaction towards a novel object. We showed that the minipigs fed a high fat/cholesterol, low carbohydrate diet were less aggressive, showed more non-agonistic social contact and had fewer and less severe skin lesions and were less fearful of a novel object than minipigs fed low fat, high carbohydrate diets. These results found in a porcine model could have important implications for general health and wellbeing of humans and show the potential for using dietary manipulations to reduce aggression in human society.     

Characterisation of Gut Microbiota in Ossabaw and G?ttingen Minipigs as Models of Obesity and Metabolic Syndrome

Background

Recent evidence suggests that the gut microbiota is an important contributing factor to obesity and obesity related metabolic disorders, known as the metabolic syndrome. The aim of this study was to characterise the intestinal microbiota in two pig models of obesity namely Göttingen minipigs and the Ossabaw minipigs.

Methods and Findings

The cecal, ileal and colonic microbiota from lean and obese Osabaw and Göttingen minipigs were investigated by Illumina-based sequencing and by high throughput qPCR, targeting the 16S rRNA gene in different phylogenetic groups of bacteria. The weight gain through the study was significant in obese Göttingen and Ossabaw minipigs. The lean Göttingen minipigs’ cecal microbiota contained significantly higher abundance of Firmicutes (P<0.006), Akkermensia (P<0.01) and Methanovibribacter (P<0.01) than obese Göttingen minipigs. The obese Göttingen cecum had higher abundances of the phyla Spirochaetes (P<0.03), Tenericutes (P<0.004), Verrucomicrobia (P<0.005) and the genus Bacteroides (P<0.001) compared to lean minipigs. The relative proportion of Clostridium cluster XIV was 7.6-fold higher in cecal microbiota of obese Göttingen minipigs as compared to lean. Obese Ossabaw minipigs had a higher abundance of Firmicutes in terminal ileum and lower abundance of Bacteroidetes in colon than lean Ossabaw minipigs (P<0.01). Obese Ossabaws had significantly lower abundances of the genera Prevotella and Lactobacillus and higher abundance of Clostridium in their colon than the lean Ossabaws. Overall, the Göttingen and Ossabaw minipigs displayed different microbial communities in response to diet-induced obesity in the different sections of their intestine.

Conclusion

Obesity-related changes in the composition of the gut microbiota were found in lean versus obese Göttingen and Ossabaw minipigs. In both pig models diet seems to be the defining factor that shapes the gut microbiota as observed by changes in different bacteria divisions between lean and obese minipigs.     

Gastrointestinal Acute Radiation Syndrome in G?ttingen Minipigs (Sus Scrofa Domestica)

In the absence of supportive care, exposing Göttingen minipigs to γ-radiation doses of less than 2 Gy achieves lethality due to hematopoietic acute radiation syndrome. Doses of 2 to 5 Gy are associated with an accelerated hematopoietic syndrome, characterized by villus blunting and fusion, the beginning of sepsis, and a mild transient reduction in plasma citrulline concentration. We exposed male Göttingen minipigs (age, 5 mo; weight, 9 to 11 kg) to γ-radiation doses of 5 to 12 Gy (total body; ⁶⁰Co, 0.6 Gy/min) to test whether these animals exhibit classic gastrointestinal acute radiation syndrome (GI-ARS). After exposure, the minipigs were monitored for 10 d by using clinical signs, CBC counts, and parameters associated with the development of the gastrointestinal syndrome. Göttingen minipigs exposed to γ radiation of 5 to 12 Gy demonstrate a dose-dependent occurrence of all parameters classically associated with acute GI-ARS. These results suggest that Göttingen minipigs may be a suitable model for studying GI-ARS after total body irradiation, but the use of supportive care to extend survival beyond 10 d is recommended. This study is the first step toward determining the feasibility of using Göttingen minipigs in testing the efficacy of candidate drugs for the treatment of GI-ARS after total body irradiation.Abbreviations: GI-ARS, gastrointestinal acute radiation syndromeNo drugs have been approved for the use of preventing gastrointestinal acute radiation syndrome (GI-ARS) in irradiated patients. The effect of the acute exposure of the GI tract to radiation traditionally has been associated with the inhibition of mitotic activity in intestinal crypts and the interrupted migration of GI epithelial cells from the crypts to the tips of the villi, leading to denudation of the intestinal mucosal barrier. The monolayer of epithelial cells lining the GI mucosa performs many functions of vital importance, including electrolyte transport, secretion of digestive fluids, absorption of nutrients, excretion of toxins, and providing a barrier to the luminal environment and commensal bacteria. The loss of intestinal barrier integrity results in the loss of nutrients, water, and electrolytes; increased permeability to bacteria and antigens; sepsis; inflammation; and organ dysfunction.In humans, GI-ARS usually manifests at doses exceeding 5 to 6 Gy;¹⁰ death occurs within 2 wk in the absence of treatment. Radiation-induced GI damage is accompanied by bone marrow suppression; the sequelae of GI-ARS and hematopoietic ARS partially overlap but do not necessarily develop concomitantly.²³ Characteristic of GI-ARS is the early onset of symptoms (nausea, vomiting, watery diarrhea, cramps) within a few hours, and the overt illness is marked by vomiting and diarrhea. Bacterial translocation from the intestinal tract, loss of crypts, shortening of villi, decreased citrulline levels, onset of abdominal pain, vomiting, and diarrhea are accepted markers of the GI syndrome.²² Cardiovascular collapse, fluctuations in electrolyte concentrations, severe hemorrhage, and sepsis likely contribute to acute renal and multiorgan failure.²³Well-characterized animal models are required for efficacy testing of radiation countermeasures. So far, NHP are the only large animal model well-characterized in regard to the dose–survival relationship, symptoms, vital signs, and GI histology of GI-ARS.¹¹ In NHP, the LD_50/15 for GI-ARS in the presence of medical management including blood product transfusion is estimated to be 11.33 Gy.¹¹ Animals in the cited study¹⁰ were characterized by shorter survival time compared with hematopoietic ARS, diarrhea, dehydration, and dose-dependent loss of body weight, intestinal crypts, and villi. In addition to the total body irradiation model, a partial-body irradiation model was established in NHP, by using 5% sparing of the bone marrow, to evaluate the long-term effects of radiation on the GI system and to study concomitant subsyndromes and organ injuries, including bone marrow and lungs.¹⁰ Advantages of the partial body irradiation model are the ability to assess the development of various subsyndromes over time and the development of a polypharmacy approach targeting multiple organs. In dogs, acute GI-ARS was produced consistently by a single dose of 9.38 Gy to the abdomen.²⁰ In rodents, GI damage was induced by 8 to 15 Gy total body irradiation, depending upon the species and strain,^5,6,17,18 and was characterized by accelerated death, poor nutrient absorption, crypt depletion, intestinal epithelial cells denudation, infections, diarrhea, dehydration, and weight loss. In mice, both acute and delayed effects of the GI syndrome have been identified; study endpoints evaluating medical countermeasures against GI-ARS have been proposed.^2,3 Acute effects were assessed at several levels of bone-marrow shielding and supportive care and were demonstrated by the presence of diarrhea, apoptosis, villus blunting, crypt depletion, loss of mucosal integrity, and ulcerations in the GI tract. Delayed effects included higher-than-normal levels of apoptotic and mitotic crypt cells, premature reductions in gut function, and the presence of adenomas.^2,3Additional large animal models are urgently needed to expedite drug testing and marketing under FDA regulations. Minipigs may be a potential alternative model for studying hematopoietic ARS,¹⁵ but their high sensitivity to radiation prompts doubt regarding the feasibility of GI-ARS studies in this model. In the current study, we filled an important gap in development of minipigs as a model for ARS and addressed potential concerns regarding the feasibility of inducing classic GI-ARS in minipigs before they succumb to cardiovascular or respiratory complications. In previous publications, we documented hematopoietic ARS and accelerated hematopoietic ARS at higher radiation doses.^14,15 However, despite sporadic reports of specific types of radiation-induced injury to the GI tract of swine after total body irradiation, GI-ARS in that species has not been addressed comprehensively. To this end, we used a population of minipigs that was completely different from that in the previous studies and exposed the animals to much higher radiation doses than had been used previously. The subsequent results were markedly different from those of the previous reports, including loss of GI crypts and large decreases in circulating citrulline, both of which are important signs of GI-ARS. Our results demonstrate convincingly that GI-ARS occurs in swine and place this subsyndrome in the context of others studied comprehensively in the same strain. This achievement is an important milestone in the development of this much-needed large animal model of ARS, which promises to provide an alternative to NHP and dogs.We show here that exposure of minipigs to γ irradiation in the range 5 to 12 Gy resulted in the development of signs typical of GI-ARS, and we discuss the importance of choosing the appropriate endpoint criteria for the different subsyndromes of ARS. Most minipigs died within 10 d, shifting the question from the feasibility of inducing GI-ARS in minipigs to the levels of supportive care appropriate for drug efficacy testing in this model.     

Cognitive Performance of G?ttingen Minipigs Is Affected by Diet in a Spatial Hole-Board Discrimination Test

Consumption of a high energy diet, containing high amounts of saturated fat and refined sugar has been associated with impairment of cognitive function in rodents and humans. We sought to contrast the effect of a high fat/cholesterol, low carbohydrate diet and a low fat, high carbohydrate/sucrose diet, relative to a standard low fat, high carbohydrate minipig diet on spatial cognition with regards to working memory and reference memory in 24 male Göttingen minipigs performing in a spatial hole-board discrimination test. We found that both working memory and reference memory were impaired by both diets relative to a standard minipig diet high in carbohydrate, low in fat and sugar. The different diets did not impact levels of brain-derived neurotrophic factor in brain tissue and neither did they affect circulatory inflammation measured by concentrations of C-reactive protein and haptoglobin in serum. However, higher levels of triglycerides were observed for minipigs fed the diets with high fat/cholesterol, low carbohydrate and low fat, high carbohydrate/sucrose compared to minipigs fed a standard minipig diet. This might explain the observed impairments in spatial cognition. These findings suggest that high dietary intake of both fat and sugar may impair spatial cognition which could be relevant for mental functioning in humans.     

Novel Technique for Retroperitoneal Implantation of Telemetry Transmitters for Physiologic Monitoring in G?ttingen Minipigs (Sus scrofa domesticus)

Telemetric monitoring of physiologic parameters in animal models is a critical component of chemical and biologic agent studies. The long-term collection of neurobehavioral and other physiologic data can require larger telemetry devices. Furthermore, such devices must be implanted in a location that is safe, well-tolerated, and functional. Göttingen minipigs (Sus scrofa domesticus) present an ideal large animal model for chemical agent studies due to their relatively small size, characterized health status, and ease of training and handling. We report an effective approach to implanting a novel device to measure transthoracic impedance to approximate respiratory tidal volume and rate in Suidae. We tested the approach using 24 male Göttingen minipigs. A ventral midline abdominal incision extending from the umbilicus to the prepuce was followed by a paramedian incision of the parietal peritoneum and dorsal blunt dissection to create a retroperitoneal pocket. The device was anchored inside the pocket to the internal abdominal musculature with 3-0 nonabsorbable suture, biopotential leads were routed through the abdominal musculature, and the pocket was closed with 3-0 absorbable suture. Paired biopotential leads were anchored intermuscularly at the level of the seventh rib midway between spine and sternum bilaterally to provide surrogate data for respiratory function. Postoperative recovery and gross pathology findings at necropsy were used to assess safety and refine the surgical procedure. Results demonstrated that this procedure permitted effective monitoring of complex physiologic data, including transthoracic impedance, without negatively affecting the health and behavior of the animals.Telemetric monitoring of physiologic parameters in animal models is a critical component of chemical⁹ and biologic agent studies.^32,36 The attenuation of the adverse effects of these agents by using medical countermeasures can be assessed also.^21,25,28,35 The long-term collection of neurobehavioral and other physiologic data can require large telemetry devices. Furthermore, such devices must be implanted in a location that is safe, well-tolerated, and functional. Potential complications of inserting telemetry devices include infection,²⁹ dehiscence,⁷ neoplasia,³³ and extrusion.²⁰Göttingen minipigs (Sus scrofa domesticus) present an ideal large animal model for chemical agent studies due to their relatively small size, characterized health status, and ease of training and handling, including for physiologic studies.^12,15,16,30 The lightly pigmented skin, sparse haircoat, fine intersecting lines of epidermal sulci, lipid biophysical properties, and epidermal turnover kinetics of Göttingen minipigs are similar to those in humans.^22,34 Swine in general are a particularly relevant animal model for assessing percutaneous absorption exposure to chemical agents.^8,11 Percutaneous application of agent represents the most likely real-world threat scenario for persistent chemical agents.^8,11 Extensive in vivo and in vitro studies have characterized percutaneous absorption,^10,13,31 protection,⁵ and decontamination²³ in swine models.Previously, respiratory impedance has been measured by using implantable telemetry devices in nonhuman primates,² and respiratory parameters have been determined by using external impedance-based technology in minipigs¹ and dogs.²⁴ We report an effective approach to implanting a device for measuring transthoracic impedance to approximate respiratory tidal volume and rate in minipigs.     

Regression of diet-induced atherosclerosis in G?ttingen miniature swine

The regression of diet-induced atherosclerosis in G?ttingen Miniature Swine was investigated after a 6-month induction period. At 1 month after feeding a high-cholesterol and high-fat diet, levels of beta-lipoprotein, total cholesterol, free fatty acids and phospholipid had increased rapidly and the high levels were maintained throughout the 6 month induction period. Morphological features at 6 months showed fatty streaks in the thoracic aorta and fibrous plaques in the abdominal aorta. After return to the conventional diet at 6 months, serum lipids decreased rapidly and maintained the baseline level throughout the 9 month regression period. Histopathological findings showed the regression of fatty streaks but the fibrous plaques did not regress. The present study therefore confirms the regression of fatty streaks in the aorta of G?ttingen Miniature Swine by the administration of a cholesterol lowering diet.     

Membrane-Localized Extra-Large G Proteins and Gβγ of the Heterotrimeric G Proteins Form Functional Complexes Engaged in Plant Immunity in Arabidopsis

In animals, heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches whose function tightly depends on Gα and Gβγ interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gβγ, but not Gα. We report here that the Gβγ dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gβ, and Gγ are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gβγ functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gβ-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gβγ dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Gα subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gβγ dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Gα subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.Heterotrimeric GTP-binding proteins (G proteins), classically consisting of Gα, Gβ, and Gγ subunits, are essential signal transduction elements in most eukaryotes. In animals and fungi, ligand perception by G protein-coupled receptors leads to replacement of GDP with GTP in Gα, triggering activation of the heterotrimer (Li et al., 2007; Oldham and Hamm, 2008). Upon activation, GTP-bound Gα and Gβγ are released and interact with downstream effectors, thereby transmitting signals to multiple intracellular signaling cascades. Signaling terminates when the intrinsic GTPase activity of Gα hydrolyzes GTP to GDP and the inactive heterotrimer reforms at the receptor. The large diversity of mammalian Gα subunits confers specificity to the multiple signaling pathways mediated by G proteins (Wettschureck and Offermanns, 2005). Five distinct classes of Gα have been described in animals (Gαi, Gαq, Gαs, Gα12 and Gαv), with orthologs found in evolutionarily primitive organisms such as sponges (Oka et al., 2009). Humans possess four classes of Gα involving 23 functional isoforms encoded by 16 genes (McCudden et al., 2005), while only a single prototypical Gα is usually found per plant genome (Urano et al., 2013). Multiple copies of Gα are present in some species with recently duplicated genomes, such as soybean (Glycine max) with four Gα genes (Blanc and Wolfe, 2004; Bisht et al., 2011). In the model plant Arabidopsis (Arabidopsis thaliana), a prototypical Gα subunit (GPA1) is involved in a number of important processes, including cell proliferation (Ullah et al., 2001), inhibition of inward K⁺ channels and activation of anion channels in guard cells by mediating the abscisic acid pathway (Wang et al., 2001; Coursol et al., 2003), blue light responses (Warpeha et al., 2006, 2007), and germination and postgermination development (Chen et al., 2006; Pandey et al., 2006).It is well established that heterotrimeric G proteins play a fundamental role in plant innate immunity. In Arabidopsis, two different Gβγ dimers (Gβγ1 and Gβγ2) are generally considered to be the predominant elements in G protein defense signaling against a variety of fungal pathogens (Llorente et al., 2005; Trusov et al., 2006, 2007, 2009; Delgado-Cerezo et al., 2012; Torres et al., 2013). By contrast, these studies attributed a small or no role to Gα, because mutants deficient in Gα displayed only slightly increased resistance against the fungal pathogens (Llorente et al., 2005; Trusov et al., 2006; Torres et al., 2013). The Gβγ-mediated signaling also contributes to defense against a model bacterial pathogen Pseudomonas syringae, by participating in programmed cell death (PCD) and inducing reactive oxygen species (ROS) production in response to at least three pathogen-associated molecular patterns (PAMPs; Ishikawa, 2009; Liu et al., 2013; Torres et al., 2013). Gα is not involved in PCD or PAMP-triggered ROS production (Liu et al., 2013; Torres et al., 2013). Nonetheless, Arabidopsis Gα plays a positive role in defense against P. syringae, probably by mediating stomatal function and hence physically restricting bacterial entry to the leaf interior (Zhang et al., 2008; Zeng and He, 2010; Lee et al., 2013). Given the small contribution from Gα, the involvement of heterotrimeric G proteins in Arabidopsis resistance could be explained in two ways: either the Gβγ dimer acts independently from Gα, raising a question of how is it activated upon a pathogen attack, or Gα is replaced by another protein for heterotrimer formation.The Arabidopsis genome contains at least three genes encoding Gα-like proteins that have been classified as extra-large G proteins (XLGs; Lee and Assmann, 1999; Ding et al., 2008). XLGs comprise two structurally distinct regions. The C-terminal region is similar to the canonical Gα, containing the conserved helical and GTPase domains, while the N-terminal region is a stretch of approximately 400 amino acids including a putative nuclear localization signal (Ding et al., 2008). GTP binding and hydrolysis were confirmed for all three XLG proteins, although their enzymatic activities are very slow and require Ca²⁺ as a cofactor, whereas canonical Gα utilizes Mg²⁺ (Heo et al., 2012). Several other features differentiate XLGs from Gα subunits. Comparative analysis of XLG1 and Gα at the DNA level showed that the genes are organized in seven and 13 exons, respectively, without common splicing sites (Lee and Assmann, 1999). XLGs have been reported to localize to the nucleus (Ding et al., 2008). Analysis of knockout mutants revealed a nuclear function for XLG2, as it physically interacts with the Related To Vernalization1 (RTV1) protein, enhancing the DNA binding activity of RTV1 to floral integrator gene promoters and resulting in flowering initiation (Heo et al., 2012). Therefore, it appears that XLGs may act independently of G protein signaling. On the other hand, functional similarities between XLGs and the Arabidopsis Gβ subunit (AGB1) were also discovered. For instance, XLG3- and Gβ-deficient mutants were similarly impaired in root gravitropic responses (Pandey et al., 2008). Knockout of all three XLG genes caused increased root length, similarly to the Gβ-deficient mutant (Ding et al., 2008). Furthermore, as observed in Gβ-deficient mutants, xlg2 mutants displayed increased susceptibility to P. syringae, indicating a role in plant defense (Zhu et al., 2009). Nevertheless, a genetic analysis of the possible functional interaction between XLGs and Gβ has not been established.In this report, we performed in-depth genetic analyses to test the functional interaction between the three XLGs and Gβγ dimers during defense-related responses in Arabidopsis. We also examined physical interaction between XLG2 and the Gβγ dimers using yeast (Saccharomyces cerevisiae) three-hybrid (Y3H) and bimolecular fluorescent complementation (BiFC) assays. Our findings indicate that XLGs function as direct partners of Gβγ dimers in plant defense signaling. To estimate relatedness of XLGs and Gα proteins, we carried out a phylogenetic analysis. Based on our findings, we conclude that plant XLG proteins most probably originated from a canonical Gα subunit and retained prototypical interaction with Gβγ dimers. They function together with Gβγ in a number of processes including plant defense, although they most probably evolved activation/deactivation mechanisms very different from those of a prototypical Gα.     

Osteoarthritis in the Knee Joints of G?ttingen Minipigs after Resection of the Anterior Cruciate Ligament? Missing Correlation of MRI,Gene and Protein Expression with Histological Scoring

IntroductionThe Göttingen Minipig (GM) is used as large animal model in articular cartilage research. The aim of the study was to introduce osteoarthritis (OA) in the GM by resecting the anterior cruciate ligament (ACLR) according to Pond and Nuki, verified by histological and magnetic resonance imaging (MRI) scoring as well as analysis of gene and protein expression.

Materials and Methods

The eight included skeletally mature female GM were assessed after ACLR in the left and a sham operation in the right knee, which served as control. 26 weeks after surgery the knee joints were scanned using a 3-Tesla high-field MR tomography unit with a 3 T CP Large Flex Coil. Standard proton-density weighted fat saturated sequences in coronal and sagittal direction with a slice thickness of 3 mm were used. The MRI scans were assessed by two radiologists according to a modified WORMS-score, the X-rays of the knee joints by two evaluators. Osteochondral plugs with a diameter of 4mm were taken for histological examination from either the main loading zone or the macroscopic most degenerated parts of the tibia plateau or condyle respectively. The histological sections were blinded and scored by three experts according to Little et al. Gene expression analysis was performed from surrounding cartilage. Expression of adamts4, adamts5, acan, col1A1, col2, il-1ß, mmp1, mmp3, mmp13, vegf was determined by qRT-PCR. Immunohistochemical staining (IH) of Col I and II was performed. IH was scored using a 4 point grading (0—no staining; 3-intense staining).

Results and Discussion

Similar signs of OA were evident both in ACLR and sham operated knee joints with the histological scoring result of the ACLR joints with 6.48 ± 5.67 points and the sham joints with 6.86 ± 5.84 points (p = 0.7953) The MRI scoring yielded 0.34 ± 0.89 points for the ACLR and 0.03 ± 0.17 for the sham knee joints. There was no correlation between the histological and MRI scores (r = 0.10021). The gene expression profiles as well as the immunohistochemical findings showed no significant differences between ACLR and sham knee joints. In conclusion, both knee joints showed histological signs of OA after 26 weeks irrespective of whether the ACL was resected or not. As MRI results did not match the histological findings, MRI was obviously unsuitable to diagnose the OA in GM. The analysis of the expression patterns of the 10 genes could not shed light on the question, whether sham operation also induced cartilage erosion or if the degeneration was spontaneous. The modified Pond-Nuki model may be used with reservation in the adult minipig to induce an isolated osteoarthritis.     

Histological study on the arterial wall of G?ttingen miniature swine

In order to clarify the histological aspects of arterial walls of miniature swine, 41 arterial segments removed from each of 17 G?ttingen miniature swines/csk at 6-12 months after birth were examined by light microscopy. Every segment was classified into three types--elastic, transitional, and muscular--according to its histological architecture. Each type was identified on the basis of relative volume and disposition of elastic tissues, collagenous fibers, and smooth muscles in three coats, Tunica intima, Tunica media, Tunica externa. The distribution of each type was demonstrated in a transition from the elastic to the muscular type. The transitional type was designated as a representative type of artery in transitional regions between elastic and muscular types and had some characteristic structures like a mixture of both of them. This type also had longer regions in the back district with the heart as the starting point, such as the Aorta abdominalis, than that of the front. Every artery belonging to the three types had some different structures at different levels, respectively. It was supposed that the histological arrangement of tissues in the walls of a vessel from various parts of the arterial tree would respond properly to the functional demand.     

Electrocardiographic monitoring in the Göttingen minipig

The purpose of the study reported here was to determine conditions for electrocardiographic monitoring in the G?ttingen minipig in view of its use as a second non-rodent species in toxicology studies. Electrocardiograms were recorded from conscious minipigs (6/sex) maintained in a sling. The three standard bipolar limb leads (I, II, III), the three augmented unipolar limb leads (aVR, aVL, aVF), the triangular Nehb-Sp?ri leads (dorsal, axial, ventral) and their corresponding unipolar leads were recorded, and automated analysis of amplitudes and intervals was made. Major QRS patterns were not observed for any of the bipolar and unipolar leads. For triangular leads, the amplitude of waves was higher than that for limb leads, and the rS pattern dominated for dorsal, axial ventral and aV(F)-Ventral leads. The qR pattern dominated in the aV(R)-dorsal lead, whereas consistency and dominant patterns were not observed for the aV(L)-axial lead. For limb leads, the position of the electrode affected the ECG. Electrodes placed on the cubital and stifle joints were the preferred positions since the P- and R-waves were clearly identifiable with amplitudes > 0.2 mV. Also, the T-wave amplitude was (positive or negative) > 0.2 mV in at least two leads, making the determination of the QT-interval accurate. For the triangular leads, the position of the electrode had less influence on the amplitude of deflections. However, if the axial lead is to be used for calculation of intervals and amplitudes, the xyphoid process is the preferred position. In conclusion, the triangular lead system is recommended for recording ECGs in minipigs. Limb leads could be used in connection. The cubital and stifle joints for standard limb leads and the neck, sacrum, and xyphoid process for triangular leads are the preferred positions for electrodes.     

Glucocorticosteroid-induced osteoporosis in adult primiparous Göttingen miniature pigs: effects on bone mineral and mineral metabolism

Information on the pathophysiology of glucocorticoid-induced osteoporosis (GIO) is limited, since its clinical picture often reflects a combined effect of glucocorticoids (GC) and the treated systemic disease (i.e., inflammation and immobility). In 50 healthy adult (30-mo-old) primiparous G?ttingen minipigs, we studied the short-term (8 mo, n = 30) and long-term (15 mo, n = 10) effect of GC on bone and mineral metabolism longitudinally and cross-sectionally compared with a control group (n = 10). All animals on GC treatment received prednisolone orally at a dose of 1.0 mg x kg body wt(-1) x day(-1) for 8 wk and thereafter at 0.5 mg/kg body wt(-1) x day(-1). In the short term, GC reduced bone mineral density (BMD) at the lumbar spine by -47.5 +/- 5.1 mg/cm(3) from baseline (P < 0.001), which was greater (P < 0.05) than the loss [not significant (NS)] in the control group of -11.8 +/- 12.6 mg/cm(3). Calcium absorption decreased from baseline by -2,488 +/- 688 mg/7 days (P < 0.001) compared with -1,380 +/- 1,297 mg/7 days (NS) in the control group. Plasma bone alkaline phosphatase (BAP) decreased from baseline by -17.8 +/- 2.2 U/l (P < 0.000), which was significantly different (P < 0.05) from the value of the control group of -1.43 +/- 4.8 U/l. In the long term, the loss of BMD became more pronounced and bone mineral content (BMC), trabecular thickness, mechanical stability, calcium absorption, 25-hydroxyvitamin D(3), 1,25-dihydroxyvitamin D(3), and parathyroid hormone tended to be lower compared with the control group. There was a negative association between the cumulative dose of GC and BMD, which was associated with impaired osteoblastogenesis. In conclusion, the main outcomes after GC treatment are comparable to symptoms of GC-induced osteoporosis in human subjects. Thus the adult G?ttingen miniature pig appears to be a valuable animal model for GC-induced osteoporosis.     

Discriminations, reversals, and extra-dimensional shifts in the Göttingen minipig

G?ttingen minipigs were trained on a set-shifting procedure involving discriminations, reversals, and extra-dimensional shifts. The discriminations used were black-white discriminations and right-left discriminations. The initial visual and spatial discrimination seemed equally difficult, and only for the visual modality was reversal found to be more difficult than the initial discrimination. Visual reversal was more difficult than spatial reversal, and a larger number of perseverative sessions were found for visual reversal compared to spatial reversal. The acquisition of the extra-dimensional shift from the visual to the spatial dimension was not inferior to the learning of spatial reversal. Neither was the acquisition of the extra-dimensional shift from the spatial to the visual dimension inferior to the learning of visual reversal. Thus, no evidence was found for attention to stimulus dimensions in discrimination learning of the pigs.     

Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

Heterotrimeric G proteins are conformational switches that turn on intracellular signaling cascades in response to the activation of G-protein-coupled receptors. Receptor activation by extracellular stimuli promotes a cycle of GTP binding and hydrolysis on the G protein α-subunit (Gα). Important conformational transitions occurring during this cycle have been characterized from extensive crystallographic studies of Gα. However, the link between the observed conformations and the mechanisms involved in G-protein activation and effector interaction remain unclear. Here we describe a comprehensive principal component analysis of available Gα crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of Gα to GTP binding and hydrolysis. Our studies reveal details of activating conformational changes as well as the intrinsic flexibility of the α-helical domain that includes a large-scale 60° domain opening under nucleotide-free conditions. This result is consistent with the recently reported open crystal structure of Gs, the stimulatory G protein for adenylyl cyclase, in complex with the α2 adrenergic receptor. Sets of unique interactions potentially important for the conformational transition are also identified. Moreover simulations reveal nucleotide-dependent dynamical couplings of distal regions and residues potentially important for the allosteric link between functional sites.Heterotrimeric G proteins undergo cycles of GTP-dependent conformational rearrangements and alterations of their oligomeric αβγ form to convey receptor signals to downstream effectors that control diverse cellular processes ranging from movement to division and differentiation. Interaction with activated receptor promotes the exchange of GDP for GTP on the G protein α subunit (Gα) and its separation from its βγ subunit partners (Gβγ). Both isolated Gα and Gβγ then interact with downstream effectors. GTP hydrolysis deactivates Gα, which reassociates with Gβγ, becoming ready to restart the cycle. Each of these stages has been subjected to extensive crystallographic studies with high-resolution structures of Gα in complex with GDP, GTP analog, Gβγ, and, most recently, the G-protein-coupled receptors now available. These studies have provided extensive mechanistic insight. However, a number of important questions remain, including:

• How do the distinct conformations evident in the accumulated structures interconvert?
• How do disease-associated mutations affect the fidelity of these transitions?
• And, critically, how do distal functional sites responsible for nucleotide and protein partner binding allosterically coordinate their activities?

Here we describe a comprehensive analysis of the accumulated Gα crystallographic structures supplemented with extensive conventional (cMD) and accelerated molecular dynamics (aMD) simulations (1) that together map the structural and dynamical features of Gα in different nucleotide states. These enhanced sampling simulations reveal the spontaneous interconversion between GDP and GTP conformations and also characterize large-scale opening motions of the α-helical domain (HD) that were not accessible to previous simulation studies (2–5). Furthermore, the current simulations results reveal a distinctive pattern of collective motions that provide evidence for a nucleotide-dependent network of dynamic communication between the active site and the receptor and effector binding sites.Principal component analysis of 53 Gα experimental structures homologous to transducin (Gαt) reveals that the major variation in accumulated structures is the concerted association/disassociation of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII). An additional small-scale (<10°) rotation of the HD relative to the main catalytic Ras-like domain (RasD) is also apparent (see Fig. S1 in the Supporting Material). The distinct conformation of SI–SIII regions gives rise to nucleotide-associated segregation of GDP- and GTP-analog-bound experimental structures along the PC1-PC2 plane. Interestingly, both GDP- and GTP-bound structures display a skewed distribution along the PC1-PC2 plane that arises from HD rotation. In comparison, the distribution of the GTP-bound structures becomes more restricted and the skew decreases when the mapping is based on a principle component analysis that excludes the HD region (see Fig. S1).Recently, the HD region of Gαs (the α-subunit of the stimulatory G protein for adenyl cyclase) was shown to adopt a dramatically more open conformation in a crystal structure complex with the β2 adrenergic receptor (β2AR) (6). This clam-shell-like 127° opening in the absence of nucleotide and presence of receptor is consistent with electron microscopy (7) and double electron-electron resonance analysis (8). These results, together with recent hydrogen-deuterium exchange mass spectrometry data (9), indicate that there may be additional functional motions and inherent flexibility in the ensemble of native states beyond those apparent in the accumulated crystal structures of Gαt (9). To address this question, we performed multiple 100-ns aMD simulations of nucleotide-free Gαt. These simulations reveal a spontaneous large-scale opening and closing motion of larger magnitude (>60°) than those evident in the distribution of crystallographic structures (Fig. 1 A and see Fig. S2). In addition, the trajectory reveals two dominant modes of HD opening: an out-of-plane shifting (PC1 in Fig. S3) and an in-plane rotation (PC2 in Fig. S3). It is also notable that nucleotide-free aMD simulations sample both active (GTP-like) and inactive (GDP-like) structures (see Fig. S2) in an analogous manner to the spontaneous GDP to GTP interconversion sampled for Ras and Rho small G proteins with similar methods (10–12).Open in a separate windowFigure 1Nucleotide-associated differences in flexibility and dynamic coupling. (A) Mapping aMD simulation trajectories (blue points) onto the principal components obtained from analysis of Gα crystallographic GDP-bound (green) and GTP-analog bound (red) experimental structures. (Orange) Open β2AR-Gαs complex structure. (B) Results of dynamic coupling analysis mapped onto the average structure for each nucleotide state. (Spheres) Nodes for the nucleotide; the protein cartoon is colored by community structure. (C) Community network graph. (Circles) Communities, colored as in panel B. Radius of the circle indicates the number of residues in the community. Thickness of linking lines is determined by the maximum betweenness of the respective intercommunity edges (see the Supporting Material). (Red, blue, and green edges) Major topological difference between states.The low sequence identity between Gαt and Gαs (44.5%), as well as the absence of the receptor and Gβγ in the simulations, may explain the difference between the predicted ∼60° Gαt-HD rotation and that displayed in the β2AR-Gαs crystallographic structure (see Fig. S3). It is notable that, although the amplitude is much smaller, aMD simulations with bound nucleotide display similar dominant HD motions to those observed in the nucleotide-free simulations (see Fig. S4). This suggests that the interdomain flexibility of RasD and HD is likely an intrinsic feature of Gαt regardless of nucleotide state.The transition between distinct conformations (structural clusters; see Fig. S5) was observed to correspond to significant dynamical changes in side-chain contacts (see Fig. S6). Specifically, we found sequential contacts breaking during the HD in-plane rotation motion starting from the region between HD helix αD and RasD helix αG toward that between HD helix αE and RasD SIII and the P-loop. In comparison, for the out-of-plane shift, we found simultaneous breaking and formation of contacts in the region containing the loop between helices αB and αC, the N-terminus of αA, αE, and αF of HD; α1, SI, and the loop between strand β6 and helix α5 of RasD. Interactions highlighted in these regions as potentially important for the conformational transitions include D137::K276, S140::K273, S140::D227, Q143::R238, N145::E39, and D146::K266, the effect of which can be further evaluated by mutagenesis experiments and simulations.Dynamic network analysis methods developed by Sethi et al. (13) were used to examine whether the motions of one residue were correlated to the motions of another (distant) residue. In this approach, a weighted graph is constructed where each residue represents a node and the weight of the connection between nodes represents their respective correlation value. A clustering of edges is then used to define local communities of highly correlated residues that represent substructures that are highly intraconnected, but loosely interconnected. Applying this approach to multiple 40-ns cMD simulations initiated from GTP-, GDP-, and aMD-derived APO conformations revealed a consistent community composition as well as a distinct pattern of intercommunity connection between nucleotide states (Fig. 1, B and C).The dynamics of the RasD region can be decomposed into two main communities that stem from the nucleotide base and phosphate regions in GDP and GTP states: The first community is composed of residues from the P-loop, helix α1, strands β1–β3, and the phosphates of the nucleotide (orange in Fig. 1, B and C). The second community comprises residues from helix αG, strands β4–β6, and the nucleotide base region (tan in Fig. 1, B and C). This dynamic partitioning of the central β-sheet and central role of the nucleotide is consistent with the bilobal structure and dynamics previously reported for Ras (14). In the presence of GTP, the first community includes or is dynamically coupled to SI, SII, and SIII regions (see the orange node and the red edge in Fig. 1 C). Removal of the γ-phosphate of GTP disrupts this region, leading to decoupling of the switch regions from the nucleotide. Also evident for GDP states is an apparent tighter coupling of RasD and HD regions (blue edges in Fig. 1 C). We note that these findings are robust to the choice of initial simulation conditions and are observed in both cMD and aMD simulations (see Fig. S7 and Fig. S8). Nucleotide-free Gαt simulations display an altered dynamical network with respect to those of nucleotide bound states. In particular, RasD and HD regions lose connecting edges consistent with the large-scale opening of these domains (e.g., SIII-HD green edges in Fig. 1 C).A number of residues highlighted here as potentially important for mediating the coupling between prominent communities (see Table S1 in the Supporting Material) have been shown by previous mutagenesis studies to affect GDP release. For example, the double mutation A322S/R174M was found to significantly enhance the rate of GDP release (15). The current results indicate that these positions are involved in coupling the nucleotide and RasD. Also, mutations R144A and L232Q caused a faster basal GDP release rate in Gαi1 (16). The current analysis indicates that the equivalent positions in Gαt (S140 and M228) couple the RasD and HD, and suggests that their mutation could promote domain-domain motions. We also note the apparent coupling of α5 with the nucleotide base and Ploop-β1 with the phosphate regions of GDP. These direct connections of the receptor connecting N- and C-terminus to GDP are suggestive of potential routes for receptor-mediated GDP release. We expect further study of these sites and of receptor-bound dynamics to be informative in this regard.In conclusion, simulations suggest a flexible HD in Gαt similar to that found for Gαs. In particular, in the absence of nucleotide we observed the spontaneous large-scale opening and closing of HD relative to RasD, which was unseen in previous computational studies. Moreover, we found that the functional states of Gαt are associated with the distinct dynamical couplings of functional regions including SI–SIII, P-loop, α5, and the HD region. Finally, our results indicate that nucleotide may not directly induce large-scale conformational changes but, instead, act as a modulator of intrinsically accessible conformations and as a central participant in their associated dynamical couplings.     

Gender-associated differences in metabolic syndrome-related parameters in Göttingen minipigs

Gender-associated differences in pathophysiology and treatment of disease are an evolving area in human medicine that should be addressed in animal models. The aim of this study was to characterize gender differences in metabolic parameters of G?ttingen minipigs and to determine which gender has the metabolic profile that is most appropriate as a model for human metabolic syndrome. Blood samples were collected from fasted, lean male and female G?ttingen minipigs at 8 wk and 8 mo of age. Samples were analyzed for glucose, fructosamine, insulin, C-peptide, glucagon, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-c), free fatty acids, leptin, testosterone, and 17beta-estradiol. Insulin sensitivity and beta cell function were estimated by homeostasis model assessment and degree of obesity by measuring the abdominal circumference. Male minipigs had higher concentrations of both testosterone and estradiol. Female minipigs had a larger abdominal circumference and higher concentrations of C-peptide, insulin, triglyceride, total cholesterol, HDL-c and leptin but a lower concentration of free fatty acids and lower HDL-c:total cholesterol ratio. Compared with male minipigs, female minipigs were more insulin-resistant and had a higher beta-cell function. No gender-associated differences were found in any of the other investigated parameters. In conclusion, female minipigs were more obese and insulin-resistant and had a more atherogenic plasma profile than did their male counterparts and therefore may be better models for metabolic syndrome. Their high concentrations of both testosterone and estradiol may protect male minipigs from obesity and metabolic disturbances.     

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

Are larks future-oriented and owls present-oriented? Age- and sex-related shifts in chronotype–time perspective associations

The chronotype (morningness/eveningness) relates to individual differences in circadian preferences. Time perspective (past, present, future) refers to the preference to rely on a particular temporal frame for decision-making processes and behavior. First evidence suggests that future time perspective is associated with greater morningness and present time perspective with greater eveningness. However, little is known about how chronotype–time perspective relationships may alter over the life span. This present study investigated links between chronotype and time perspective more thoroughly by taking age and sex into account as well. Seven hundred six participants aged between 17 and 74 completed German adaptations of the Morningness--Eveningness Questionnaire (MEQ) and Zimbardo Time Perspective Inventory (ZTPI). Controlling for age and sex, relationships between morningness and future time perspective as well as between eveningness and present time perspective were replicated. These findings were supported by significant associations between time perspective and midpoint of sleep. Future time perspective was linked to earlier midpoints of sleep, indicating an early chronotype. Present time perspective was associated with later midpoints of sleep, indicating a late chronotype. However, age and sex had an impact on the chronotype–time perspective relationships. In all age groups, male larks were more future-oriented and less present-oriented, male owls more present-oriented and less future-oriented. The same conclusion could be drawn for female adolescents and young adults. For female adults above 30, there was no interrelationship between morningness and future time perspective but between eveningness and past time perspective. Female adult owls were more present-oriented as well as more past-oriented. Female adult larks were less present-oriented and less past-oriented. Findings are discussed in the light of neuroendocrine and serotonergic functioning.