Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

The perception of gravity by a plant

Ever since geotropism was first studied in plants, attempts have been made to create model systems which might simulate the perception by a plant of a gravitational change. The most resilient of these models, the so-called statolith theory, has now enjoyed a run of over 75 years and demonstrates its viability by reappearing in many different forms. It has shown its value by anticipating the now well understood graviperception mechanism in the Chara rhizoid and this will be described. However, it is unlikely that many features of this relatively simple system can be translated to the higher plant. We now know precisely, at least in many primary roots, the distribution and approximate numbers of the cells that perceive gravity. There is no reason to assume that an identical system operates in shoots, since it is now clear that the fundamental hormonal bases of these two systems are different. We also know much about the ultrastructure of many geoperceptive cells, but apart from speculative models a satisfactory explanation of this very rapid and flexible system eludes us. A possible model system is proposed and ways of testing it in zero gravity are suggested.     

The effect of exposure to oestrous ewes on rams' sexual behaviour, plasma testosterone concentration and ability to stimulate ovulation in seasonally anoestrous ewes

Ruminants eat a variety of foods from different locations in the environment. While water, cover, social interactions, and predators are all likely to influence choice of foraging location, differences in macronutrient content among forages may also cause ruminants to forage in different locations even during a meal. We hypothesized that lambs forage at locations containing foods that complement their basal diet and meet their nutritional needs. Based on this hypothesis, we predicted that lambs (n=12) fed a basal diet low in protein and high in energy would forage where a high-protein food (Food P) was located, and that lambs (n=12) fed a basal diet low in energy and high in protein would forage where a high-energy food (Food E) was located. Food P was a ground mixture of blood meal (50%), grape pomace (30%), and alfalfa (20%) that contained 47% crude protein (CP) and 2.211 Mcal/kg digestible energy (DE). Food E was a ground mixture of cornstarch (50%), grape pomace (30%), and rolled barley (20%) that contained 6% CP and 3.07 Mcal/kg DE. Food P provided 212 g CP/Mcal DE, whereas Food E provided 20 g CP/Mcal DE. Lambs growing at a moderate rate require 179 g CP and 3.95 Mcal DE. During Trial 1, we determined if lambs foraged to correct a nutrient imbalance, and if they preferred a variety of foods (Foods P and E) to only one food at a location (Food P or E). During Trial 2, we determined if nutrient-imbalanced lambs foraged in the location with the food that corrected the imbalance when the location of the foods changed daily. During Trial 3, lambs were offered familiar foods (Foods P and E) at the location furthest - and novel foods (wheat and soybean meal) at the location nearest - the shelter of their pen. During all three trials, lambs foraged most at the location with the food that contained the highest concentration of the macronutrient lacking in their basal diet, but they always ate some of both foods. Lambs did not feed exclusively at the location with a variety of foods (P and E). Rather, they fed at the location nearest the shelter that contained the macronutrient lacking in their diet. As availability of the food with the needed macronutrient declined in one location, lambs moved to the nearest location that had food with the needed macronutrient. When food that complemented their basal diet was moved to a different location, lambs foraged in the new location. Collectively, these results show that lambs challenged by imbalances in energy or protein selected foods and foraging locations that complemented the nutrient content of their macronutrient imbalanced basal diets.     

Diversity and characterization of bacterial communities of five co‐occurring species at a hydrothermal vent on the Tonga Arc

Host–symbiont relationships in hydrothermal vent ecosystems, supported by chemoautotrophic bacteria as primary producers, have been extensively studied. However, the process by which densely populated co‐occurring invertebrate hosts form symbiotic relationships with bacterial symbionts remains unclear. Here, we analyzed gill‐associated symbiotic bacteria (gill symbionts) of five co‐occurring hosts, three mollusks (“Bathymodiolus” manusensis, B. brevior, and Alviniconcha strummeri) and two crustaceans (Rimicaris variabilis and Austinograea alayseae), collected together at a single vent site in the Tonga Arc. We observed both different compositions of gill symbionts and the presence of unshared operational taxonomic units (OTUs). In addition, the total number of OTUs was greater for crustacean hosts than for mollusks. The phylogenetic relationship trees of gill symbionts suggest that γ‐proteobacterial gill symbionts have coevolved with their hosts toward reinforcement of host specificity, while campylobacterial Sulfurovum species found across various hosts and habitats are opportunistic associates. Our results confirm that gill symbiont communities differ among co‐occurring vent invertebrates and indicate that hosts are closely related with their gill symbiont communities. Considering the given resources available at a single site, differentiation of gill symbionts seems to be a useful strategy for obtaining nutrition and energy while avoiding competition among both hosts and gill symbionts.     

Redox regulation of cAMP-dependent protein kinase signaling: kinase versus phosphatase inactivation

Many components of cellular signaling pathways are sensitive to regulation by oxidation and reduction. Previously, we described the inactivation of cAMP-dependent protein kinase (PKA) by direct oxidation of a reactive cysteine in the activation loop of the kinase. In the present study, we demonstrate that in HeLa cells PKA activity follows a biphasic response to thiol oxidation. Under mild oxidizing conditions, or short exposure to oxidants, forskolin-stimulated PKA activity is enhanced. This enhancement was blocked by sulfhydryl reducing agents, demonstrating a reversible mode of activation. In contrast, forskolin-stimulated PKA activity is inhibited by more severe oxidizing conditions. Mild oxidation enhanced PKA activity stimulated by forskolin, isoproterenol, or the cell-permeable analog, 8-bromo-cAMP. When cells were lysed in the presence of serine/threonine phosphatase inhibitor, NaF, the PKA-enhancing effect of oxidation was blunted. These results suggest oxidation of a PKA-counteracting phosphatase may be inhibited, thus enhancing the apparent kinase activity. Using an in vivo PKA activity reporter, we demonstrated that mild oxidation does indeed prolong the PKA signal induced by isoproterenol by inhibiting counteracting phosphatase activity. The results of this study demonstrate in live cells a unique synergistic mechanism whereby the PKA signaling pathway is enhanced in an apparent biphasic manner.     

Evaluation of Buprenorphine in a Postoperative Pain Model in Rats

We evaluated the commonly prescribed analgesic buprenorphine in a postoperative pain model in rats, assessing acute postoperative pain relief, rebound hyperalgesia, and the long-term effects of postoperative opioid treatment on subsequent opioid exposure. Rats received surgery (paw incision under isoflurane anesthesia), sham surgery (anesthesia only), or neither and were treated postoperatively with 1 of several doses of subcutaneous buprenorphine. Pain sensitivity to noxious and nonnoxious mechanical stimuli at the site of injury (primary pain) was assessed at 1, 4, 24, and 72 h after surgery. Pain sensitivity at a site distal to the injury (secondary pain) was assessed at 24 and 72 h after surgery. Rats were tested for their sensitivity to the analgesic and locomotor effects of morphine 9 to 10 d after surgery. Buprenorphine at 0.05 mg/kg SC was determined to be the most effective; this dose induced isoalgesia during the acute postoperative period and the longest period of pain relief, and it did not induce long-term changes in opioid sensitivity in 2 functional measures of the opioid system. A lower dose of buprenorphine (0.01 mg/kg SC) did not meet the criterion for isoalgesia, and a higher dose (0.1 mg/kg SC) was less effective in pain relief at later recovery periods and induced a long-lasting opioid tolerance, indicating greater neural adaptations. These results support the use of 0.05 mg/kg SC buprenorphine as the upper dose limit for effective treatment of postoperative pain in rats and suggest that higher doses produce long-term effects on opioid sensitivity.Relief of postoperative pain is mandated in the Guide for the Care and Use of Animals¹⁸ and the Public Health Service Policy¹⁷ and is a major objective of laboratory animal medicine. Buprenorphine is one of the most commonly used opioid analgesics for postoperative pain in laboratory animals, mainly because of its long duration of action.¹⁰ The typical recommended dose range of buprenorphine in rats is 0.02 to 0.05 mg/kg SC.¹⁰ The upper end of this range, although effective at relieving acute postoperative pain in rats, is associated with side effects such as enhanced postoperative pain after the drug has worn off (rebound hyperalgesia),²³ respiratory depression,²¹ nausea or gastrointestinal distress and pica,²⁵ and neural adaptations (for example, sensitization) that may lead to long-term changes in neural function in the central nervous system and consequent changes in behavior.¹⁴ Central sensitization is a well-studied neural adaptation expressed in the brain and spinal cord and induced by nociceptive stimulation (that is, pain-induced by surgical manipulation) that manifests as hyperalgesia (decreased pain threshold to noxious stimuli) and allodynia (appearance of pain-like responses to nonnoxious tactile stimuli) during the recovery period.^16,29 Central sensitization contributes to persistent pain during the postoperative recovery period (that is, maintenance of increased pain sensitivity during tissue recovery) and chronic pain in some pathologic conditions (that is, persistent pain sensitivity after full tissue recovery). Central sensitization also accounts for the spread of hyperalgesia and allodynia to noninjured areas of the body distal to the injury.³¹ This phenomenon is referred to as ‘secondary pain’ (secondary hyperalgesia and allodynia), because it is not directly associated with the primary injury site.Opioid analgesics inhibit pain by acting on the nervous system to block transduction of pain signals traveling in sensory neurons toward the central nervous system and by facilitating activity of the descending pain inhibition neural pathway.¹⁶ Opioid analgesics also induce neural adaptations in the nervous system, phenomena that underlie the pronounced changes in behavior associated with addiction to narcotics.² Notably, opioid analgesics have been shown to enhance central sensitization initiated by pain transmission.^6,8,14,20 This property means that opiate analgesics facilitate both the inhibition of pain and central sensitization that leads to the enhancement of pain. Because central sensitization is a neural adaptation, the interaction of opiates on this pain mechanism outlasts the presence of the drug; in contrast, opiate effects on pain inhibition are limited to the presence of the drug. This arrangement is thought to account for rebound pain, that is, increased pain sensitivity after the opiate analgesic has worn off. Opiate side effects can compromise the success of recovery by increasing the level of distress experienced during recovery (for example, inducing nausea) and possibly increasing the duration of distress during recovery (for example, allowing for rebound pain). Moreover, and of importance specifically to laboratory animal medicine, the general neural adaptations induced by even a single dose of an opiate analgesic²⁶ may induce changes in the nervous system that alter and therefore compromise the validity of the animal model under study (for example, opioid mechanisms involved in behavioral control).We previously evaluated the feasibility of oral administration of buprenorphine.^15,25 As a basis for comparison, we used the ‘gold-standard’ postoperative buprenorphine dose of 0.05 mg/kg SC. The results of those studies showed that oral administration of buprenorphine was not feasible because the dose necessary to produce analgesia comparable to the standard dose of 0.05 mg/kg SC was 10 times the oral dose recommended in the literature and because the resulting concentration of oral buprenorphine was too bitter for rats to ingest voluntarily in a volume of flavored foodstuff that they could eat in a single meal.^15,25 We also observed that both subcutaneous and oral buprenorphine caused conditioned aversion to flavors,²⁵ suggestive of gastrointestinal distress⁵, with a greater effect for the oral route. Our conclusions and the associated clinical recommendation were limited by our presumption that buprenorphine at 0.05 mg/kg SC was the ideal postsurgical dose.An assessment of the literature that established this dose identified 2 problems. First, little or no research had directly assessed the effect of buprenorphine on pain sensitivity in animals in the hyperalgesic state that characterized the postoperative period,²³ and to our knowledge, no study has directly assessed the dose–response function of postsurgical buprenorphine on hyperalgesia. We hypothesized that endogenous opioids activated during the postoperative period²⁴ might act synergistically with buprenorphine to allow adequate relief of postoperative pain with a lower dose of buprenorphine than is necessary in an algesiometric test, thereby making predictions and extrapolations from algesiometric tests inaccurate. Second, we found that little consideration had been given to the consequences of other physiologic effects of buprenorphine on the recovery process (for example, gastrointestinal distress⁵, rebound hyperalgesia, and allodynia). As stated earlier, recent research on central sensitization has determined that although opioid analgesics inhibit pain sensation acutely, they also enhance neural adaptations that account for rebound pain and other long-term chronic pain conditions.^16,28,29,31 We hypothesized secondarily that a lower dose of buprenorphine, if effective acutely, would result in reduced side effects and be less likely to initiate or enhance neural adaptations, such as rebound hyperalgesia and allodynia.The current study had 2 goals. The first was to establish the minimum dose of buprenorphine needed to relieve acute postoperative pain effectively in rats. As a starting point, we defined effective relief of acute pain as the induction of isoalgesia during the postoperative period; isoalgesia is the normal level of pain sensation, in contrast to analgesia (absence of pain sensation) or hypoalgesia (lower-than-normal pain sensation). The second goal was to evaluate the effect of postoperative buprenorphine on factors that slow recovery (that is, rebound hyperalgesia and allodynia) or create long-term changes (that is, sensitization or tolerance to opiates). We tested our hypothesis by using various doses of buprenorphine in a rat model of incisional pain.^3,4,31 This model was selected because it induces cutaneous and muscular pain common to most surgery and generates mild to moderate persistent pain so that both the acute inhibitory effects of the buprenorphine (that is, pain relief) and the lasting effects of buprenorphine (that is, rebound hyperalgesia) could be studied.     

Selenium persistency and speciation in the tissues of lambs following the withdrawal of dietary high-dose selenium-enriched yeast

The objective was to determine the concentration of total selenium (Se) and the proportion of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in post mortem tissues of lambs in the 6 weeks period following the withdrawal of a diet containing high-dose selenised yeast (HSY), derived from a specific strain of Saccharomyces cerevisae CNCM (Collection Nationale de Culture de Micro-organism) I-3060. Thirty Texel × Suffolk lambs used in this study had previously received diets (91 days) containing either HSY (6.30 mg Se per kg dry matter (DM)) or an unsupplemented control (C; 0.13 mg Se per kg DM). Following the period of supplementation, all lambs were then offered a complete pelleted diet, without additional Se (0.15 mg Se per kg DM), for 42 days. At enrolment and 21 and 42 days later, five lambs from each treatment were blood sampled, euthanased and samples of heart, liver, kidney and skeletal muscle (longissimus dorsi and psoas major) tissue were retained. Total Se concentration in whole blood and tissues was significantly (P < 0.001) higher in HSY lambs at all time points that had previously received long-term exposure to high dietary concentrations of SY. The distribution of total Se and the proportions of total Se comprised as SeMet and SeCys differed between tissues, treatment and time points. Total Se was greatest in HSY liver and kidney (22.64 and 18.96 mg Se per kg DM, respectively) and SeCys comprised the greatest proportion of total Se. Conversely, cardiac and skeletal muscle (longissimus dorsi and psoas major) tissues had lower total Se concentration (10.80, 7.02 and 7.82 mg Se per kg DM, respectively) and SeMet was the predominant selenised amino acid. Rates of Se clearance in HSY liver (307 μg Se per day) and kidney (238 μg Se per day) were higher compared with HSY cardiac tissue (120 μg Se per day) and skeletal muscle (20 μg Se per day). In conclusion, differences in Se clearance rates were different between tissue types, reflecting the relative metabolic activity of each tissue, and appear to be dependent on the proportions of total Se comprised as either SeMet or SeCys.