An animal model for oroantral communications: a pilot study with Göttingen minipigs

A pilot study was performed to investigate whether the G?ttingen minipig is a suitable animal model for creating and closing oroantral communications (OACs) and to test whether these defects can be closed with a biodegradable polyurethane (PU) foam. In three adult minipigs, an OAC was created on both sides of the maxilla. The left side was closed by a standard surgical buccal flap procedure, the right side by applying a PU foam. The pigs were killed after two weeks, one month and three months, respectively. Postmortem and histological examination showed that an OAC was created in only one of six cases. In the remaining cases, the infraorbital canal was perforated instead of the floor of the maxillary sinus. It was concluded that the G?ttingen minipig is not a suitable animal model for OAC investigations. As a result, the closure of OACs with a biodegradable PU could not be evaluated.     

Three-dimensional electron microscopic imaging of membrane invaginations in Escherichia coli overproducing the chemotaxis receptor Tsr

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.     

Rap1 regulates E-cadherin-mediated cell-cell adhesion

The small GTPase Rap is best characterized as a critical regulator of integrin-mediated cell adhesion, although its mechanism of action is not understood. Rap also influences the properties of other cell-surface receptors and biological processes, although whether these are a consequence of effects on integrins is not clear. We show here that Rap also plays an important role in the regulation of cadherin-mediated cell-cell adhesion in epithelial cells. Expression of constitutively active Rap1A restored cadherin-mediated cell-cell contacts in mesenchymal Ras-transformed Madin-Darby canine kidney cells, resulting in reversion to an epithelial phenotype. Activation of endogenous Rap via the Rap exchange factor Epac1 also antagonized hepatocyte growth factor-induced disruption of adherens junctions. Inhibition of Rap signaling resulted in disruption of epithelial cell-cell contacts. Rap activity was required for adhesion of cells to recombinant E-cadherin extracellular domains, i.e. in the absence of integrin-mediated adhesion. These findings suggest that Rap signaling positively contributes to cadherin-mediated adhesion and that this occurs independently of effects on integrin-mediated adhesion. Our results imply that Rap may function in a broader manner to regulate the function of cell-surface adhesion receptors.     

A guinea pig model of acute and chronic asthma using permanently instrumented and unrestrained animals

To investigate mechanisms underlying allergen-induced asthmatic reactions, airway hyperresponsiveness and remodeling, we have developed a guinea pig model of acute and chronic asthma using unanesthetized, unrestrained animals. To measure airway function, ovalbumin (IgE)-sensitized animals are permanently instrumented with a balloon-catheter, which is implanted inside the pleural cavity and exposed at the neck of the animal. Via an external cannula, the balloon-catheter is connected to a pressure transducer, an amplifier, an A/D converter and a computer system, enabling on-line measurement of pleural pressure (P(pl))-closely correlating with airway resistance-for prolonged periods of time. Using aerosol inhalations, the method has been successfully applied to measure ovalbumin-induced early and late asthmatic reactions and airway hyperresponsiveness. Because airway function can be monitored repeatedly, intra-individual comparisons of airway responses (e.g., to study drug effects) are feasible. Moreover, this model is suitable to investigate chronic asthma and airway remodeling, which occurs after repeated allergen challenges. The protocol for establishing this model takes about 4 weeks.     

Dietary protein regulates hepatic constitutive protein anabolism in rats in a dose-dependent manner and independently of energy nutrient composition

We had previously observed that drastic increases in protein consumption greatly modified hepatic protein anabolism in rats, but the confounding effects of other macronutrient changes or a moderate protein increase to generate the same modifications have not yet been established. This study examined the metabolic and hormonal responses of rats subjected to 14-day isoenergetic diets containing normal, intermediate, or high-protein levels (NP: 14% of energy, IP: 33%, HP: 50%) and different carbohydrate (CHO) to fat ratios within each protein level. Fasted or fed rats (n = 104) were killed after the injection of a flooding dose of (13)C-valine. The hepatic protein content increased in line with the dietary protein level (P < 0.05). The hepatic fractional synthesis rates (FSR) of protein were significantly influenced by both the protein level and the nutritional state (fasted vs. fed) (P < 0.0001) but not by the CHO level, reaching on average 110%/day, 92%/day, and 83%/day in rats fed the NP, IP, and HP diets, respectively. The FSR of plasma albumin and muscle did not differ between diets, while feeding tended to increase muscle FSR. Proteolysis, especially the proteasome-dependent system, was down-regulated in the fed state in the liver when protein content increased. Insulin decreased with the CHO level in the diet. Our results reveal that excess dietary protein lowers hepatic constitutive, but not exported, protein synthesis rates, independently of the other macronutrients, and related changes in insulin levels. This response was observed at the moderate levels of protein intake (33%) that are plausible in a context of human consumption.     

Learning and anticipatory behaviour in a “sit-and-wait” predator: The Atlantic halibut

We studied the learning capacities and anticipatory behaviour in a “sit-and-wait” predatory fish, the Atlantic halibut, Hippoglossus hippoglossus. In Experiment 1 two groups of halibut received series of light flashes (conditioned stimulus, CS) that started before delivery of food (unconditioned stimulus, US) and persisted until after food delivery, i.e. delay conditioning. Control groups received unpaired CS and US presentations. The anticipatory behaviour of delay conditioned halibut consisted mainly of take-offs towards the surface shortly after onset of the CS. In Experiment 2 six groups of halibut were trained in three trace conditioning procedures: Two groups with 20 s, two groups with 60 s and two groups with 120 s trace interval. Learning was evident in the 20 and 60 s trace groups and in one of the 120 s trace groups. In contrast to delay conditioning the anticipatory behaviour of trace conditioned halibut was characterized by subtle movements near the tank floor with orientation towards the CS. The cautious responses of halibut after trace conditioning differed markedly from what is observed in other fish species and are suggested to reflect a “sit-and-wait” foraging strategy that requires the predator to remain undetected until the prey is within lunging range.     

Evolution by recombination and transspecies polymorphism in the MHC class I gene of Xenopus laevis

The patterns of major histocompatibility complex (MHC) evolution involve duplications, deletions, and independent divergence of loci during episodes punctuated by natural selection. Major differences in MHC evolution among taxa have previously been attributed to variation in linkage patterns of class I and class II MHC genes. Here we characterize patterns of evolution in the MHC class Ia gene of Xenopus laevis in terms of polymorphism, recombination, and extent of transspecies polymorphism. We also compare these patterns to see if a correlation exists with linkage or separation of the MHC class I and class II regions as seen in amphibians and teleost fishes. In X. laevis, we find high levels of polymorphism. Also, genetic exchange is relatively frequent and occurs in intron II, reshuffling allelic forms of exons 2 and 3. Evolutionary relationships among class I alleles show an intermingling of alleles from divergent Xenopus species rather than a species-specific clustering. Results indicate that the patterns of evolution are similar to those found in salmonid fishes and are different from the mode of evolution seen in primates. Similar patterns of class Ia evolution in salmonid fishes and X. laevis suggest that nonlinkage of class I and class II regions alone is insufficient to explain some patterns of MHC evolution in salmonids.     

In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-gamma-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-gamma production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-gamma-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-gamma, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.     

Polymorphism, natural selection, and structural modeling of class Ia MHC in the African clawed frog (Xenopus laevis)

In the African clawed frog (Xenopus laevis), two deeply divergent allelic lineages of multiple genes of the class I MHC region have been discovered. For the MHC class I UAA locus, functional differences and the molecular basis for lineages maintenance are unknown. Alleles of linked class I region genes also exhibit strong disequilibrium with specific MHC alleles, but the underlying cause is not clear. We use MHC class Ia sequence data to estimate substitution rates and investigate structural differences between allelic lineages from protein models. Results indicate the operation of natural selection, and differences in the steric properties in the F pocket of the peptide-binding region among lineages. Variability in this pocket likely enables allelic lineages to bind very different sets of peptides and to interact differently with MHC chaperones in the endoplasmic reticulum. These results constitute evidence of the molecular evolutionary basis for 1) the maintenance of allelic lineages, 2) functional differences among lineages, and 3) strong linkage disequilibrium of allelic variants of class I region genes in X. laevis.Electronic Supplementary Material Supplementary material is available for this article at