Life cycle assessment of energy self-sufficiency systems based on agricultural residues for organic arable farms.

The agricultural industry today consumes large amounts of fossil fuels. This study used consequential life cycle assessment (LCA) to analyse two potential energy self-sufficient systems for organic arable farms, based on agricultural residues. The analysis focused on energy balance, resource use and greenhouse gas (GHG) emissions. A scenario based on straw was found to require straw harvest from 25% of the farm area; 45% of the total energy produced from the straw was required for energy carrier production and GHG emissions were reduced by 9% compared with a fossil fuel-based reference scenario. In a scenario based on anaerobic digestion of ley, the corresponding figures were 13%, 24% and 35%. The final result was sensitive to assumptions regarding, e.g., soil carbon content and handling of by-products.

Intestinal tissue transglutaminase in coeliac disease of children and adults: ultrastructural localization and variation in expression.

BACKGROUND: Tissue transglutaminase is the main antigen for the anti-endomysial antibodies used for diagnosis of coeliac disease and can with some specificity in vitro deamidate gliadins generating potent epitopes. The intestinal levels and the ultrastructural localization of tissue transglutaminase in normal and affected persons were investigated to provide further information on its role in this disease. Intestinal biopsies were taken from normal and coeliac children and adults. METHODS: The level of transglutaminase was analysed by means of a quantitative enzymatic assay and its ultrastructural localization by immunogold electronmicroscopy using a monoclonal antibody against tissue transglutaminase. RESULTS: In relation to normal individuals, the enzymatic activity of tissue transglutaminase in adult coeliac patients was increased. The enzyme was found in the enterocytes and in increased amount just beneath the enterocytes, where cytosolic and nuclear labelling of distinct elongated cells was seen in addition to extracellular labelling close to collagen fibrils. In children, the enzymatic activity and the immunogold labelling could not be shown to be related to disease. In all cases the enzyme activity was EDTA-sensitive. CONCLUSIONS: The increased amount of tissue transglutaminase activity in coeliac adults was shown to be due to the appearance of the enzyme in enterocytes and increased expression in the lamina propria. No evidence was found to support the idea of a changed localization or changed amounts as primary elements in coeliac disease pathogenesis, nor for the involvement of non-calcium dependent microbial transglutaminases.

Gliadin is a good substrate of several transglutaminases: possible implication in the pathogenesis of coeliac disease.

BACKGROUND: Deamidation of distinct glutamines in HLA-DQ2 restricted gliadin epitopes, considered critical in the pathogenesis of coeliac disease (CD), can be mediated by tissue transglutaminase (tTG). To elucidate the possible role of other transglutaminases in CD we investigated whether different mammalian, microbial and vegetable transglutaminases can use gliadin as substrate. METHODS: Studies in which small amounts of transglutaminase have been measured have led to our modifying a microtitre plate assay. We used proteolytically digested gliadin as solid phase substrate and Europium-labelled streptavidine to quantify the biotinylated product covalently linked by the enzyme to the plate. RESULTS: The modified assay is ultrasensitive and quantitative, measuring guinea pig liver transglutaminase concentrations between 0.5 and 50 ng/well. The specific activities of the enzymes (counts/min/mg) against gliadin and N,N-dimethylcasein, respectively, are: tTG 9800/4900, Factor XIII 97330/55620, epidermal transglutaminase 47650/50770, streptoverticillium transglutaminase 4290/2200, phytophora cactorum transglutaminase 6910/4110. For the first time, we have detected transglutaminase activity in bean sprouts, spinach leaves and green peas, which are commonly used Vegetables. CONCLUSION: Gliadin is a good substrate for endogenous, microbial and plant transglutaminases. An interesting altemative is that gliadins are deamidated by microbial or food transglutaminases in the intestinal lumen. The assay described provides an ultrasensitive method for measuring small amounts of transglutaminase and is considered a helpful tool in further studies of the possible role of transglutaminases in the pathogenesis of CD.

Interaction between the homeodomain proteins Cdx2 and HNF1alpha mediates expression of the lactase-phlorizin hydrolase gene.

Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1alpha (HNF1alpha), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1alpha physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1alpha. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1alpha. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.

Transcriptional regulation of pig lactase-phlorizin hydrolase: involvement of HNF-1 and FREACs.

BACKGROUND & AIMS: One-kilobase sequence of the upstream fragment of the pig lactase-phlorizin hydrolase gene has been shown to control small intestinal-specific expression and postweaning decline of lactase-phlorizin hydrolase in transgenic mice. The aim of this study was to identify the regulatory DNA elements and transcription factors controlling lactase-phlorizin hydrolase expression. METHODS: The activity of different lactase-phlorizin hydrolase promoter fragments was investigated by transfection experiments using Caco-2 cells. Electrophoretic mobility shift assays and supershift analyses were used to characterize the interaction between intestinal transcription factors and the identified regulatory elements. RESULTS: Functional analysis revealed three previously undescribed regulatory regions in the lactase-phlorizin hydrolase promoter: a putative enhancer between -894 and -798 binding hepatocyte nuclear factor (HNF)-1 at position -894 to -880; a repressor-binding element between -278 to -264 to which an HNF-3-like factor is able to bind; and an element between -178 to -164 that binds an activating transcription factor. CONCLUSIONS: Identification of three new regulatory regions and HNF-1 and HNF-3-like transcription factor as players in the regulation of lactase-phlorizin hydrolase gene transcription has an impact on the understanding of the molecular mechanisms behind age-dependent, tissue-specific, differentiation-dependent, and regional regulation of expression in the intestine.

Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition.

Coeliac disease probably results from a T-cell response to wheat gliadin and is associated to HLA-DQ2. No gliadin epitopes recognized by intestinal T cells have yet been identified, limiting our understanding of the pathogenesis. Gut-lesion-derived DQ2-restricted T cells from coeliac disease patients were used to identify an epitope within a purified gamma-type gliadin. The structure of the epitope was characterized by mass spectrometry and verified by synthesis. The epitope (QPQQSFPEQQ) results from deamidation of a distinct glutamine in the native structure. This deamidation is important for binding to DQ2 and T-cell recognition. Other gut-derived T cells fail to recognize the epitope, although deamidation of unfractionated gliadin enhances the response of all gut-derived DQ2-restricted T cells isolated from several patients. Several DQ2-restricted T-cell epitopes exist, but for all of them deamidation of glutamine residues appears to be critical for creation of active epitopes. Native gliadin has few negatively charged residues but is very rich in glutamine. After deamidation gliadin becomes a rich source of DQ2 epitopes thus providing a link between DQ2, gliadin and coeliac disease. The necessity for modification may have general immunological relevance.

Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.

The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

The HOXC11 homeodomain protein interacts with the lactase-phlorizin hydrolase promoter and stimulates HNF1alpha-dependent transcription.

The lactase-phlorizin hydrolase (LPH) gene is expressed specifically in the enterocytes of the small intestine. LPH levels are high in newborn mammals, but decrease after weaning. We have previously suggested that the promoter element CE-LPH1, located at -40 to -54, plays an important role in this down-regulation, because the DNA binding activity of a nuclear factor that binds to this site is present specifically in small intestinal extracts and is down-regulated after weaning. In an effort to clone CE-LPH1-binding factors, a yeast one-hybrid genetic selection was used, resulting in the isolation of a partial cDNA encoding the human homeodomain protein HOXC11. The full-length HOXC11 sequence was obtained by rapid amplification of cDNA ends. It was shown in a yeast assay and by electrophoretic mobility shift assay that HOXC11 binds to the CE-LPH1 element with similar specificity to the endogenous intestinal factor. Two HOXC11 transcript sizes were identified by Northern blot analysis. The larger transcript (2.1 kilobase pairs) is likely to contain a translational start site in good context and is present in HeLa cells. The shorter 1.7-kilobase pair transcript, present in HeLa and Caco-2 cells, probably encodes a protein lacking 114 amino acids at the N-terminal end. Both forms of HOXC11 potentiate transcriptional activation of the LPH promoter by HNF1alpha. The expression of HOXC11 mRNA in human fetal intestine suggests a role in early intestinal development.

The TATA-less, GC-rich porcine dipeptidylpeptidase IV (DPPIV) promoter shows bidirectional activity.

Dipeptidylpeptidase IV (DPPIV) is an exopeptidase highly expressed in the brush-border membrane of the small intestine and in the proximal renal tubules. In this paper the 5'-flanking region of the DPPIV gene containing the promoter was sequenced and functionally characterized. The porcine DPPIV promoter lacks a consensus TATA-box, but contains two TATA-like sequences. Evidence for multiple transcription initiation sites was found. Different deletion constructs of the DPPIV 5'-flanking region in front of the CAT gene were analyzed for transient CAT-expression after transfection of the intestinal Caco-2 cell line. These experiments showed that a 89 base pair construct (-91 to -3 relative to the translation initiation site) is sufficient for promoter activity. In the reverse orientation this construct also stimulates transcription with a similar effectivity indicating that the DPPIV promoter has a bidirectional function. The bidirectional function was further demonstrated by the introduction of the -91 to -3 construct into the bidirectional vector system pLUC/CAT-3. In the hepatoma cell line HepG2 two selected deletion constructs (-857 to -3; -282 to -3) were analyzed in the normal orientation using the CAT gene as a reporter gene. The transfection experiments showed that deletion of the region -857 to -282 raised the promoter activity 3-fold. The GC-rich 5'-flanking region was further analyzed and we demonstrate that the DPPIV promoter contains a region with the characteristics of an unmethylated CpG island.

The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment.

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.

Heterogeneous reactivity patterns of HLA-DQ-restricted, small intestinal T-cell clones from patients with celiac disease.

BACKGROUND & AIMS: T-cell immune reactions toward wheat gliadins seem important in the pathogenesis of celiac disease. We have previously shown that gliadin-specific T-cell clones (TCCs) from the small intestinal mucosa of patients with celiac disease are predominantly restricted by the celiac disease-associated HLA-DQ2 and HLA-DQ8 molecules, suggesting a link between the HLA association and immunopathogenesis. The aim of the present study was to investigate the nature of the stimulating gliadin antigens. METHODS: Three different pools of gliadins and one purified alpha-type and two purified gamma-type gliadin preparations were prepared by ion exchange chromatography and tested for recognition by a panel of small intestinal gliadin-specific TCCs. RESULTS: Evidence suggested that enzymatic digestion and heating of the gliadins influenced TCC stimulation. Most of the TCCs recognized all three gliadin pools, but some had distinct reactivity patterns. Thirteen of 21 TCCs responded to one or more of the three purified gliadins discerning highly discriminative patterns. CONCLUSIONS: Small intestinal, gliadin-specific T cells from patients with celiac disease show diverse reactivity patterns. Stimulation of large numbers of different gliadin-specific T cells in the small intestinal mucosa of patients with celiac disease may occur; this may be an important immunopathogenic step in the disease.

Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2.

Lactase-phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase-phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity.

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms of the glycoprotein aminopeptidase N can be synthesized and the effects of altered glycosylation can be studied. It is demonstrated that aminopeptidase N carries "mucin-type" O-glycans and that this is predominantly located in the stalk, which connects the catalytic headgroup to the membrane anchor. Normally glycosylated aminopeptidase N is present in the plasma membrane of the ldl(D) cells. This is also the case for the non-O-glycosylated and defectively N-glycosylated forms. This is in line with the finding that the intracellular transport APN is unaffected by the absence of O-glycosylation or by changes in N-glycosylation as the various glycosylated forms of aminopeptidase N are normally converted from the high-mannose form to the complex glycosylated form. Enzymatic activity is not influenced by the changes in glycosylation.

Pseudopregnancy induces the expression of hepatocyte nuclear factor-1 beta and its target gene aminopeptidase N in rabbit endometrium via the epithelial promoter.

The rabbit endometrium is an excellent model system allowing experimental manipulation of aminopeptidase N (APN) mRNA expression in vivo. By RNase mapping and sequencing of cloned PCR-amplified primer-extended RNA, it was demonstrated that endometrial APN expression is directed by the epithelial APN promoter and is increased in human-choriogonadotropin-induced pseudopregnancy. Cloning and sequencing of the rabbit APN epithelial promoter revealed conservation of the upstream footprint (UF), hepatocyte nuclear factor-1 (HNF1) and Sp1 elements known to be present in the pig and human promoters as well. The pseudopregnancy-induced APN expression was found to be accompanied by a parallel increase in the level of the transcription factor HNF1 beta, whereas a much smaller increase in Sp1 and UF-binding proteins was observed. This indicates that HNF1 beta acts as a switch triggering the pregnancy-induced APN expression. The sequence of the UF element suggests members of the nuclear hormone-receptor superfamily as possible UF-binding proteins, and competition experiments suggest that the chicken ovalbumin upstream promoter transcription factor functions as such in the rabbit endometrium.

Transcytosis of aminopeptidase N in Caco-2 cells is mediated by a non-cytoplasmic signal.

In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P. (1990) Cell 60, 429-437). The aim of this study was to determine the importance of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from the basolateral to the apical plasma membrane, whereas no transcytosis of two secretory forms could be detected, showing that the transmembrane domain is important for efficient transcytosis to take place. A significant difference in transcytosis kinetics of the human and the porcine wild type aminopeptidase N was observed. This indicates that transcytosis of aminopeptidase N from the basolateral to the apical membrane does not occur by default transport but involves an active sorting mechanism.

Thermal aspects of vehicle comfort.

The combined thermal effects of convection, radiation and conduction in a vehicle compartment need special measuring equipment accounting for spatial and temporal variations in the driver space. The most sophisticated equipment measures local heat fluxes at defined spots or areas of a man-shaped manikin. Manikin segment heat fluxes have been measured in a variety of vehicle climatic conditions (heat, cold, solar radiation etc.) and compared with thermal sensation votes and physiological responses of subjects exposed to the same conditions. High correlation was found for segment fluxes and mean thermal vote (MTV) of subjects for the same body segments. By calibrating the manikin under homogenous, wind still conditions, heat fluxes could be converted (and normalised) to an equivalent homogenous temperature (EHT). Regression of MTV-values on EHT-values was used as basis for the derivation of a comfort profile, specifying acceptable temperature ranges for 19 different body segments. The method has been used for assessment of the thermal climate in trucks and crane cabins in winter and summer conditions. The possibility for spatial resolution of thermal influences (e.g. by solar radiation or convection currents) appeared to be very useful in the analysis of system performance. Ventilation of driver's seats is a technical solution to reducing insulation of thigh, seat and back areas of the body. Constructions, however, may vary in efficiency. In one system seat ventilation allowed for almost 2 degrees C higher ambient conditions for unchanged general thermal sensation, in addition to the pronounced local effect. In a recent study the effects of various technical measures related to cabin design and HVAC-systems have been investigated.(ABSTRACT TRUNCATED AT 250 WORDS)