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Transgenic rice lines expressing maize C1 and R-S regulatory genes produce various flavonoids in the endosperm.

Flavonoids, compounds that possess diverse health-promoting benefits, are lacking in the endosperm of rice. Therefore, to develop transgenic lines that produce flavonoids, we transformed a white rice cultivar, Oryza sativa japonica cv. Hwa-Young, with maize C1 and R-S regulatory genes. Expression of these transgenes was restricted to the endosperm using the promoter of a rice prolamin gene. The pericarp of the C1/R-S homozygous lines became dark brown in accordance with their maternal genotype, whereas the endosperm turned chalky, similar to the opaque kernel phenotype. Analysis via high-performance liquid chromatography (HPLC) revealed that numerous kinds of flavonoids were produced in these transgenic kernels. To identify individual flavonoids, the number of HPLC peaks was reduced through moderate acid hydrolysis, followed by ethyl acetate partitioning. Amongst the major flavonoids, dihydroquercetin (taxifolin), dihydroisorhamnetin (3'-O-methyl taxifolin) and 3'-O-methyl quercetin were identified through liquid chromatography/mass spectrometry/mass spectrometry and nuclear magnetic resonance analyses. Fluorescence labelling with diphenylboric acid showed that the flavonoids were highly concentrated in the cells of four to five outer endosperm layers. More importantly, a high fluorescence signal was present in the cytosol of the inner endosperm layers. However, the overall signal in the inner layers was significantly lower because starch granules and protein bodies occupied most of the cytosolic space. Our estimate of the total flavonoid content in the transgenic kernels suggests that C1/R-S rice has the potential to be developed further as a novel variety that can produce various flavonoids in its endosperm.

Chromatography, High Pressure Liquid↗

Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease.

OBJECTIVES: Exposure to gliadin and related prolamins and appropriate HLA-DQ haplotype are necessary but not sufficient for contracting celiac disease (CD). Aberrant innate immune reactions could be contributing risk factors. Therefore, jejunal biopsies were screened for bacteria and the innate immune status of the epithelium investigated. METHODS: Children with untreated, treated, challenged CD, and controls were analyzed. Bacteria were identified by scanning electron microscopy. Glycocalyx composition and mucin and antimicrobial peptide production were studied by quantitative RT-PCR, antibody and lectin immunohistochemistry. RESULTS: Rod-shaped bacteria were frequently associated with the mucosa of CD patients, with both active and inactive disease, but not with controls. The lectin Ulex europaeus agglutinin I (UEAI) stained goblet cells in the mucosa of all CD patients but not of controls. The lectin peanut agglutinin (PNA) stained glycocalyx of controls but not of CD patients. mRNA levels of mucin-2 (MUC2), alpha-defensins HD-5 and HD-6, and lysozyme were significantly increased in active CD and returned to normal in treated CD. Their expression levels correlated to the interferon-gamma mRNA levels in intraepithelial lymphocytes. MUC2, HD-5, and lysozyme proteins were seen in absorptive epithelial cells. beta-defensins hBD-1 and hBD-2, carcinoembryonic antigen (CEA), CEA cell adhesion molecule-1a (CEACAM1a), and MUC3 were not affected. CONCLUSIONS: Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-gamma production by intraepithelial lymphocytes.

Adolescent↗

In vitro inhibition of diphtheria toxin action by ammonium salts and amines.

Kim, K. (University of Washington, Seattle), and N. B. Groman. In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J. Bacteriol. 90:1552-1556. 1965.-An inhibitor for diphtheria toxin action on HeLa cells was demonstrated in the growth supernatant fractions of both toxinogenic and nontoxinogenic strains of Corynebacterium diphtheriae and in the Mueller and Miller medium in which these organisms were grown. The inhibitor in the growth supernatant fractions of the nontoxinogenic strain was dialyzable, stable to autoclaving, and stable on storage in the refrigerator for a period of many months, but was destroyed by ashing. When the components of Mueller and Miller medium were analyzed, only the Casamino Acids proved inhibitory. Further study with artificial mixtures of amino acids revealed that glutamine alone inhibited toxin. It was subsequently shown that ammonium salts and the aliphatic amines, glycamine and prolamine, could also function as inhibitors. Histamine and 16 amino acids tested individually were ineffective. The effectiveness of the amines and the ineffectiveness of sodium or potassium ions indicates that there is a specific requirement for inhibition.

Amines↗

Gliadin uptake in human enterocytes. Differences between coeliac patients in remission and control individuals.

The pepsin trypsin digest of the wheat prolamin gliadin (PT-gliadin) is deleterious to the small intestinal mucosa of coeliac patients. The handling of PT-gliadin by the intestinal epithelium in coeliac patients in remission and control individuals was investigated by in vivo instillation of PT-gliadin. The uptake of PT-gliadin was monitored by immunofluorescence microscopy of intestinal biopsy specimens, using affinity purified PT-gliadin antibodies. Control individuals show weak staining in the apical region of the enterocytes thereby showing an uptake of PT-gliadin. Coeliac patients have a conspicuous fluorescence in relation to the lateral membrane/intercellular space of enterocytes and intense staining intracellularly in the apical region. There is only weak staining in the enterocytes after the instillation was terminated, indicating an intracellular clearance. The study shows that normal enterocytes are able to take up PT-gliadin. The increased uptake in coeliac patients might be of importance for the pathogenesis either by direct toxicity or by presentation to immunocompetent cells. Furthermore, the results are in agreement with the suggestion of a functional alteration in the zonula occludens in the intestinal epithelium of coeliac patients.

Adult↗

Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon gamma.

Coeliac disease is precipitated in susceptible subjects by ingestion of wheat gluten or gluten related prolamins from some other cereals. The disease is strongly associated with certain HLA-DQ heterodimers, for example, DQ2 (DQ alpha 1*0501, beta 1*0201) in most patients and apparently DQ8 (DQ alpha 1*0301, beta 1*0302) in a small subset. Gluten specific T cell clones (TCC) from coeliac intestinal lesions were recently established and found to be mainly restricted by HLA-DQ2 or HLA-DQ8. Antigen induced production of cytokines was studied in 15 TCC from three patients, 10 being DQ2 and five DQ8 restricted. Cell culture supernatants were prepared by stimulation with gluten peptides in the presence of DQ2+ or DQ8+ Epstein-Barr virus transformed B cells as antigen presenting cells (APC). Supernatants were analysed for cytokines by bioassays, ELISA, and CELISA. Cellular cytokine mRNA was analysed semi-quantitatively by slot blotting and polymerase chain reaction (PCR). All TCC were found to secrete interferon (IFN) gamma, often at high concentrations (> 2000 U/ml); some secreted in addition interleukin (IL) 4, IL 5, IL 6, IL 10, tumour necrosis factor (TNF), and transforming growth factor (TGF) beta. The last TCC thus displayed a Th0-like cytokine pattern. However, other TCC produced IFN gamma and TNF but no IL 4, or IL 5, compatible with a Th1-like pattern. In conclusion, most DQ8 restricted TCC seemed to fit with a Th0 profile whereas the DQ2 restricted TCC secreted cytokines more compatible with a Th1 pattern. The TCC supernatants induced upregulation of HLA-DR and secretory component (poly-Ig receptor) in the colonic adenocarcinoma cell line HT-29.E10, most probably reflecting mainly the high IFN gamma concentrations. This cytokine, particularly in combination with TNF alpha, might be involved in several pathological features of the coeliac lesion. The characterised cytokine profiles thus support the notion that mucosal T cells activated in situ by gluten in a DQ restricted fashion play a central part in the pathogenesis of coeliac disease.

Base Sequence↗

A moderately repetitive DNA sequence in alfalfa is transcribed in a floral-specific manner.

Based on DNA sequence analysis of 5 clones of repetitive DNA from alfalfa (Medicago sativa), we propose the existence of a dispersed middle repetitive element about 3400 bp long with a copy number in the range of 2-3 x 10(3) per haploid genome. The average A+T content of the sequences was 54.6%, compared with 61.4% for the alfalfa genome. Sequence homologies between overlapping regions of the clones ranged from 85 to 89.5% with an average of 86.6%; sequence divergence was due largely to single base pair changes, with deletions or insertions occurring randomly across sequences. An open reading frame (ORF) in one clone, RPE15, contained homologies to cereal prolamin genes and a legumin box was located upstream of the coding region. A Northern blot of RNA from various alfalfa tissues, probed with the above clone containing this ORF, showed an extensive heterodispersed pattern of hybridization in the late stages of flower bud development but in no other tissues.

Base Composition↗

Influence of pancreatic duct occlusion on islet hormones in peripheral and portal plasma and in the pancreas of the mini-pig.

In a total of 18 'Göttingen' mini-pigs we studied basal glucose in the peripheral plasma, and the hormones insulin, glucagon, and somatostatin in the peripheral and portal plasma, as well as in extracts of pancreatic tissue, both in animals subjected to pancreatic duct occlusion with prolamine (Occ pigs) 9 months previously and in controls. Additionally, in the pancreas the relative frequency of A-, B-, D- and PP-cells was determined by immunocytochemistry. In peripheral blood of Occ pigs glucose, insulin, and somatostatin were unchanged, while glucagon was decreased. Also after occlusion the portal plasma revealed an increase in insulin but unchanged glucagon and somatostatin, while in the pancreatic tissue insulin and glucagon were statistically unchanged, but somatostatin was reduced. The relative frequency of A-, B-, D- and PP-cells in the pancreatic islets was comparable in both control and Occ pigs. It is concluded that also in the pig pancreatic duct occlusion leads to atrophy of the exocrine pancreas, but leaves undisturbed basal blood glucose, insulin, glucagon and islet cells.

Amylases↗

In vitro activation of adenylate cyclase of atrophic celiac intestinal mucosa by wheat gliadin-derived peptides.

In order to demonstrate that gliadin peptides may interact with cell membranes of celiac small intestinal mucosa, the capacity of these peptides to activate the cell membrane enzyme adenylate cyclase was tested. The addition of peptides from bread wheat purified A-gliadin and whole gliadin (proteins that are toxic for celiac patients) enhanced the adenylate cyclase activity of crude cell membrane preparations obtained from atrophic small intestinal mucosa of celiac patients. No activation of adenylate cyclase of this tissue was observed with peptides from proteins nontoxic for celiac patients (bread wheat albumin and maize prolamin). Gliadin peptides did not activate adenylate cyclase of morphologically normal small intestinal mucosa from normal subjects or from celiac patients in remission. These results, therefore, suggest that peptides from bread wheat gliadin may interact with cell membrane of atrophic small intestinal mucosa of celiac patients.

Adenylyl Cyclases↗

Enhanced peptide-binding capacities of small intestinal brush border membranes in celiac disease.

In pathogenesis of celiac disease, the significance of prolamin peptide interactions with enterocytes is controversial. Changes in cellular metabolism induced by gliadin peptides, as well as uptake and presentation by enterocytes, are discussed. We analyzed peptide binding to enterocytic membranes as a potential key event. Binding capacities of brush border membranes isolated from small intestinal biopsies of untreated (n = 49) and treated celiac patients on a gluten-free diet (n = 30), as well as control subjects (n = 43), were measured with a dot blot chemiluminescence assay. Synthetic gliadin peptides comprising amino acid position 8-19 (G XIV) and 30-41 (G XI) of alpha-gliadins, a peptic-tryptic digest of gliadin (PT-GLI), and a synthetic zein peptide were used. Comparing treated celiac patients with controls, we observed significantly enhanced membrane-binding of PT-GLI [mean 122.4 densitometric units/microg (95% confidence interval 116.0-128.9) vs 108.9 (102.1-115.7)] and of zein peptide [50.2 (38.4-61.9) vs 28.8 (13.4-44.2)], but only slightly increased binding of the synthetic gliadin peptides G XIV [65.5 (60.6-70.5) vs 62.4 (56.3-68.5) and G XI [75.2 (69.8-80.6) vs 65.9 (55.2-76.5)]. Independent of patient group, membrane-binding capacities for celiac-active gliadin peptides exceeded those of the zein peptide. Thus, interaction of gliadin peptides with the apical enterocytic membrane was not found exclusively in celiac disease. Furthermore, increased binding capacities in treated celiac disease were not confined to celiac-active peptides. Quantitative differences in gliadin peptide binding as a primary characteristic in celiac disease might contribute to pathogenetic effects exerted on small intestinal epithelial cells.

Adolescent↗

Changes in grain size and grain storage protein of rice (Oryza Sativa L.) in response to elevated UV-B radiation under outdoor conditions.

Variation in growth, grain size and grain storage protein content of rice (Oryza sativa L.) in response to elevated UV-B radiation under sunlight was examined in a cool rice-growing region of Miyagi Prefecture, Japan, in 1999, 2001 and 2002. Tiller number, dry mass, panicle number, grain yield and grain size significantly decreased under elevated UV-B radiation in 2001 and 2002. The effects of elevated UV-B radiation on the reduction of each growth parameter were greatly enhanced by daily lower temperature during the ripening stage in those two years. On the contrary, total grain nitrogen content and grain storage protein content significantly increased under elevated UV-B radiation in 2001 and 2002. Among grain storage proteins, glutelin content significantly increased but albumin-globulin and prolamin contents did not. It was thus evident that not only grain size but also grain storage protein of rice was markedly influenced due to elevated UV-B radiation.

Climate↗

Computational analysis of the amino acid residue sequences of amaranth and some other proteins.

Amaranth belongs to a nutritious class of pseudocereals. The well balanced amino acid composition of amaranth compared with those of major cereals would indicate that it deserves a quantitative study of its chemical properties. This work was undertaken to compare Amaranthus (A.) caudatus with a number of other plants on the basis of the sequences of various proteins and the composition of their alcohol-soluble protein mixture and glutelins. Alcohol-soluble proteins were extracted with 55% isopropanol (2-ProOH) + 5% 2-mercaptoethanol (2-ME) and glutelin fractions were obtained with borate buffer + 3% 2-ME + 0.5% sodium dodecyl sulfate (SDS), pH 10. Protein fractions were then electrophoreded on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). FASTA and TFASTA programmes were used for comparison of amino acid sequences. Dot matrix analysis and secondary structure predictions which were drawn by Plot-structure, were taken from the GCG package. Electrophoretic tests failed to indicate significant correlation between prolamins from cereals and other plants with the alcohol-soluble fractions from amaranth, proving that these proteins cannot represent the main fraction in amaranth. On the other hand, glutelins shared some common electrophoretic bands with other cereals and showed some identity by SDS-PAGE. Amino acid sequences of A. caudatus (100% identity) had degrees of similarity in the range of 71.4 to 52.2% with rice, garden pea, jobs' tears, maize, and yam. Rice glutelin had similarity in the range of 93.3% to 44.8% with oats, soybean, and pea. Secondary structures of A. caudatus (using conservative amino acid replacement), jobs' tears and rice glutelins, oat globulin, and pea legumin sequences were predicted. Some relationship was shown among electrophoretic patterns of alcohol-soluble proteins and glutelins of A. caudatus.

Amino Acid Sequence↗

Rice allergenic proteins, 14-16 kDa albumin and alpha-globulin, remain insoluble in rice grains recovered from rice miso (rice-containing fermented soybean paste).

The rice grains (RG) and rice seed proteins remaining in rice miso were investigated with a view point to the potential allergenicity of rice miso. RG ranging from 36 to 180 mg dry weight per g dry miso were separated from several samples of commercially available rice miso. Scanning electron microscopy of the recovered RG indicated that starch granules disappeared almost completely while protein bodies remained intact in RG. Most of the major seed proteins were extracted from RG by heating with 1% SDS/2% 2-mercaptoethanol and detected by SDS-polyacrylamide gel electrophoresis. Major rice allergenic proteins, 14-16 kDa albumin (Alb14-16) and alpha-globulin (alpha-Glb) were also detected by immunoblotting using the specific antisera, and their contents were estimated to be 1.7 to 9.0 and 1 to 7 mg protein per g dry RG respectively. However, the major rice proteins, including glutelin and prolamin, in RG were insoluble in salt, alcohol, and urea solutions, but soluble in 6 M guanidine hydrochloride (Gu-HCl). By immunoblotting and ELISA, no Alb14-16 and only a slight amount of alpha-Glb were detected even in the 6 M Gu-HCl fraction, indicating that these major allergenic proteins are denatured and are present in an insoluble form in rice miso.

Albumins↗

Inhibitory plant serpins with a sequence of three glutamine residues in the reactive center.

Serpins appear to be ubiquitous in eukaryotes, except fungi, and are also present in some bacteria, archaea and viruses. Inhibitory serpins with a glutamine as the reactive-center P1 residue have been identified exclusively in a few plant species. Unique serpins with a reactive center sequence of three Gln residues at P3-P1 or P2-P1' were isolated from barley and wheat grain, respectively. Barley BSZ3 was an irreversible inhibitor of chymotrypsin, with a second-order association rate constant for complex formation k(a)' of the order of 10(4) M(-1) s(-1); however, only a minor fraction of the serpin molecules reacted with chymotrypsin, with the majority insensitive to cleavage in the reactive center loop. Wheat WSZ3 was cleaved specifically at P8 Thr and was not an inhibitor of chymotrypsin. These reactive-center loops may have evolved conformations that are optimal as inhibitory baits for proeinases that specifically degrade storage prolamins containing Gln-rich repetitive sequences, most likely for digestive proteinases of insect pests or fungal pathogens that infect cereals. An assembled full-length amino acid sequence of a serpin expressed in cotton boll fiber (GaZ1) included conserved regions essential for serpin-proteinase interaction, suggesting inhibitory capacity at a putative reactive center P2-P2' with a sequence of four Gln residues.

Amino Acid Sequence↗

Cholecystokinin and glucose-induced insulinaemia in dogs with and without pancreatic acinar atrophy.

The entero-insular hormonal axis was studied in eleven conscious Beagle dogs, loaded with glucose orally and intravenously. In five of them, exocrine pancreatic atrophy was induced by pancreatic duct occlusion with prolamine, and documented by means of the p-amino-benzoic acid test. After oral glucose, the duct-occluded dogs displayed higher blood glucose (log area 4.12 +/- 0.07 versus 3.76 +/- 0.10; p less than 0.01), less plasma insulin (log area 3.56 +/- 0.08 versus 3.99 +/- 0.08; p less than 0.01) and less cholecystokinin-like immunoreactivity (log area 2.64 +/- 0.09 versus 3.10 +/- 0.14; p less than 0.01) than controls. In controls, the peripheral venous insulin concentrations were higher after oral than after isoglycaemic intravenous glucose, and this difference was no longer demonstrable in duct-occluded dogs. In the latter, gel permeation chromatography of pool plasma after oral glucose revealed a relative decrease of cholecystokinin-like immunoreactivity species, which eluted at the positions of sulphated cholecystokinin octapeptide, cholecystokinin-33 and cholecystokinin-39, and at a position intermediate between these two. Also in the duct-occluded animals, intravenous infusion of sulphated cholecystokinin octapeptide, in addition to oral glucose, resulted in an increase in plasma insulin (log area 3.83 +/- 0.10 versus 3.64 +/- 0.06; p less than 0.01) and an improvement in oral glucose tolerance. It is concluded that in the dog 1) the absence of pancreatic acinar tissue is associated with a loss of gastrointestinal factors mediating glucose-induced insulin secretion, and 2) reduction of circulating endogenous cholecystokinin species may account at least in part for this defect.

Animals↗

Magnetic resonance imaging and computed tomography in long-term-functioning duct-occluded pancreas allotransplants.

Duct-occluded segmental-pancreas transplants develop progressive fibrotic atrophy, even though endocrine function seems to be unaffected. To determine end-stage size of the graft and to evaluate magnetic resonance imaging (MRI) and computed tomography (CT), these imaging techniques were applied in eight patients with well-functioning intraperitoneal prolamine-injected segmental-pancreas transplants for 79, 48, 35, 20, 19, 19, 18, and 10 mo. MRI was performed on a 1.5 Tesla system (Philips Gyroscan S15). T1- and T2-weighted images were acquired. CT (Siemens Somatom 2) was done before and after intravenous contrast agent. The graft was visualized in seven of eight patients with both techniques. Visualization with MRI (vs. CT) was considered excellent in 2 (vs. 1), good in 3 (vs. 6), and poor in 2 (vs. 0). The three grafts with function longer than 2 yr measured 3-4 cm in length; the remaining grafts measured 3-6 cm. Because of a marked decrease in size the transplants were no longer localized in Douglas' pouch but adjacent to or on top of the uterus or bladder, the position depending on the volume of these organs. The allografts exhibited an inhomogeneous structure with casual cystic degeneration visible with MRI due to a high signal intensity on T2-weighted images. This study suggests that shrinkage of the duct-occluded pancreatic segment due to exocrine atrophy may be terminated after approximately 2 yr. It is concluded that thereafter an overshooting fibrosis causing late endocrine graft failure may not be anticipated.

Adult↗

Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization.

Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant alpha-gliadin (r-alpha-gliadin) showing the most conserved sequence among the fraction of alpha-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-alpha-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-alpha-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120-139) and p23 (aa 220-239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-alpha-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-gamma secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.

Amino Acid Sequence↗

[Amino acid composition of rice grain proteins].

The composition of the major reserve proteins of rice grain--globulins, prolamines and glutelins--was examined in four rice varieties (Dubovsky 129, Kuban 3, Alakul, Ushtobinsky). Globulins proved to be most heterogeneous whereas glutelins appeared to be least heterogeneous. In regards to the ratio of components globulins showed high variability and glutelins displayed high stability.

Amino Acids↗

[Celiac disease. Recent findings on its pathogenesis, diagnosis and clinical presentation].

Coeliac disease is a gluten-sensitive enteropathy which results in a permanent malabsorption of nutrients in that portion of the small intestine (the jejunum) that is damaged. A genetic, inheritable disease, it is directly related to ingestion of certain wheat proteins especially found in rye secalins, barley hordeins and, in a much lower amount, oat avenins. A fundamental role in the pathological response is played by grain prolamins (gliadins). The actual damage to intestinal mucosa is almost certainly mediated by the immune system but its mechanism has not been so far clarified. Coeliac disease incidence rate is ever increasing among children and adolescents and it is rather frequently reported as relapsing in the third and fourth decade. The most distressing problems of malabsorption syndrome are diarrhea, weight loss, meteorism, abdominal pain, vomiting and asthenia; nonetheless, not all patients report symptoms. Both diagnosis and differential diagnosis--intestinal lymphoma, refractory sprue--prove difficult: a diagnosis of gluten intolerance can be made through careful consideration of a series of laboratory findings which are being improved by researchers in order to avoid delays for patients with probable gluten-sensitive enteropathy with non-specific symptoms. Although there may be many clinical signs and laboratory tests indicating probable malabsorption, the likely gold standard of diagnosing coeliac disease remains to be the jejunal biopsy.

Autoimmune Diseases↗