OBJECTIVE

Celiac disease (CD) prevalence is higher in women with infertility. Our study aims were to evaluate the prevalence of undiagnosed CD in Arab infertile women and to explore the usefulness of using more than one serological marker in the diagnostic screening for CD in this population.

METHODS

Women with unexplained infertility (n = 192) and age-matched healthy controls (n = 210) were prospectively enrolled. Serum was tested for human tissue transglutaminase antibodies (TTG), antiendomysial antibodies (EMA), and immunoglobulin A. Intestinal biopsy was offered to women with positive serology or immunoglobulin A (IgA) deficiency.

RESULTS

CD was diagnosed in five infertile women (2.65%) and in one control (0.5%) (p = 0.11). Gastrointestinal complaints were present in 60% (three of five) of women with CD and 11.8% (22 of 187) of women without CD (p = 0.017). Anemia was reported in 80% of infertile women with CD and 4.8% of infertile women without CD (p = 0.0001).

CONCLUSIONS

Undiagnosed CD is prevalent in Arab infertile women as well as in Arab women in general. CD in Arab infertile women is frequently associated with gastrointestinal complaints and anemia. EMA testing is sufficient in suspected cases.

As many as one in every 100 to 200 persons in the United States has celiac disease, a condition resulting from an inappropriate immune response to the dietary protein gluten. The manifestations of celiac disease range from no symptoms to overt malabsorption with involvement of multiple organ systems and an increased risk of some malignancies. When celiac disease is suspected, initial testing for serum immunoglobulin A (IgA) tissue transglutaminase (tTG) antibodies is useful because it offers adequate sensitivity and specificity at a reasonable cost. A positive IgA tTG result should prompt small bowel biopsy with at least four tissue samples to confirm the diagnosis. However, 3 percent of patients with celiac disease have IgA deficiency. Therefore, if the serum IgA tTG result is negative but clinical suspicion for the disease is high, a serum total IgA level may be considered. Screening of asymptomatic patients is not recommended. The basis of treatment for celiac disease is adherence to a gluten-free diet, which may eliminate symptoms within a few months. Patients should also be evaluated for osteoporosis, thyroid dysfunction, and deficiencies in folic acid, vitamin B12, fat-soluble vitamins, and iron, and treated appropriately. Serum IgA tTG levels typically decrease as patients maintain a gluten-free diet.

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen and contains double stranded DNA genome with approximately 230 kbp. Because of its huge size, comparative genomic studies of HCMV genome have been limited. In this study it was attempted to obtain and analyze the full genome sequence from clinical isolate from Korea. The strain JHC was isolated from Korean patient undergoing bone marrow transplantation who exhibited resistance to ganciclovir treatment (Lee et al., 2005). The virus was plaque-purified, and the full genome sequence was determined by pyrosequencing technique. The JHC genome was found to contain 235,476 bp and 165 open reading frames (ORFs). Comparison with the full genome nucleotide sequences of 11 other HCMV strains suggest that JHC is not closely related with any other strains at genome level. As expected, JHC lacked IRL sequences found in lab-adapted AD169-varUK strain and this region was replaced by ORFs UL133-UL150 as in other clinical isolates. Two ORFs (UL1 and UL119) of the strain JHC were found to be truncated due to early stop codons, and RL6 contains an unusual start codon TTG. The strain JHC contains all the genetic information for micro RNAs known to be present in HCMV.

BACKGROUND

We measured anti-transglutaminase (anti-tTG) antibody in the culture medium of intestinal biopsy specimens from patients with suspected celiac disease (CD) and evaluated the relationship between antibody production and severity of intestinal mucosal damage.

METHODS

We performed diagnostic testing for CD on 273 consecutive patients. In addition to routine histologic evaluation of duodenal biopsy specimens, we assayed anti-tTG antibodies in serum and in the culture medium of duodenal biopsy specimens.

RESULTS

CD was diagnosed in 191 of the 273 patients. Sensitivity and specificity of the serum anti-endomysium (EmA) and anti-tTG assays were 83% and 85% and 99% and 95%, respectively, and both had 88% diagnostic accuracy. EmA and anti-tTG assayed in the culture medium had 98% sensitivity, 100% specificity, and 98% diagnostic accuracy (vs serum assays; P <0.0001). Twenty-nine CD patient specimens (16%) were negative for serum anti-tTG and EmA; for 24 of these patients, anti-tTG assay of the culture medium was positive. The CD patients whose biopsy specimens were positive for serum antibodies showed the following intestinal histologies: total villous atrophy, 35%; severe villous atrophy, 25%; mild atrophy, 25%; villi with no atrophy but with increased intraepithelial lymphocytes, 15%. None of the CD patients whose specimens were negative for serum antibodies showed total or severe villous atrophy; 77% had mild villous atrophy, and 23% had no villous atrophy but had increased intraepithelial lymphocyte counts. Mild villous atrophy was also seen in specimens from approximately 15% of patients without CD.

CONCLUSIONS

Anti-tTG assay of the culture medium of biopsy specimens can improve the accuracy of CD diagnosis in patients negative for serum antibodies.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the transamidation of specific polypeptide-bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium-activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine-5'-O-(3-thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH-SY5Y cells with 1 nM maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an approximately 50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.

Celiac disease (CD) is defined as a permanent intolerance to ingested wheat gliadins and other cereal prolamins, occurring in genetically susceptible people. Persistent elevation of serum aminotransferase activity is expression of liver damage related to CD, which occurs in two distinctive forms. The most frequent is a mild asymptomatic liver injury, with a moderate increase of serum aminotransferase activities and a mild inflammatory portal and lobular infiltrate on liver biopsy (celiac hepatitis), reversible on a gluten-free diet (GFD). More rarely, severe and progressive inflammatory liver damage, induced by an autoimmune process and identified as autoimmune hepatitis (AIH), can develop and it is generally unaffected by gluten withdrawal. Surveys that included only pediatric patients report a wide range of prevalence of CD in AIH of 11.5-46% (mean 21.5%). CD and AIH share selected combinations of genes coding for class II human leukocyte antigens, which could explain their coexistence. Increased intestinal permeability and circulation of anti-tissue transglutaminase (tTG) have also been considered as further potential causes of liver damage in CD patients. tTG in the liver and in other extraintestinal tissues could modify other external- or self-antigens and generate different neo-antigens, which are responsible for liver injury in patients with CD. Patients with AIH represent a population at high risk for developing CD; screening for CD should be integrated into the diagnostic routine of all patients with AIH, with or without gastrointestinal manifestations, before starting immunosuppressive treatments. The only currently available treatment for CD is the GFD and the supportive nutritional care for iron, calcium, and vitamin deficiencies. Due to the difficulties of a GFD, in the past decade researchers have become increasingly interested in therapeutic alternatives to continuous or intermittent use of a GFD in patients with CD. Interventions addressed to correct the defect in the intestinal barrier are currently at the most advanced stage of clinical trials. The impact of a GFD on the outcome of AIH is not clear but it seems to be ineffective in the treatment of AIH. The early detection and treatment of CD, however, may prevent progression to end-stage liver failure.

In the present study, we sequenced the complete mt genome (14,022 bp) of parasitic nematode Contracaecum rudolphii B and its structure and organization compared with Anisakis simplex s.l. The mt genome of C. rudolphii B is slightly longer than that of A. simplex s.l. (13,916 bp). C. rudolphii B mt genome is circular, and consists of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA. This genome contains a high A+T (70.5%) content. The mt gene order for C. rudolphii B is the same as those for A. simplex s.l., but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of C. rudolphii B are TTG and ATT. Six protein-coding genes use TAA as a stop codon whereas five genes use T and one genes use TAG as a termination codon. This pattern of codon usage reflects the strong bias for A and T in the mt genome of C. rudolphii B. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (Bayes, ML and MP), all revealed distinct groups with high statistical support, indicating that C. rudolphii B and A. simplex s.l. is distinct but closely related species. These data provide additional novel mtDNA markers for studying the molecular epidemiology and population genetics of the C. rudolphii B, and should have implications for the molecular diagnosis, prevention and control of anisakidosis in humans and animals.

OBJECTIVE

Celiac disease is increasingly recognized worldwide, but guidelines on how to detect the condition and diagnose patients are unclear. In this study the prevalence and predictors of celiac disease were prospectively determined in a cross-sectional sample of Lebanese patients undergoing esophagogastroduodenoscopy (EGD).

METHODS

Consecutive consenting patients (n = 999) undergoing EGD answered a questionnaire and had blood taken for serologic testing. Endoscopic markers for celiac disease were documented and duodenal biopsies were obtained. The diagnosis of celiac disease was based on abnormal duodenal histology and positive serology. Risk factors were used to classify patients to either high or low risk for celiac disease. Independent predictors of celiac disease were derived via multivariate logistic regression.

RESULTS

Villous atrophy (Marsh 3) and celiac disease were present in 1.8 % and 1.5 % of patients, respectively. Most were missed on clinical and endoscopic grounds. The sensitivity of tissue transglutaminase (tTG) testing for the diagnosis of villous atrophy and celiac disease was 72.2 % and 86.7 %, respectively. The positive predictive value of the deamidated gliadin peptide (DGP) test was 34.2 % and that of a strongly positive tTG was 80 %. While the strongest predictor of celiac disease was a positive tTG (odds ratio [OR] 131.7, 95 % confidence interval [CI] 29.0 - 598.6), endoscopic features of villous atrophy (OR 64.8, 95 %CI 10.7 - 391.3), history of eczema (OR 4.6, 95 %CI 0.8 - 28.8), anemia (OR 6.7, 95 %CI 1.2 - 38.4), and being Shiite (OR 5.4, 95 %CI 1.1 - 26.6) significantly predicted celiac disease. A strategy of biopsying the duodenum based on independent predictors had a sensitivity of 93 % - 100 % for the diagnosis of celiac disease, with an acceptable (22 % - 26 %) rate of performing unnecessary biopsies. A strategy that excluded pre-EGD serology produced a sensitivity of 93 % - 94 % and an unnecessary biopsy rate of 52 %.

CONCLUSIONS

An approach based solely on standard clinical suspicion and endoscopic findings is associated with a significant miss rate for celiac disease. A strategy to biopsy based on the derived celiac disease prediction models using easily obtained information prior to or during endoscopy, maximized the diagnosis while minimizing unnecessary biopsies.

Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.

OBJECTIVE

: Several serologic assays are commercially available to aid in the diagnosis of gluten-sensitive enteropathy (GSE). Our objective in this study was to assess the performance of a novel combined antigen-screening assay for GSE.

METHODS

: Deidentified sera from 111 pediatric patients suspected of having celiac disease (CD), 130 adults diagnosed with dermatitis herpetiformis (DH), and 77 pediatric and 49 adult normal controls were included in the study. Sera from 10 patients submitted to our laboratory for GSE testing with IgA deficiency and IgG antibodies against 1 or more of the traditional serologic markers associated with GSE were also included. All sera were screened for antibodies (IgA and IgG) against tissue transglutaminase (tTG) and deamidated gliadin peptides (DGP) by enzyme immunoassay (EIA) in a single test well. In addition, all sera were assessed for each individual marker and isotype using separate EIAs.

RESULTS

: The IgA/IgG anti-tTG/DGP EIA screen was 92.6% sensitive and 94.3% specific in pediatric CD and detected 1 patient (Marsh 3c) who was IgA anti-tTG negative; this patient was not IgA deficient (<7.0 mg/dL). All 10 IgA-deficient sera gave positive results by the tTG/DGP EIA screen. Sensitivity and specificity of the tTG/DGP EIA screen in retrospective and prospective DH were 65% and 100% versus 62% and 100%, respectively.

CONCLUSIONS

: The new IgA/IgG anti-tTG/DGP EIA screen was slightly more sensitive than IgA anti-tTG alone in pediatric CD. This novel screening assay may allow the current recommendation of measuring total serum IgA in suspected GSE patients to be eliminated.

BACKGROUND

Tissue transglutaminase enzyme-linked immunosorbent assay (tTG-ELISA) has recently been proposed as a simple and fast screening test for celiac disease (CD). The rate of false-positive and false-negative tests with tTG-ELISA, however, has not been definitively established. Therefore, the aim of our study was to investigate anti-tTG antibodies (TGA) not only in untreated patients with CD and in healthy controls, but also in a large group of patients with other autoimmune diseases.

METHODS

The presence of TGA was investigated in sera from 111 patients with untreated CD, 96 patients with other autoimmune conditions (28 with autoimmune liver disease, 46 with insulin-dependent diabetes mellitus, 10 with inflammatory bowel syndrome, 12 with type 1 polyglandular syndrome) and from 100 healthy controls using guinea pig tTG-ELISA (gp-TG/ELISA) and highly purified recombinant human tTG-ELISA (h-TG/ELISA). Western blotting with guinea pig tTG was also performed.

RESULTS

Ninety-four patients with CD who tested positive for antiendomysial antibodies (AEA) and one who tested negative for AEA showed antibodies against the gp-TG. Among the controls, 50% of patients with autoimmune liver disease and 6.5% of patients with insulin-dependent diabetes mellitus tested positive with gp-TG/ELISA. Western blotting experiments revealed that the high rate of positive tests observed using ELISA among the control group sera is attributable to impurities in the gp-TG preparation. However, h-TG/ELISA tests were positive for the sera from all patients who tested positive for AEA and from one control who tested negative for AEA, whereas h-TG/ELISA tests were negative for all CD patients who tested negative for AEA and for other controls who tested negative for AEA.

CONCLUSIONS

The frequency of false-negative and false-positive tests represents the major limit to the use of gp-tTG/ELISA. However, because h-TG/ELISA is both simple and fast, it could be used in large screening programs for CD.

We have cloned the entire mitochondrial genome of Locusta migratoria in four fragments and characterised by restriction mapping. In addition, we have sequenced a 1,095 kb region containing the ND-1 (URF-1) gene. The inferred primary structure of the protein is highly homologous to its Drosophila counterpart (68%). The gene is flanked at the 5' end by the tRNA(CUNleu) gene, interrupted by the sequence TTG. The 3' end is flanked by the tRNA(UCNser) gene, followed by a sequence homologous to the 3' end of D. yakuba cytochrome b. The relative position of the genes is conserved between Locusta and Drosophila, thus indicating conservation of mitochondrial gene order in insects.

This study concerns the role of apoptosis in the growth of human neuroblastomas transplanted into immunodeficient SCID mice. Human neuroblastoma cell lines may consist of one or more distinct phenotypes including the neural 'N-type' and flat substrate-adherent 'S-type'. A differential phenotype-specific proliferation was apparent among S- and N-type cell clones transplanted into SCID mice when compared with the wild-type SK-N-BE(2) cell line. This differential growth capacity of the tumours was correlated with spontaneous apoptosis. Another SK-N-BE(2)-derived cell line (TGA), displaying high levels of apoptosis upon stable transfection with the full length 'tissue' transglutaminase (tTG) cDNA, was unable to induce tumour development when xenografted into SCID mice. To support these observations, the expression of apoptosis-related genes (i.e., bcl-2, p53, and tTG) in the various neuroblastomas was also investigated.

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme catalyzing the formation of covalent crosslinks between peptide-bound glutamine and lysine residues. Lens crystallins, including alphaB-crystallin and several beta-crystallins, are in vitro substrates for tTG. In both human and bovine fetal lens extracts treated with commercially available guinea pig liver tTG we detected the formation of high molecular weight (HMW) aggregates containing crosslinked betaB(2)- and betaA(3)-crystallin. More interestingly, 2D-gel electrophoresis combined with mass spectrometry analysis revealed that glutamines present in the N-terminal arms of betaB(2)- and betaB(3)-crystallins deamidate readily in the presence of tTG. We found that both tTG-catalyzed crosslinking and deamidation disrupt the beta-crystallin complex, suggesting that these tTG-catalyzed modifications can influence the macromolecular assembly of lens crystallins. These data together suggest that tTG can contribute to the age-related deamidation of glutamine residues of lens crystallins.

We investigated the presence of IgA anti-tissue transglutaminase (tTG) antibodies in untreated coeliac disease (CD) and other gastrointestinal diseases, and compared IgA tTG concentrations with anti-endomysium (EMA) immunofluorescent findings. The study included 116 untreated CD patients (74 female, 42 male, age range 15-78 years, median 47 years), 82 treated CD patients, 65 patients with normal duodenal histology, 260 disease control samples and 29 healthy volunteers. IgA anti-tTG, EMA, and anti-gliadin (AGA) antibodies were measured. Serum total IgA was measured in the CD patients. Two IgA-deficient untreated CD patients were excluded. IgA EMA and IgA AGA were positive in 99 (87%) and 69 (61%), respectively, of the 114 untreated CD patients. Elevated IgA anti-tTG were found in 92/114 (81%) untreated coeliacs, 1/82 (1%) treated coeliacs, 2/65 (3%) non-coeliacs, 10/260 (4%) disease controls and 2/29 (7%) volunteers. Four of the untreated CD patients, with a normal serum total IgA concentration, were negative for all the serological tests. IgA anti-tTG concentrations were significantly higher in untreated coeliacs (median 10200 units/ml) than in other groups (Mann-Whitney, p<0.00001) and compared well with IgA EMA titres (r(2)=0.54; p<0.0001).

BACKGROUND

Tissue transglutaminase has recently been identified as the main autoantigen recognized by antiendomysial antibodies in celiac disease. Serum immunoglobulin (Ig)A antibodies to tissue transglutaminase (tTG-ab) determined by an enzyme-linked immunosorbent assay (ELISA) technique have been reported to correlate closely with IgA antiendomysial antibodies (EMA). The purpose of this study was to assess the sensitivity, specificity, and predictive value of tTG-ab measured by a commercially available ELISA technique, compared with those of EMA and IgA antigliadin antibodies (AGA) for the diagnosis of celiac disease.

METHODS

Twenty-seven serum samples were obtained from patients with untreated celiac disease, 37 from patients who had had gluten withdrawn from their diets for varying time spans, and 34 from control subjects without celiac disease. All were younger than 14 years. Presence of tTG-ab and AGA was determined by ELISA and of EMA by indirect immunofluorescence.

RESULTS

Twenty-six of 27 serum samples obtained from patients at the time of diagnosis of celiac disease were AGA positive. All 27 (concordance rate 100%) were positive for EMA and tTG-ab. Of the 34 control subjects, 1 was for AGA and 2 for tTG-ab. All 34 were negative for EMA. Sensitivity, specificity, positive predictive value, and negative predictive value within this group were, for tTG-ab: 100%, 94%, 93%, and 100%, respectively; for EMA: all four indexes were 100%; and for AGA: 96%, 97%, 96%, and 97%, respectively. Of the 37 with treated celiac disease, 2 were AGA positive, 9 were EMA positive, and 6 were tTG-ab positive. The concordance rate between EMA and tTG-ab was 100% in the group with untreated celiac disease, 94% in the control subjects, and 76% in the group with treated celiac disease.

CONCLUSIONS

Immunoglobulin A antibodies to tissue transglutaminase are new, highly sensitive, and specific markers of celiac disease. They can be determined easily by an accurate, comparatively cheap technique and thereby may advantageously replace the EMA marker traditionally used.

Celiac disease is an autoimmune illness characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals. The immune response is based on both cellular and humoral components, although the former seem to be more important in the pathogenesis. The autoantibody response is directed at the enzyme tissue transglutaminase, tTG or TG2, which possibly play a role in the onset of the disease. In this study we sought to develop an animal model in which to analyze the immunological regulation and significance of anti-TG2 antibodies, by expressing specific human single-chain antibody fragments in mice using adeno-associated virus vectors. Upon vector injection in the skeletal muscles, high and persistent systemic levels of anti-TG2 antibodies were obtained. Mice injected with vectors encoding antibodies also recognizing rodent TG2, also developed a strong anti-idiotypic response. This finding raises the question of whether an anti-idiotypic response to anti-TG2 antibodies is a factor associated with celiac disease.

OBJECTIVE

Immune system of some autistic patients could be abnormally triggered by gluten/casein assumption. The prevalence of antibodies to gliadin and milk proteins in autistic children with paired/impaired intestinal permeability and under dietary regimen either regular or restricted is reported.

METHODS

162 ASDs and 44 healthy children were investigated for intestinal permeability, tissue-transglutaminase (tTG), anti-endomysium antibodies (EMA)-IgA, and total mucosal IgA to exclude celiac disease; HLA-DQ2/-DQ8 haplotypes; total systemic antibodies (IgA, IgG, and IgE); specific systemic antibodies: α-gliadin (AGA-IgA and IgG), deamidated-gliadin-peptide (DGP-IgA and IgG), total specific gliadin IgG (all fractions: α, β, γ, and ω), β-lactoglobulin IgG, α-lactalbumin IgG, casein IgG; and milk IgE, casein IgE, gluten IgE,-lactoglobulin IgE, and α-lactalbumin IgE.

RESULTS

AGA-IgG and DPG-IgG titers resulted to be higher in ASDs compared to controls and are only partially influenced by diet regimen. Casein IgG titers resulted to be more frequently and significantly higher in ASDs than in controls. Intestinal permeability was increased in 25.6% of ASDs compared to 2.3% of healthy children. Systemic antibodies production was not influenced by paired/impaired intestinal permeability.

CONCLUSIONS

Immune system of a subgroup of ASDs is triggered by gluten and casein; this could be related either to AGA, DPG, and Casein IgG elevated production or to impaired intestinal barrier function.

Human alpha(2)-antiplasmin (alpha(2)AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in alpha(2)AP, only Gln(2) is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (k(cat)/K(m)((app))) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type alpha(2)AP (WT-alpha(2)AP) and Q2A mutant alpha(2)AP (Q2A-alpha(2)AP). [(14)C]Methylamine incorporation showed the k(cat)/K(m)((app)) of FXIIIa to be 3-fold greater than that of tTG for WT-alpha(2)AP. With FXIIIa or tTG catalysis, [(14)C]methylamine was incorporated into Q2A-alpha(2)AP, indicating that WT-alpha(2)AP has more than one Gln cross-linking site. To identify transglutaminase-reactive sites in WT-alpha(2)AP or Q2A-alpha(2)AP, each was labeled with 5-(biotinamido)pentylamine by FXIIIa or tTG catalysis. After each labeled alpha(2)AP was digested by trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gln residues were labeled with the following reactivities: Gln(2)>> Gln(21)>> Gln(419)>> Gln(447). Q(2)A-alpha(2)AP was also labeled at Gln(21)>> Gln(419)>> Gln(447), but became cross-linked to fibrin by FXIIIa or tTG at approximately one-tenth the rate for WT-alpha(2)AP. These results show that alpha(2)AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gln substrate sites in alpha(2)AP and yields the same order of reactivities.

Nitric oxide (NO) is an endogenous second messenger which acts as a potent vasodilator, anti-inflammatory, anti-thrombotic and pro-angiogenic agent in the vasculature. Recent studies revealed that the effects of NO on blood vessels are mediated in part by its ability to regulate protein trafficking machinery and vesicle-based exocytosis in vascular cells. Specifically, NO-dependent S-nitrosylation of N-ethylmaleimide sensitive factor (NSF), an ATPase that enables membrane fusion, was shown to inhibit exocytosis of vesicular secretory compartments such as endothelial Weibel-Palade bodies, platelet alpha granules and cytolytic granules from activated lymphocytes. Tissue transglutaminase (tTG or TG2) is a multifunctional protein synthesized and secreted by various cell types in the vasculature, which is involved in multiple vascular diseases, including atherosclerosis, vascular calcification and age-dependent aortic stiffening. Our recent findings indicate that tTG is delivered to the cell surface and the extracellular matrix (ECM) via a non-classical ER/Golgi-independent secretion pathway, which depends on the recycling endosomes and the NSF function. Here we report that NO attenuates the unconventional secretion of tTG in human aortic endothelial cells. NO-dependent downregulation of extracellular tTG levels via inhibition of its secretion might be a part of general physiological mechanism which limits externalization of adhesive, pro-inflammatory and thrombogenic proteins in the vasculature.

TRANSPARENT TESTA GLABRA (<em>TTG</em>) proteins that contain the WD40 protein interaction domain are implicated in many signalling pathways in plants. The salicylic acid (SA) signalling pathway regulates the resistance of plants to pathogens through defence responses involving pathogenesis-related (PR) gene transcription, activated by the NPR1 (nonexpresser of PR genes 1) protein, which contains WD40-binding domains. We report that tobacco (Nicotiana tabacum) Nt<em>TTG</em>2 suppresses the resistance to viral and bacterial pathogens by repressing the nuclear localisation of NPR1 and SA/NPR1-regulated defence in plants. Prevention of Nt<em>TTG</em>2 protein production by silencing of the Nt<em>TTG</em>2 gene resulted in the enhancement of resistance and PR gene expression, but Nt<em>TTG</em>2 overexpression or Nt<em>TTG</em>2 protein overproduction caused the opposite effects. Concurrent Nt<em>TTG</em>2 and NPR1 gene silencing or Nt<em>TTG</em>2 silencing in the absence of SA accumulation compensated for the compromised defence as a result of the NPR1 single-gene silencing or the absence of SA. However, Nt<em>TTG</em>2 did not interact with NPR1 but was able to modulate the subcellular localisation of the NPR1 protein. In the absence of Nt<em>TTG</em>2 production NPR1 was found predominantly in the nucleus and the PR genes were expressed. By contrast, when Nt<em>TTG</em>2 accumulated in transgenic plants, a large proportion of NPR1 was retained in the cytoplasm and the PR genes were not expressed. These results suggest that Nt<em>TTG</em>2 represses SA/NPR1-regulated defence by sequestering NPR1 from the nucleus and the transcriptional activation of the defence-response genes.

Coeliac disease (CD) is an enteropathy induced in genetically susceptible individuals by gluten components, gliadin, hordein and secalin, polypeptides present in cereals such as wheat, barley and rye, respectively. Although the disease starts as intolerance to gliadins, antibodies to tissue transglutaminase (tTG) in the gut epithelium are characteristic of the disease. Whereas serum autoantibodies against tTG (tTGA) are highly specific for CD, antibodies to gliadin are less informative as they can also be detected in other enteropathies, and even in healthy individuals. However, it was shown recently that antibodies to certain gliadin peptides occur with high specificity in CD patient sera. We developed a solid phase lanthanide-based immunofluorometric assay for simultaneous detection of serum IgA and IgG antibodies to a synthetic peptide derived from gamma gliadin of wheat comprising amino acids 86-103. Three glutamine residues of this native 18-mer peptide were replaced by glutamic acids and the peptide was biotinylated. Sera from 87 individuals who had undergone duodenal biopsy and were diagnosed with CD and from 81 healthy individuals were analysed for the presence of both IgA and IgG anti-gliadin peptide antibodies. The performance of the peptide AGA assay was excellent, showing a specificity and sensitivity of 90% and 92% for IgA, and 98% and 75% for IgG, respectively. The corresponding values for conventional anti-gliadin antibody (AGA) enzyme-linked immunosorbent assay (ELISA) tests were 72% specificity and 87% sensitivity for IgA, and 64% specificity and 78% sensitivity for IgG. In a prospective study, almost all the tTGA-positive sera drawn from children who later developed CD were also positive for gliadin peptide antibodies.

BACKGROUND

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease in the western population and expanding disease in the world. Pathological changes in fatty liver are like alcohol liver damage, which can lead to end-stage liver disease. The prevalence of NAFLD in obese or overweight people is higher than general population, and it seems that people with high Body Mass Index (BMI) or abnormality in some laboratory tests are more susceptible for severe fatty liver and high grade of NAFLD in ultrasonography (U.S).

OBJECTIVE

This study aimed to evaluate the correlation of BMI and laboratory tests with NAFLD in ultrasonography.

METHODS

During a multi-step process, we selected two-hundred and thirteen cases from four hundred and eighteen patients with NAFLD. Laboratory tests performed included: ALT, AST, FBS, Triglyceride and cholesterol levels, hepatitis B surface antigen, hepatitis C antibody, ceruloplasmin, serum iron, TIBC, transferrin saturation, ferritin, AMA, ANA, ANTI LKM1, serum protein electrophoresis, TSH, anti TTG (IgA). BMI and ultrasonography for 213 patients were performed, and then data was analyzed. These parameters and grades of ultrasonography were compared with the values obtained using one way ANOVA. An ordinal logistic regression model was used to estimate the probability of ultrasonography grade. The Statistical Package for the Social Science program (SPSS, version 16.0) was used for data analysis.

RESULTS

Two-hundred and thirteen cases including 140 male and 73 female, were studied. In general, 72.3% of patients were overweight and obese. Post-hoc tests showed that only BMI (P < 0.001) and TG (P < 0.011) among variables had statistically significant associations with ultrasonography grade (USG), and ordinal logistic regression model showed that BMI and AST were the best predictors.

CONCLUSIONS

Our results suggest that in patients with NAFLD, BMI and TG are most effective factors in severity of fatty liver disease and ultrasonography grade (USG). On the other hand, BMI as a predictor can be helpful. But, AST has not been a reliable finding, because it changes in many conditions.

BACKGROUND

The phytohormone auxin mediates a stunning array of plant development through the functions of AUXIN RESPONSE FACTORs (ARFs), which belong to transcription factors and are present as a protein family comprising 10-43 members so far identified in different plant species. Plant development is also subject to regulation by TRANSPARENT TESTA GLABRA (<em>TTG</em>) proteins, such as Nt<em>TTG</em>2 that we recently characterized in tobacco Nicotiana tabacum. To find the functional linkage between <em>TTG</em> and auxin in the regulation of plant development, we performed de novo assembly of the tobacco transcriptome to identify candidates of Nt<em>TTG</em>2-regulated ARF genes.

RESULTS

The role of Nt<em>TTG</em>2 in tobacco growth and development was studied by analyzing the biological effects of gene silencing and overexpression. The Nt<em>TTG</em>2 gene silencing causes repressive effects on vegetative growth, floral anthocyanin synthesis, flower colorization, and seed production. By contrast, the plant growth and development processes are promoted by Nt<em>TTG</em>2 overexpression. The growth/developmental function of Nt<em>TTG</em>2 associates with differential expression of putative ARF genes identified by de novo assembly of the tobacco transcriptome. The transcriptome contains a total of 54,906 unigenes, including 30,124 unigenes (54.86%) with annotated functions and at least 8,024 unigenes (14.61%) assigned to plant growth and development. The transcriptome also contains 455 unigenes (0.83%) related to auxin responses, including 40 putative ARF genes. Based on quantitative analyses, the expression of the putative genes is either promoted or inhibited by Nt<em>TTG</em>2.

CONCLUSIONS

The biological effects of the Nt<em>TTG</em>2 gene silencing and overexpression suggest that Nt<em>TTG</em>2 is an essential regulator of growth and development in tobacco. The effects of the altered Nt<em>TTG</em>2 expression on expression levels of putative ARF genes identified in the transcriptome suggest that Nt<em>TTG</em>2 functions in relation to ARF transcription factors.