Members of the human TTG/RBTN family, now renamed 'LMO' for LIM-only proteins, encode proteins with two tandem copies of a LIM motif. There are three members of this family, two have been isolated at the sites of chromosomal translocations in T-cell leukaemia. The function of the LIM motifs is at present unknown. We found that the LMO-2 gene is highly conserved between mammals, Drosophila and yeast. As a first step to obtain a model system for studying the function of the LIM motifs we have isolated the Drosophila homologue Dlmo. In contrast to mammals Drosophila appears to have only one lmo gene. A 2087 bp cDNA clone was isolated from a larval cDNA library, encoding a protein of 266 amino acids. A second transcript with an alternative 5' end was identified in RNA from embryos. The Drosophila lmo protein consists of two tandem copies of the conserved LIM domain characteristic of the human LMO family and an extended amino and carboxy terminus, which is not present in the human proteins. The amino acid sequence similarity with human LMO-1 and LMO-2 in LIM 1 is 79% and 69% and in LIM-2 90% and 60%, respectively. In addition a short stretch of 25 nucleotides with a homology of 83% between LMO-2 and Dlmo is found in the 3' UTR. Dlmo, like LMO-1, has an intron after the second LIM encoding region, which is not present in LMO-2. It is expressed maternally and at a high level in early embryogenesis as well as in adults. Interestingly we observed that the Dlmo protein is immunologically related to LMO-2 and can be detected by immunohistochemistry in early cellular blastoderm embryos. The gene was localised to a genetically well characterized region (17C on the X chromosome) opening the way for identification of mutations.

Several in vivo and in vitro experimental model systems demonstrate a direct relationship between the expression and activity of tissue transglutaminase [tTG; also called transglutaminase type 2 (TGase 2)] and programmed cell death or apoptosis. This is based on mRNA and protein studies, sense and antisense transfection, identification of N epsilon-(gamma-glutamyl)-lysine cross-links in extracted apoptotic bodies, and in blue mouse experiments. In the epidermis, apoptosis occurs under particular conditions in the proliferative basal layer with the involvement of the tTG enzyme. However, in epidermal keratinocytes other TGases (TGase 1, TGase 3, and perhaps TGase X) are normally activated in a terminal differentiation program, called cornification, that leads to cell death. These cells perform their functions after death, providing an elastic physical and permeability barrier to the skin. In fact, TGase 1 mutations cause the skin disease lamellar ichthyosis. Because all TGases share strong similarities in structure and function, being involved in mechanisms of cell death, this chapter describes the current assays for TGases at the mRNA, protein, and enzymatic levels. We also describe procedures to produce, purify, and characterize recombinant TGases, to identify mutation in disease, to isolate cross-linked bodies, and to analyze the N epsilon-(gamma-glutamyl)-lysine isopeptide cross-links. Finally, we discuss general rules for the interpretation and comparison of these events in cell death.

BACKGROUND

Many studies have shown that the prevalence of celiac disease (CD) is increasing in the patients with irritable bowel syndrome (IBS), but the present evidences are controversy and all of the researches don't suggest evaluation of the patients with IBS for the CD.

METHODS

In the cross-sectional study, 1000 patients that affected by IBS with the predominant feature of diarrhea who referred to the gastroenterology clinic were evaluated during 2009-2012 years. Blood samples were taken from the patients for ELISA of IgA tissue transglutaminase (TTG) Ab. Then biopsy was taken from the second part of duodenum of all of patients by endoscopy and the sample was referred to pathologist for histopathology evaluation in order to confirm diagnosis.

RESULTS

About half of the patients with IBS were women (50.3%) and the mean age ± Standard deviation of the men and women was 29.59±11.41 and 28.42±11.73, respectively. The mean titer of TTG IgA Ab in the women and men affected by IBS was 5.25±17.77 and 7.22±25.4, respectively. Seventy six cases (7.6%) that affected by IBS had high serum level of Ab titer (TTG IgA.Ab≥10) that including 41 women and 35 men. In the patients with high serum level of Ab titer, 57 cases (75%) were affected by celiac disease (based on histopathology report). Therefore, the prevalence of CD was 5.7% among patients with IBS.

CONCLUSIONS

In the current study the incidence of CD was evaluated 5.7 cases per each 100 people with IBS. It's suggested that all of the patients with diarrhea predominant IBS and high serum level of TTG IgA Ab must be examined for evaluation of CD.

The nucleotide sequences of 16S rDNAs (coding for the small subunit ribosomal RNAs) were used to identify Xylella fastidiosa, a nutritionally fastidious plant pathogenic bacterium. The near-complete 16S rDNAs from nine strains of Xyl. fastidiosa, including seven pathotypes and one strain of Xanthomonas campestris pv. campestris, were amplified through PCR with two conserved primers (forward primer 5'-AGA GTT TGA TCC TGG CTC AG-3' and reverse primer 5'-AAG GAG GTG ATC CAG CC-3') and sequenced. The 16S sequences were compared with all eukaryote and prokaryote DNA entries in GenBank database. A Xyl. fastidiosa 16S rDNA sequence, M26601, was determined to be the most similar to all the near-complete (1537 bp) and partial 5' end sequences from Xyl. fastidiosa, but not those from the Xanthomonas strain. A 20-bp oligonucleotide (5'-TTG GTA GTA ATA CCA TGG GT-3') was found to be highly characteristic of Xyl. fastidiosa. Since the 16S rDNA of Xyl. fastidiosa strains are highly homologous and characteristically different from other bacteria, including the most closely related Xanthomonas, 16S rDNA sequences can be used as signature characters to identify this bacterium.

Tissue transglutaminase (tTG) is upregulated in various cells undergoing apoptosis. To investigate the transcriptional regulation of tTG a mouse strain carrying a beta-galactosidase reporter gene under the control of a 3.8 kilobase fragment of the tTG promoter was characterised. The transgene construct was shown to be expressed in the apoptotic regions of the mouse embryo. Here we report that the regulation of the transgene is also apoptosis-linked in adult animals. The transgene is induced in endocrine apoptosis involving mammary gland involution and corpus luteum regression. Induction of the reporter gene is detectable during in vivo but not in vitro apoptosis of thymocytes induced by the glucocorticoid receptor, the nur77, p53 and the retinoid receptor gamma mediated pathways. Additionally, the lacZ expression mimics the activation of the endogenous promoter in tissues characterised by high apoptotic turnover. These results suggest that the apoptosis-specific transcriptional regulation of tTG is mediated through elements of a 3.8 kb promoter and may require cosignals available only in tissue environment. Cell Death and Differentiation (2000) 7, 1225 - 1233.

Tissue transglutaminase (tTG) is a novel G-protein that previous studies showed can couple ligand-bound activated alpha(1B) adrenoreceptors to phospholipase C-delta, resulting in phosphoinositide (PI) hydrolysis. In human neuroblastoma SH-SY5Y cells we found that although endogenous tTG can facilitate alpha(1B) adrenoreceptor-stimulated PI hydrolysis, its contribution is minor compared with the classical heterotrimeric G-protein G(q/11). Further, we show that the alpha(1B) adrenoreceptor recruits tTG to the membrane and that this recruitment is enhanced by agonist occupancy of the receptor. In addition, the effects of tTG on signalling are bimodal. At low expression levels, tTG enhanced alpha(1B) adrenoreceptor-stimulated PI hydrolysis, whereas at higher expression levels tTG attenuated significantly this response. These findings are the first to demonstrate that a protein can both facilitate and attenuate receptor-stimulated PI hydrolysis.

BACKGROUND

Liver transplantation (LT) in cirrhotics is characterized by severe coagulopathy, associated with a well documented heparin-like effect (HLE) seen by thromboelastography (TEG) after reperfusion. The amount of HLE present in patients with acute liver failure (ALF) and its role in their bleeding tendency before LT has not been investigated.

OBJECTIVE

To investigate the presence and extent of HLE in patients with ALF undergoing LT and to compare the extent of HLE in this group with a group of cirrhotics undergoing LT.

METHODS

Ten consecutive ALF and 10 cirrhotic patients undergoing LT were included. TEG (with and without heparinase I), surrogate total thrombin generation (TTG) derived by TEG and haemodynamic variables were recorded for every stage of the LT. HLE was defined as a correction of r+k times on TEG of more than 50% by the addition of heparinase I.

RESULTS

Before incision, patients with ALF showed a significantly greater HLE compared with patients with cirrhosis (r+k time: 66 min corrected to 29 vs 45 min corrected to 32 min, P=0.001). After reperfusion, all the patients showed extensive HLE, without any difference between the two groups. Despite the greater HLE, patients with ALF showed similar TTG compared with the cirrhotic group. By the end of the operation, the extent of the HLE was greatly reduced in both the groups.

CONCLUSIONS

Before transplantation, patients with ALF have a greater HLE than patients with liver cirrhosis. However, this did not affect the thrombin generation calculated by TEG and resolved after transplantation.

Patients with neurological disease of unknown etiology sometimes present with antigliadin and antitissue transglutaminase antibodies. The association between these antibodies and multiple sclerosis has been previously suggested. The purpose of this study was to determine the prevalence of these antibodies in multiple sclerosis patients. We determined the level of serum immunoglobulin A and immunoglobulin G antigliadin and antitissue transglutaminase antibodies in 98 patients with multiple sclerosis. We found a highly significant increase in titers of immunoglobulin G antibodies against gliadin and tissue transglutaminase in the multiple sclerosis patients. Seven patients had a positive IgG AGA, whereas only 2 controls presented positive titers (P = 0.03). Four patients had positive IgG anti-tTG while all the controls tested negative (P = 0.02). However, immunoglobulin A antibodies against gliadin and tissue transglutaminase were not statistically higher in the multiple sclerosis group in comparison to the control group. Our findings support the associations between antibodies against gliadin and tissue transglutaminase to multiple sclerosis. The specific role of these antibodies in the pathogenesis of multiple sclerosis remains uncertain and requires additional research. A gluten free diet should be considered in specific cases of patients who present with gluten antibodies.

OBJECTIVE

To compare the frequency of gluten sensitivity in patients with multiple sclerosis (MS) and healthy controls.

METHODS

The patients were 161 clinically definite MS patients who referred to neurology outpatient clinic of Nemazee Hospital, Shiraz, south of Iran from March 2004 to October 2005. IgG and IgA antigliadin antibodies were measured by enzyme immuno assay (EIA) method. The test of IgA antitranstissue glutaminase (tTG) and duodenal biopsy were carried out in patients with either IgA or IgG AGA positive sera. Antigliadin antibodies were also measured for 166 age and sex matched control group.

RESULTS

Neither IgG nor IgA antigliadin antibodies showed significant differences between MS patients and controls. Anti-tTG antibody and histopathologic studies were negative in all patients with positive IgG or IgA antigliadin antibodies results. Mean values of IgG and IgA antigliadin antibodies in MS patients with different sex, age, course, and functional systems involvement were not significantly different.

CONCLUSIONS

Gluten sensitivity is not associated with MS in Iran.

BACKGROUND

Celiac disease (CD) is an autoimmune disorder, characterized by the presence of gastrointestinal and multisystem symptoms, which occasionally mimic those of Irritable Bowel Syndrome (IBS) and Fibromyalgia Syndrome (FMS). To assess the effectiveness of a Gluten-Free Diet (GFD) in seven adult female screening-detected CD subjects, categorized as severe IBS and FMS patients.

METHODS

All subjects showed villous atrophy in duodenal biopsies, were HLA-DQ2/DQ8-positive, and fulfilled the Rome III and ACR 1990 criteria respectively for IBS and FMS classification. GFD effectiveness was assessed at baseline and after 1 year, examining the score changes in the Tender Points (TPs) test, Fibromyalgia Impact Questionnaire (FIQ), Health Assessment Questionnaire (HAQ), Short Form Health Survey (SF-36), Visual Analogue Scales (VAS) for gastrointestinal complaints, pain and tiredness, drug prescriptions and tissue-Trans-Glutaminase (tTG) serum levels.

RESULTS

At baseline, all patients had poor Quality of Life and VAS scores, a high number of TPs and drug prescriptions, and increased tTG levels. After 1 year of GFD, all outcome measures significantly improved, with a decrease of 51-60% in TPs, FIQ, HAQ, and VAS scales, and in the number of prescribed drugs, accompanied by an increase of 48-60% in SF-36 Physical and Mental Component Summary scores, and a decrease of tTG to normal values.

CONCLUSIONS

Results of this pilot study show that the adherence to a GFD by CD-related IBS/FMS patients can simultaneously improve CD and IBS/FMS symptoms, and indicate the merit of further research on a larger cohort.

BACKGROUND

Irritable bowel syndrome (IBS) and fibromyalgia syndrome (FMS) are two common central sensitization disorders frequently associated in the same patient, and some of these patients with IBS plus FMS (IBS/FMS) could actually be undiagnosed of coeliac disease (CD). The present study was an active case finding for CD in two IBS cohorts, one constituted by IBS/FMS subjects and the other by people with isolated IBS.

METHODS

A total of 104 patients (89.4% females) fulfilling the 1990 ACR criteria for FMS and the Rome III criteria for IBS classification and 125 unrelated age- and sex-matched IBS patients without FMS underwent the following studies: haematological, coagulation and biochemistry tests, serological and genetic markers for CD (i.e., tissue transglutaminase 2 (tTG-2) and major histocompatibility complex HLA-DQ2/HLA-DQ8), multiple gastric and duodenal biopsies, FMS tender points (TPs), Fibromyalgia Impact Questionnaire (FIQ), Health Assessment Questionnaire (HAQ), 36-Item Short Form Health Survey (SF-36) and Visual Analogue Scales (VASs) for tiredness and gastrointestinal complaints.

RESULTS

As a whole, IBS/FMS patients scored much worse in quality of life and VAS scores than those with isolated IBS (P < 0.001). Seven subjects (6.7%) from the IBS/FMS group displayed HLA-DQ2/HLA-DQ8 positivity, high tTG-2 serum levels and duodenal villous atrophy, concordant with CD. Interestingly enough, these seven patients were started on a gluten-free diet (GFD), showing a remarkable improvement in their digestive and systemic symptoms on follow-up.

CONCLUSIONS

The findings of this screening indicate that a non-negligible percentage of IBS/FMS patients are CD patients, whose symptoms can improve and in whom long-term CD-related complications might possibly be prevented with a strict lifelong GFD.

OBJECTIVE

The aim of this study was to evaluate hypothetical screening strategies in a Swedish celiac disease (CD) mass screening.

METHODS

Of 10,041 Swedish sixth graders born in 1993 invited to a population-based CD mass screening, 7208 participated. Anti-tissue transglutaminase (tTG) immunoglobulin (Ig) A were analyzed in all children and total serum IgA (s-IgA) in 7161 children. Additional analyses of tTG-IgG, endomysial antibodies (EMA) IgA and IgG, and human leukocyte antigen (HLA) alleles were performed according to a standardized protocol. Children with elevated levels of serological markers were recommended to undergo a small intestinal biopsy to verify diagnosis, and 153 children with CD were thus identified. Sensitivity, specificity, positive predictive values (PPVs) and negative predictive values (NPVs) were calculated and receiver operating characteristic curves were plotted.

RESULTS

By lowering the cutoff for tTG-IgA, 17 additional cases of CD were identified at the cost of 32 biopsies. All children with tTG-IgA >50 U/mL (10 times the recommended upper limit of normal) had gluten enteropathy. Area under the receiver operating characteristic curve for tTG-IgA was 0.988. All cases carried HLA-DQ2 or HLA-DQ8, as did 53% of the controls. For different hypothetical screening strategies, sensitivity, specificity, PPV, and NPV ranged between 87.6% and 100%, 99.5% and 99.9%, 79.7% and 89.7%, and 99.7% and 100%, respectively. Efforts to increase sensitivity by lowering tTG-IgA cutoff would result in increased number of small intestinal biopsies and lower PPV. Sequential testing for both EMA and HLA-DQ genotyping would reduce the number of negative small intestinal biopsies.

CONCLUSIONS

tTG-IgA is a robust marker when used in CD mass screening and its performance can be enhanced by sequential testing for EMA or HLA-DQ genotyping.

A cyclodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at -108 to -67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (Mr) and enzymatic properties as follows: Mr, 80,000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55 degrees C; and stability below 45 degrees C. The yields of the products from starch as the substrate were 25% for beta- and 5% for gamma-cyclodextrin.

Celiac disease is an intestinal disorder that develops as a result of interplay between genetic and environmental factors. HLA genes along with non-HLA genes predispose to the disease. Linkage studies have failed to identify chromosomal regions other than the HLA region which have major effects, indicating the existence of multiple non-HLA predisposing genes with modest effects. Association studies have shown that CTLA4 or a closely located gene is one of these genes. The primary HLA association in the majority of celiac disease patients is with DQ2 (DQA1*05/DQB1*02) and in the minority of patients with DQ8 (DQA1*0301/DQB1*0302). Gluten reactive CD4+ T cells can be isolated from small intestinal biopsies of celiac patients but not from controls. DQ2 or DQ8, but not other HLA molecules carried by patients, present peptides to these T cells. A number of distinct T cell gluten epitopes exist, most of them posttranslationally modified by deamidation. DQ2 and DQ8 bind the epitopes such that the glutamic acid residues created by deamidation are accommodated in pockets that have a preference for negatively charged side chains. There is evidence that deamidation in vivo is mediated by the enzyme tissue transglutaminase (tTG). Overall, the results point to control of the immune response to gluten by intestinal T cells restricted by the DQ2 or DQ8 molecules. This is likely to be a critical checkpoint for the development of celiac disease and could explain the dominant genetic role of HLA in this disorder. The products of the other predisposing genes may participate in pathway(s) that lead(s) to lesion formation. The minor genetic effects of the non-HLA genes could indicate a lack of critical checkpoints along these pathways, or that there are several pathways leading to the lesion formation.

Tissue transglutaminase (tTG) and keratinocyte transglutaminase (kTG), as well as the cross-linked envelopes (CLE) that they form, have been associated with squamous differentiation and programmed cell death in epithelial cells. When interferon-beta (IFN-beta) was used to stimulate differentiation and programmed cell death in the human lung cancer cell lines NCI-H596 and NCI-H226, the cells underwent a decrease in cellular density. In NCI-H596 IFN-beta caused an increase in kTG activity and DNA fragmentation in the lower density cells, which were significantly slower growing than control cells. However, in the higher density cells, which were only slightly slower growing than control cells, IFN-beta caused an increase in tTG activity and CLE competence. Dual-parameter flow cytometry demonstrated that IFN-beta-induced squamous differentiation preceded programmed cell death. Treatment of NCI-H596 cells with monodansylcadaverine, a transglutaminase inhibitor, prevented the increase in CLE competence, but did not inhibit DNA fragmentation. These results suggest that IFN-beta can induce NCI-H596 cells to enter multiple cell death pathways and that these pathways are not only differentiation related, but may also be growth driven.

Tissue transglutaminase (tTG) is known to participate in multiple cellular processes, including apoptosis, cellular adhesiveness etc. Alterations of tTG expression could contribute to the development of several categories of diseases, including AIDS, cancer etc. The aim of the study was to test the pattern and relevance of tTG expression in a subset of breast carcinomas. RT-PCR has detected tTG-specific RNA message in 11 out of 25 (44%) breast cancer samples. tTG message was detected in 6/8 (75%) breast carcinomas with high apoptotic index, but only in 5/17 (29%) with the low one (p = 0.03). Immunohistochemical analysis revealed that only 15% of breast carcinomas displayed tTG protein in tumor cells, while the staining of the stromal components occurred in approximately one-half of the tumours tested. Surprisingly, there was no significant association between tTG RNA expression and protein positivity. Moreover, there was no evident relationships between tTG immunostaining and apoptotic index or clinical parameters of breast neoplasms. There are at least 2 alternative explanations for the poor concordance between RNA and protein data. It is likely that the sensitivity of immunohistochemistry is not sufficient to detect functionally relevant tTG enzyme in all breast cancer sections. Otherwise, tTG RNA expression does not always lead to accumulation of its product in the tumor cells, but reflects the transcriptional activation of other pro-apoptotic genes due to common triggering mechanisms.

OBJECTIVE

To indirectly determine if tissue transglutaminase (tTG)-specific T cells play a crucial role in the propagation of celiac disease.

METHODS

Anti-deamidated gliadin peptide (DGP) and anti-tTG IgA and IgG were measured in the sera of celiac patients (both untreated and treated). The correlations were determined by Spearman's rank correlation test.

RESULTS

In celiac patients, we found a very significant correlation between the production of DGP IgA and IgG (r = 0.75), indicating a simultaneous and ongoing production of these two isotypes reminiscent of oral vaccination studies. However, there was far less association between the production of tTG IgA and tTG IgG in celiac patients (r = 0.52). While tTG IgA was significantly correlated with DGP IgA (r = 0.80) and DGP IgG (r = 0.67), there was a weak correlation between production of anti-tTG IgG and the production of anti-DGP IgA (r = 0.38) and anti-DGP IgG (r = 0.43).

CONCLUSIONS

These data demonstrate that the production of anti-tTG IgA is directly correlated to the production of anti-DGP IgG and IgA, whereas anti-tTG IgG is only weakly correlated. This result therefore supports the hapten-carrier theory that in well-established celiac patients anti-tTG IgA is produced by a set of B cells that are reacting against the complex of tTG-DGP in the absence of a tTG-specific T cell.

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31-43 (but not peptide 57-68) uptake by cells, thereby impairing the ability of p31-43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31-43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.

OBJECTIVE

To identify the prevalence of gluten sensitivity in a healthy adult Saudi population within a low endemic area of celiac disease using IgA tissue transglutaminase antibody. The study was conducted as a prospective pilot study for Saudi attendees of a blood donation centre at King Faisal Specialist Hospital & Research Centre in Jeddah, Saudi Arabia.

METHODS

Individuals were invited to participate in the study and screened for gluten sensitivity using immunoglobulin A tissue transglutaminase antibody (IgA TTG) along with serum IgA level. Descriptive data was presented and expressed as mean value; correlation between variables was estimated using Pearson correlation, and nonparametric data using Pearson rho correlation (level of P value <0.05 is considered to be statistically significant).

RESULTS

Two hundred and four individuals (122 males and 82 females, mean age 35 years) attending the blood donation centre were screened. Three individuals tested positive for IgA TTG showing normal IgA level (1 female and 2 males) with a 1.5% prevalence in the cohort.

CONCLUSIONS

Positive celiac screening is present at a low prevalence rate in our adult population, in which the individuals' age and their serum IgA levels are not associated with the positivity level. A study on a larger scale with the application of histologic confirmation of positive cases is needed.

OBJECTIVE

To evaluate and compare the sensitivity and specificity of the new serologic marker human antitissue transglutaminase antibodies (IgA anti-tTG) with those of antiendomysium (IgA EMA) and antigliadin antibodies (IgA and IgG AGA) for the diagnosis of celiac disease (CD).

METHODS

The level of IgA antibodies to tTG in serum was determined by an enzyme-linked immunosorbent assay (ELISA) test using recombinant human tTG as the antigen; IgA EMA, by indirect immunofluorescence; and IgA and IgG AGA, by ELISA. Sixty-eight serum samples from 59 patients with CD were studied-30 patients had untreated CD, 22 were on gluten-free diets, and 16 had been reintroduced to gluten-and compared with serum samples from 116 children examined for failure to thrive, short stature, various digestive diseases, or other non-CD conditions.

RESULTS

Twenty-eight of 30 patients with CD had anti-tTG (the 2 patients whose results were negative were 1 patient with IgA deficiency and 1 infant); 27 of 30 patients had IgA EMA (1 child was IgA anti-tTG positive and IgA EMA negative); 18 of 30 had IgA AGA; and 28 of 30 had IgG AGA. On gluten-free diets, 4 of 22 patients had anti-tTG but none had IgA EMA or IgA AGA. On normal diets, 15 of 15 children who had relapsed had anti-tTG; 9, IgA EMA; 4, IgA AGA; and 8, IgG AGA (1 child did not relapse). In subjects without CD, 3 of 116 had anti-tTG; 12, IgG AGA; and 1, IgA AGA, but none had IgA EMA. In the 3 children who had anti-tTG, CD could be excluded. The positive predictive value of IgA anti-tTG was 90% and the negative predictive value, 98%. In comparison, results for IgA EMA were 100% and 97%, IgA AGA 94% and 90%, and IgG AGA 70% and 98%, respectively.

CONCLUSIONS

The presence of human anti-tTG is a reliable indicator for the diagnosis and follow-up of CD.

In this research, it has been aimed to evaluate the improvement effects of alpha lipoic acid (ALA), ascorbic acid-6-palmitate (AA6P), fish oil (FO), and their combination (COM) on some biochemical properties in erythrocytes of streptozotocin (STZ)-induced diabetic male rats. According to experimental results, glutathione (GSH) level in erythrocytes decreased in diabetes (P < 0.01), D + ALA, and D + AA6P groups (P < 0.001). Malonaldehyde (MA) level increased in diabetes (P < 0.05), D + FO, and D + COM groups (P < 0.001), but its level in D + AA6P and D + ALA groups was lower in diabetes group (P < 0.01). Total lipid level in diabetes and diabetes plus antioxidant administered groups were higher than control. Total cholesterol level was high in diabetes and D + ALA groups (P < 0.05), but its level reduced in D + FO compared to control and diabetes groups, P < 0.05, < 0.001, respectively. Total triglyceride (TTG) level was high in the D + ALA (P < 0.05) and D + COM (P < 0.001) groups. In contrast, TTG level in blood of diabetes group was higher than diabetes plus antioxidant and FO administered groups (P < 0.001). According to gas chromatography analysis results, while the palmitic acid raised in diabetes group (P < 0.05), stearic acid in D + FO, D + ALA, and diabetes groups was lower than control (P < 0.05), oleic acid reduced in D + COM and D + FO groups, but its level raised in D + AA6P and D + ALA groups (P < 0.01). As the linoleic acid (LA) elevated in ALA + D, D + AA6P, and diabetes groups, linolenic acid level in diabetes, D + AA6P, and D + FO groups was lower than control (P < 0.001). Arachidonic acid (AA) decreased in D + ALA, D+ AA6P, and diabetes groups (P < 0.01), but its level in D + COM and D + FO was higher than control (P < 0.05). Docosahexaenoic acid (DHA) increased in D + AA6P and D + COM (P < 0.05). While the total saturated fatty acid level raised in diabetes group, its level reduced in D + ALA and D + FO groups (P < 0.05). In contrast, total unsaturated fatty acid level in D + ALA and D + FO groups was higher than control (P < 0.05). In conclusion, present data have confirmed that the combination of the ALA, AA6P, and FO have improvement effects on the recycling of GSSG to reduced GSH in erythrocytes of diabetic rats, and in addition to this, oxidative stress was suppressed by ALA and AA6P, and unsaturated fatty acid degree was raised by the effects of ALA and FO.

Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS- and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTG-catalyzed reaction as the root cause of DLK down-regulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.

OBJECTIVE

Thyroxine-binding globulin (TBG) encoded by the TBG gene on chromosome Xq22 is the major transport protein, carrying approximately 75% of circulating T4. Inherited defects in TBG are associated with three phenotypes based on the level of TBG in serum of affected hemizygous males: complete TBG deficiency (TBG-CD), partial TBG deficiency (TBG-PD) and TBG excess (TBG-E). In this study, we report two unrelated Han Chinese males with complete TBG deficiency who carry different mutations in the TBG gene.

METHODS

Two index cases of Han males who were diagnosed as having TBG deficiency on the basis of undetectable serum TBG and an additional 75 (50 males and 25 females) normal Han Chinese.

METHODS

Serum thyroid hormones were measured by chemiluminescent immunoassay, thyroid autoantibodies by an agglutination test, and TSH receptor antibody and TBG by radioimmunoassay. Genomic DNA extraction, polymerase chain reaction (PCR) and DNA sequence analysis of the TBG gene were performed with standard methods.

RESULTS

One index case had one missense mutation in his copy of the gene, a G ->> A transition in codon 52 that results in the replacement of serine by asparagine, and a known polymorphism in codon 283 (TTG ->> TTt) that results in the replacement of leucine by phenylalanine. The allelic frequency of TBG-Poly allele in 75 normal Han Chinese (100 chromosomes) was 31%. A second index case was hemizygous for a nonsense mutation in codon 280 of exon 3 (TGG ->> TGa). This mutation, located in the C-terminal of TBG, predicts a markedly truncated protein.

CONCLUSIONS

This is the first report of complete thyroxine-binding globulin deficiency (TBG-CDT1 and TBG-CDT2) due to TBG gene mutations in Taiwan.

A new stable mutant of Arabidopsis thaliana with a spotted pigment in the seed coat, named anthocyanin spotted testa (ast), was induced by carbon ion irradiation. The spotted pigmentation of ast mutant was observed in immature seeds from 1-2 days after flowering (DAF), at the integument of the ovule, and spread as the seed coat formed. Anthocyanin accumulation was about 6 times higher in ast mutant than in the wild-type at 6 DAF of the immature seeds, but was almost the same in mature dry seeds. A higher anthocyanin accumulation was not observed in the seedlings, leaves or floral buds of ast mutant compared with the wild-type, which suggests that a high accumulation of anthocyanins is specific to the seed coat of the immature ast seeds. Reciprocal crosses between ast mutant and the wild-type indicated that ast is a single recessive gene mutation and segregates as a delayed inheritance. The results of crossing with tt7 and ttg mutants also confirmed that the AST gene is probably a regulatory locus that controls flavonoid biosynthesis. A mapping analysis revealed that the gene is located on chromosome I and is closely linked to the SSLP DNA marker nga280 with a distance of 3.2 cM. AST has been registered as a new mutant of Arabidopsis.