Hepatopancreatic parvovirus is an emerging disease in crustacean aquaculture. Consequently, methods of detection are needed that enable the sensitive detection and confirmation of the virus better than currently used methods such as histology and conventional polymerase chain reaction (PCR). A TaqMan based real-time PCR assay was developed for the detection of the Australian isolate of hepatopancreatic parvovirus which is only 85% similar to its nearest known relative. The TaqMan assay was developed within the capsid protein region of the genome and is optimised to detect as little as 10 copies of the targeted sequence per PCR vial. The hepatopancreatic parvovirus primers and probe were HPV140F 5'-CTA CTC CAA TGG AAA CTT CTG AGC-3', HPV140R 5'-GTG GCG TTG GAA GGC ACT TC-3' and HPV140probe 5'-FAM TAC CGC CGC ACC GCA GCA GC TAMRA-3', respectively. The assay was specific for the hepatopancreatic parvovirus strain from Australian Penaeus merguiensis as it did not detect related crustacean and canine parvoviruses from Australia. In addition, the very low homology of the target sequence with published sequences from the Thai and Korean strains of hepatopancreatic parvovirus and other prawn viruses such as WSSV, suggested this assay would be specific for the Australian hepatopancreatic parvovirus isolate. Furthermore, it detected hepatopancreatic parvovirus in 22/22 wild-caught P. merguiensis clinical samples and 473/545 (87%) farmed P. merguiensis. This assay has the potential to be used for diagnostic purposes and in robotic applications, particularly for the detection and quantitation of low-grade infections.

OBJECTIVE

Type1 diabetes mellitus may be associated with celiac disease. The prevalence of celiac disease as determined by screening among adult patients with type 1 diabetes is high with rates of 1.07.8% in Europe and U.S.A. The aims of the study are to determine the prevalence of celiac disease in adults with type 1 diabetes in Tunisia.

METHODS

348 consecutive adult patients with type1 diabetes were investigated prospectively and screened for celiac disease. The mean age was 28.45+/-10.74 years old. There were 176 females and 172 males. For the screening of celiac disease, we used immunoglobulin A (IgA) anti-endomysium (EMA) antibodies determined by an indirect immunofluorescence method. Anti-transglutaminase (tTG) antibodies were determined by an ELISA method. Those patients with positive results for anti EMA and or tTG were proposed for duodenal biopsy.

RESULTS

14 patients were positive for anti EMA and had high or a weak positive level of tTG antibodies. One patient from this group was already known to have celiac disease. Only 8 patients consented to biopsy and morphological changes were consistent with celiac disease in all cases. Prevalence of biopsy-proven celiac disease was 2.3% (95% CI=1.0-4.5%).

CONCLUSIONS

The present study confirms that celiac disease of adults is prevalent in type 1 diabetic patients in Tunisia. Serological screening for celiac disease in type 1 diabetes is important because many patients are asymptomatic and most are detected by the screening.

A prospective, multicenter study of patients with Ullrich-Turner syndrome (UTS) was conducted to estimate the prevalence of autoantibodies to tissue transglutaminase (tTg), thyroid stimulating hormone receptor (TSH-R), thyroglobulin (TG) and thyroid peroxidase (TPO) in relation to adult height after long-term growth hormone (GH) treatment. Out of 347 near-adult >> 16 years) patients with UTS from 96 German centers, whose longitudinal growth was documented within the Pharmacia International Growth Study (KIGS), 188 returned for a standardized follow-up visit at a median chronological age of 18.7 (16.0-23.6) years (bone age>> 15 years). Serum samples of 120 patients were obtained for central measurements of TSH, thyroxine (T4) and free T4 and autoantibodies by standard immunoassays. Information regarding thyroid disease, karyotype and anthropometric data was extracted from the KIGS database. Thirty-six percent of the patients with UTS had positive TG and/or TPO autoantibodies and 4% had positive tTg autoantibodies, whereas 2% had positive TG and/or TPO autoantibodies as well as positive tTg autoantibodies. TSH-R autoantibodies were undetectable in all patients. The detection of autoantibodies was unrelated to a specific karyotype. Median height standard deviation scores (SDS, UTS) at start of GH treatment (0.43; -1.07, 1.85) and at follow-up (1.36; -0.11, 2.57) were comparable in all patients independent of their antibody status. The total deltaheight SDS, however, was higher in patients with negative autoantibody titers (1.08; -0.03, 2.25) compared to those with positive antibody titers (0.68; -0.44, 1.82; p < 0.01). Our study confirms the high prevalence of autoantibodies in patients with UTS predisposing them to autoimmune thyroid disease and celiac disease, and indicates for the first time that autoimmune pathologies may interfere with GH therapy and thus compromise final height. Therefore, medical care for patients with UTS should routinely include screening for these autoimmune disorders in order to assure early detection and appropriate treatment.

BACKGROUND

Dermatitis herpetiformis (DH) is a rare gluten-sensitive blistering itchy skin disease, strictly related to coeliac disease (CD). Direct immunofluorescence, demonstrating IgA granular deposits localized either in the dermal papillae or along the dermo-epidermal junction, is currently the gold standard for diagnosis of DH. It has been shown that DH immunocomplexes contain epidermal transglutaminase (eTG) and that sera from patients with DH contain antibodies specifically directed against eTG.

OBJECTIVE

We studied the usefulness of serum eTG antibodies in discriminating between DH, CD and other gastrointestinal and dermatologic diseases.

METHODS

eTG antibodies were tested in 308 adult patients' sera: 44 patients with untreated dermatitis herpetiformis (UDH), 99 patients with untreated coeliac disease (UCD), 70 dermatological controls and 95 gastrointestinal controls.

RESULTS

In UDH eTG antibody levels were significantly higher than in DH patients on gluten-free diet, UCD, gastrointestinal controls and dermatological controls. In UCD eTG antibodies strongly correlated with tissue transglutaminase (tTG) antibodies, whereas in UDH no significant correlation was observed.

CONCLUSIONS

Serum IgA eTG antibody determination can efficiently distinguish UDH from other dermatological itchy diseases and is highly sensitive to gluten-free diet.

The antigen for immunoglobulin (Ig) A endomysium antibody (EmA), a sensitive and specific serological marker for celiac disease, has recently been described as tissue transglutaminase (tTG). The aim of this study was to compare the assays used to measure IgA EmA and IgA tTG antibody in patients with celiac disease and disease control subjects. Sera from 21 patients with untreated celiac disease, 48 patients with treated celiac disease and 128 disease control subjects were tested both for IgA EmA with the use of indirect immunofluorescence against human umbilical cord and for IgA tTG antibody with the use of ELISA. Titres of IgA tTG antibody were significantly higher in both the untreated and treated celiac groups than in the disease control group. Titres in the treated group were, however, significantly lower than in the untreated group. A reference range was calculated to include 99.8% of the disease control group in whom small bowel biopsy showed no evidence of celiac disease. One patient from the disease control group with raised IgA tTG antibody titres and positive IgA EmA was found to have celiac disease on small bowel biopsy. The sensitivity, specificity, and positive and negative predictive values of the IgA EmA assay were all 100%. The sensitivity of the IgA tTG antibody assay was 95%, specificity 100%, positive predictive value 100% and negative predictive value 97.7%. An ELISA used to measure IgA tTG antibody is an excellent tool to screen for celiac disease and may prove useful for monitoring response to treatment.

Histological confirmation of infiltrative lesions via small bowel biopsy is the gold standard for diagnosing celiac disease. Four serum antibody assays may serve as a first-step diagnostic tool to identify biopsy candidates: immunoglobulin A tissue transglutaminase (IgA tTG), IgA endomysial antibody (IgA EMA), IgA antigliadin antibody (IgA AGA), and IgG antigliadin antibody (IgG AGA). IgA tTG and IgA EMA offer the best diagnostic accuracy. Patients with selective IgA deficiency may have falsely negative IgA assays (strength of recommendation [SOR]: B, based on a systematic review, multiple small cross-sectional studies, and expert opinion).

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.

Dieterich et al show that immunoprecipitation of human fibrosarcoma cell lysates (cell line HT1080) using the IgA fraction from serum samples of patients with coeliac disease results in a single protein band with a molecular weight of 85 kDa. Immunoprecipitation occurred exclusively with 25 coeliac disease serum samples, but with none of the 25 control samples. The 85 kDa antigen was cleaved with endoproteinase Asp-N, and after amino-terminal sequence analysis, the three cleavage products tested all yielded sequences that could be assigned to tissue transglutaminase (EC 2.3.2.13; tTG). In order to prove that tTG obtained from the fibrosarcoma cells binds to the endomysial antibody fraction of coeliac serum IgA, the authors performed indirect immunofluorescence with high titre coeliac disease serum samples on monkey oesophagus with or without prior incubation of those samples with a commercially available tTG extract (Sigma, Deisenhofer, Germany). Pretreatment with tTG almost completely abolished endomysial antibody labelling. Also, when the Sigma tTG was used as antigen in an enzyme linked immunosorbent assay (ELISA), 12 coeliac disease patient samples but none of the seven control serum samples displayed increased IgA immunoreactivity. Dieterich et al conclude they have identified tTG as the unknown endomysial autoantigen.

BACKGROUND

Tissue transglutaminase (tTG) has recently been found to be the major if not the only autoantigen of gluten-sensitive enteropathy (GSE).

OBJECTIVE

To further determine the significance of this finding for diagnostic (screening) and follow-up purposes, we performed tTG-based ELISAs, and compared the results to the endomysium antibody test (EMA).

METHODS

We examined 120 serum samples from patients with celiac disease (CD) including 72 on a gluten-free diet (GFD) and eleven on a gluten challenge, 47 with dermatitis herpetiformis (DH) including 16 on a GFD and one on a gluten challenge, 96 with non-CD gastrointestinal diseases, and 117 with others; i.e. 380 serum samples altogether. Follow-up sera from 13 patients were included.

METHODS

Results of an ELISA with guinea pig liver tTG were compared with the EMA test using monkey esophagus. Inhibition of endomysial staining was performed with sera positive on the EMA test but negative with the guinea pig tTG ELISA.

RESULTS

The specificity and sensitivity of the tTG ELISA are high (98.6% and 92.5%). The serum IgA antibody titers against tTG decrease after introduction of a GFD. In one case, endomysial staining could not be inhibited.

CONCLUSIONS

Our results show that the guinea pig tTG ELISA is suitable for use as a simple diagnostic screening and follow-up method for GSE. Further studies are necessary to identify possible additional minor antigens in GSE.

A patient with clinical findings of dermatitis herpetiformis (DH), negative direct immunofluorescent (DIF) findings for junctional IgA deposits in 2 biopsy specimens, and positive for IgA endomysial (AEmA) and tissue transglutaminase (tTG) antibodies responded initially to dapsone. After dapsone had to be discontinued because of side effects, a gluten-free diet and supportive therapy controlled the disease; the AEmA and tTG antibodies became negative. Our data on 10 consecutive DH cases examined by DIF and by serum studies for AEmA and antibodies to tTG, point to frequencies of 90% DIF positive and 70% AEmA and tTG positive cases. The use of both DIF and serum tests for AEmA and tTG reveals DH cases not detected by DIF alone that respond to gluten-free diet. Findings on autoantibodies to tTG, an enzyme that metabolizes gliadin, points to a role of tTG in the immunopathology of gluten-sensitive enteropathy and helps to explain the need for a gluten-free diet in the management of DH cases.

Prior analysis on human protein-coding DNA sequences has identified local base composition as the primary predictor of synonymous codon usage. However, in many organisms, codon usage is influenced by natural selection, particularly for efficient expression of functional gene products. Because viruses are expected to evolve codon usage in the context of their host's molecular machinery, their genomes provide another window into the forces that guide their host's molecular evolution. Factor analysis was performed on codon usage of 16,654 genes annotated in Build 34 of the human genome, and the primary factor was correlated strongly with local base composition. However, two codons, AGG and TTG, rose in frequency as all other C- and G-ending codons decreased in frequency. These two codons were the only C- or G-ending codons with usages that negatively correlated with gene expression. Variation among viruses in codon usage also strongly reflects variation in base composition and, again, AGG and TTG decrease in frequency as all other C- and G-ending codons increase in frequency. It appears that usages of these two codons can not be explained by local compositional biases, implying a more direct role of natural selection on codon usage in humans.

We cloned an alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606 to overexpress it in Escherichia coli transformants. Deletion of the 5'-noncoding region as well as expression of the alpha-glucosidase gene under the control of the icp promotor of the insecticidal crystal protein gene from Bacillus thuringiensis subsp. sotto enhanced the enzyme productivity to 23.5 U/ml, which was 12,000-fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the alpha-glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the alpha-glucosidase from N-terminal serine was calculated to be 68,886 Da. Sequence analysis revealed that the SAM1606 alpha-glucosidase belonged to the alpha-amylase family. The SAM1606 alpha-glucosidase showed extremely high sequence identity (62-65%) to the Bacillus cereus and Bacillus thermoglucosidasius oligo-1,6-glucosidases, which were 72% identical to each other. Sequence identity in the suggested active site regions were essentially the same (80-82%) among these three enzymes. However, the substrate specificity of the SAM1606 alpha-glucosidase was significantly different from those of the oligo-1,6-glucosidases. The thermostability of these three alpha-glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at beta turns and coils in the enzymes.

OBJECTIVE

There have been few studies on the association between childhood autoimmune and rheumatic diseases. Therefore, this study aims to assess the frequency of autoimmune thyroiditis (AT), coeliac disease (CD) and type 1 diabetes mellitus (T1DM) in children and adolescents with juvenile idiopathic arthritis (JIA) and rheumatic fever (RF).

METHODS

This cross-sectional study includes 53 patients with JIA, 66 patients with RF and 40 healthy subjects controls. All subjects were evaluated for thyrotropin (TSH), triiodothyronine (T3), free thyroxine (FT4), antithyroglobulin (Tg) and antiperoxidase antibodies, fasting glucose, C-peptide, anti-glutamic acid decarboxylase (GAD), anti-islet cell (IA) and antitransglutaminase IgA (tTG) antibodies. Patients with thyroid dysfunction, positive anti-thyroid antibodies or tTG underwent thyroid ultrasonography and jejunal biopsy, respectively.

RESULTS

In group 1 (n=53), 21 patients presented thyroid disorders (40%; 42% oligoarticular), either subclinical hypothyroidism (13%) or positive anti-thyroid antibodies (26%, 50% oligoarticular), significantly higher than in control group (p<0.009, OR=10.5, CI 1.29-85.2). In group 2 (n=66), thyroid disorders were identified in 11 patients, four (6%) with subclinical hypothyroidism and seven (11%) with positive anti-thyroid antibodies (p=0.06, compared with the control group). There were no cases of clinical overt hypothyroidism, positive anti-GAD or anti-IA, nor changes in serum C-peptide and glycemia. CD was confirmed in one patient from each group.

CONCLUSIONS

Patients with JIA (especially the oligoarticular form) and RF should be investigated for thyroid dysfunction. Longitudinal studies could establish screening protocols for CD in patients with JIA and RF. The cost-effectiveness of T1DM screening is not justified in this population.

A recombinant Saccharomyces cerevisiae, expressing and secreting rice alpha-amylase, converts starch to ethanol. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N3-GCTTCCAA-N5-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida boidinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.

The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H2. H2 activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H2 of hupS gene expression. The transcriptional start site of the hupS promoter, determined by primer extension mapping, was located 55 nucleotides upstream from the translational start codon of the hupS gene. Regulatory sequences were identified by serial 5' deletions of the 300bp hupS promoter-regulatory region (phupS) and phupS-lacZ translational fusions. Cis-regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response regulator of the NtrC subfamily, were studied by electrophoretic mobility shift assays (EMSAs). The R. capsulatus IHF and HupR proteins were overexpressed in Escherichia coli and purified by affinity chromatography. IHF binds to a site, 5'-TCACACACCATTG, centred at -87 nt from the transcription start site. The HupR protein binds to one site within the -162 to -152 nt region, which contains the palindromic sequence 5'-TTG-R5-CAA. By the use of 5' deletions and site-directed mutagenesis of the -162/-152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H2.

BACKGROUND

Fighter pilots' muscular strength and endurance are subjected to very high demands. Pilots' fatigued muscles are at higher risk for injuries. The purpose of this study was to compare the effects of two different training methods in reducing muscular loading during in-flight and cervical loading testing (CLT).

METHODS

There were 16 volunteer Finnish Air Force cadets who were divided into 2 groups: a strength training group (STG) and a trampoline training group (TTG). During the 6-wk training period, the STG performed dynamic flexion and extension and isometric rotation exercises, and the TTG performed trampoline bouncing exercises. During in-flight and CLT, muscle strain from the sternocleidomastoid, cervical erector spinae, trapezius, and thoracic erector spinae muscles was recorded with EMG.

RESULTS

In-flight muscle strain in the STG after the training period decreased in the sternocleidomastoid 50%, cervical erector spinae 3%, trapezius 4%, and thoracic erector spinae 8%. In the TTG, the decrease was 41%, 30%, 20%, and 6%, respectively. In CLT, the results were similar. After a 3-mo follow-up period with intensive high +Gz flying, EMG during CLT was still lower than in baseline measurements.

CONCLUSIONS

Both training methods were found to be effective in reducing muscle strain during in-flight and CLT, especially in the cervical muscles. There was no statistically significant difference between the training groups. Introduced exercises expand muscles' capacities in different ways and the authors recommend both strength and trampoline training programs to be included in fighter pilots' physical education programs.

Actinobacillus pleuropneumoniae promoter-containing clones were isolated from a genomic DNA library constructed in our lVET promoter trap vector pTF86. The promoter-containing clones were identified by their ability to drive expression of the promoterless luxAB genes of Vibrio harveyi. The degree of expression was quantifiable, and only high-expression or "hot" promoters were used for this study. Nine clones were sequenced, and their transcriptional start sites were determined by primer extension. The sequences upstream of the start site were aligned, and a consensus promoter structure for A. pleuropneumoniae was identified. The consensus promoter sequence for A. pleuropneumoniae was found to be TATAAT and TTG/AAA, centered approximately 10 and 35 bp upstream of the transcriptional start site, respectively. A comparison of the A. pleuropneumoniae consensus with other prokaryotic consensus promoters showed that the A. pleuropneumoniae consensus promoter is similar to that found in other eubacteria in terms of sequence, with an identical -10 element and a similar but truncated -35 element. However, the A. pleuropneumoniae consensus promoter is unique in the spacing between the -10 and -35 elements. The promoter spacing was analyzed by site-directed mutagenesis, which demonstrated that optimal spacing for an A. pleuropneumoniae promoter is shorter than the spacing identified for Escherichia coli and Bacillus subtilis promoters.

Tissue transglutaminase (tTG) is a multifunctional enzyme with a plethora of potential applications in regenerative medicine and tissue bioengineering. In this study, we examined the role of tTG as a regulator of chondrogenesis in human mesenchymal stem cells (MSC) using nanofibrous scaffolds coated with collagen type XI. Transient treatment of collagen type XI films and 3D scaffolds with tTG results in enhanced attachment of MSC and supports rounded cell morphology compared to the untreated matrices or those incubated in the continuous presence of tTG. Accordingly, enhanced cell aggregation and augmented chondrogenic differentiation have been observed on the collagen type XI-coated poly-(L-lactide) nanofibrous scaffolds treated with tTG prior to cell seeding. These changes implicate that MSC chondrogenesis is enhanced by the tTG-mediated modifications of the collagen matrix. For example, exogenous tTG increases resistance to collagenolysis in collagen type XI matrices by catalyzing intermolecular cross-linking, detected by a shift in the denaturation temperature. In addition, tTG auto-crosslinks to collagen type XI as detected by western blot and immunofluorescent analysis. This study identifies tTG as a novel regulator of MSC chondrogenesis further contributing to the expanding use of these cells in cartilage bioengineering.

OBJECTIVE

To use functional MR imaging to measure the effect of frequency (pitch), intensity (loudness), and complexity of auditory stimuli on activation in the primary and secondary auditory cortexes.

METHODS

Multiplanar echo-planar images were acquired in healthy subjects with normal hearing to whom auditory stimuli were presented intermittently. Functional images were processed from the echo-planar images with conventional postprocessing methods. The stimuli included pure tones with a single frequency and intensity, pure tones with the frequency stepped between 1,000, 2,000, 3,000, or 4,000 Hz, and spoken text. The pixels activated by each task in the transverse temporal gyrus (TTG) and the auditory association areas were tabulated.

RESULTS

The pure tone task activated the TTG. The 1,000-Hz tone activated significantly more pixels in the TTG than did the 4,000-Hz tone. The 4,000-Hz tone activated pixels primarily in the medial TTG, whereas the 1,000-Hz tone activated more pixels in the lateral TTG. Higher intensity tones activated significantly more pixels than did lower intensity tones at the same frequency. The stepped tones activated more pixels than the pure tones, but the difference was not significant. The text task produced significantly more activation than did the pure tones in the TTG and in the auditory association areas. The more complex tasks (stepped tones and listening to text) tended to activate more pixels in the left hemisphere than in the right, whereas the simpler tasks activated similar numbers of pixels in each hemisphere.

CONCLUSIONS

Auditory stimuli activate the TTG and the association areas. Activation in the primary auditory cortex depends on frequency, intensity, and complexity of the auditory stimulus. Activation of the auditory association areas requires more complex auditory stimuli, such as the stepped tone task or text reading.

The calcium-dependent enzyme tissue transglutaminase (tTG) is associated with diverse biological functions, such as induction of apoptosis, modeling of the extracellular matrix, receptor-mediated endocytosis, cell growth and differentiation, cell adhesion and signal transduction. Also, it may deamidate glutamine residues to glutamic acid and catalyze cross-linking of proteins. In this study, we have investigated the impact of tTG for posttranslational modifications and cross-linking of the immunodominant T-cell epitope CII260-270 and their effects on the collagen-induced arthritis, an animal model for rheumatoid arthritis. By using mass spectrometry analysis and hybridoma assays, we have demonstrated that tTG could perform both types of modifications (deamidation and cross-link formation) on the immunodominant T-cell epitope CII259-273. Replacement of the glutamine at position 267 with glutamic acid leads to a decreased binding affinity to MHC II. T cells recognized both non-modfied (Q(267)) and modified (E(267)) CII259-273-peptides. We also show that administration of tTG leads to increased incidence, severity and histopathological manifestations of collagen-induced arthritis in mice. Moreover, we conclude that both processes, deamidation and cross-linking, are involved in the tTG-catalyzed reactions, and in vivo administration of tTG enhances arthritis severity and joint destruction in mice.

In coeliac disease, gliadin peptides p56-88, p57-68 and p31-49 have been demonstrated to be involved in the pathogenic damage of the small intestine via their immunogenicity or toxicity to epithelial cells. To try to understand the mechanism of their toxicity, we investigated the effect of synthetic peptides (p31-49, p56-88, p57-68, p69-82) and of their deamidated analogues on Caco2 and FHs 74 Int cell toxicity and tissue tranglutaminase activity. Apoptosis, necrosis and cell viability were assessed by flow cytometry, and peptide deamidation was determined indirectly by measuring its capacity to inhibit tTG activity. The results showed that p56-88 and p57-68 reduced cell growth and concomitantly inhibited tTG activity in both cell types. This effect was abolished when Caco2 cells were treated with antibodies to tTG. Deamidated peptide p57-68 (E(65)) lost practically all of its inhibitory effect on cell growth and on tTG activity. Cellular toxicity was also observed with p31-49, which was not a substrate for tTG. p69-82 was not cytotoxic but became so when glutamine 72 was substituted by glutamic acid. These findings provide evidence for the existence of three types of toxicity among gliadin peptides: (i) peptides that are intrinsically toxic and are not substrates of tTG; (ii) peptides that are non-toxic but become so when they act as substrates of tTG; and (iii) peptides that are non-toxic and are not substrates of tTG but become so when deamidated. A mechanism other than that involving tTG could be responsible for the deamidation of glutamine residues of gliadin in the intestinal tract.

Tissue transglutaminase (tTg) has been identified as an autoantigen in coeliac disease (CD). There is a marked homology between different forms of transglutaminase, such as tTg and coagulation factor XIII. We compared titres of both IgA- and IgG-antibodies against these two antigens in 20 CD patients, 20 endomysial antibody (EMA)-negative controls and a group with inflammatory bowel disease (34 with Crohn's disease and 23 with ulcerative colitis). IgA-antibodies against tTg correlated with EMA titres and had high sensitivity and specificity in screening for CD. Only in two CD patients were high titres found of IgA-antibodies against factor XIII, non-reactive with tTg. Both lacked bleeding tendency. The presence of IgG-antibodies against tTg, in contrast, had low sensitivity and specificity in screening for CD and were frequently seen in inflammatory bowel disease. Similarly, factor XIII IgG-antibodies displayed a non-specific pattern with modestly elevated titres in patients with Crohn's disease and in both EMA-negative and positive patients. Despite a marked homology with tTg, the occurrence of high titre IgA-antibodies against factor XIII is infrequent in CD, but may-when present-be the result of epitope spreading. The presence of IgG-antibodies in CD and inflammatory bowel disease illustrates the complexity of autoantibody reactions in gastrointestinal disease.

The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.

Most neurodegenerative pathologies stem from the formation of aggregates of mutant proteins, causing dysfunction and ultimately neuronal death. This study was aimed at elucidating the role of the protein factors that promote aggregate formation or prevent the process, respectively, glyceraldehyde-3-dehydrogenase (GAPDH) and tissue transglutaminase (tTG) and Hsp70 molecular chaperone. The siRNA technology was used to show that the inhibition of GAPDH expression leads to a 45-50% reduction in the aggregation of mutant huntingtin, with a repeat of 103 glutamine residues in a model of Huntington's disease (HD). Similarly, the blockage of GAPDH synthesis was found for the first time to reduce the degree of aggregation of mutant superoxide dismutase 1 (G93A) in a model of amyotrophic lateral sclerosis (ALS). The treatment of cells that imitate HD and ALS with a pharmacological GAPDH inhibitor, hydroxynonenal, was also shown to reduce the amount of the aggregating material in both disease models. Tissue transglutaminase is another factor that promotes the aggregation of mutant proteins; the inhibition of its activity with cystamine was found to prevent aggregate formation of mutant huntingtin and SOD1. In order to explore the protective function of Hsp70 in the control of the aggregation of mutant huntingtin, a cell model with inducible expression of the chaperone was used. The amount and size of polyglutamine aggregates were reduced by increasing the intracellular content of Hsp70. Thus, pharmacological regulation of the function of three proteins, GAPDH, tTG, and Hsp70, can affect the pathogenesis of two significant neurodegenerative diseases.