Active polar transport establishes directional auxin flow and the generation of local auxin gradients implicated in plant responses and development. Auxin modulates gravitropism at the root tip and root hair morphogenesis at the differentiation zone. Genetic and biochemical analyses provide evidence for defective basipetal auxin transport in trh1 roots. The trh1, pin2, axr2 and aux1 mutants, and transgenic plants overexpressing PIN1, all showing impaired gravity response and root hair development, revealed ectopic PIN1 localization. The auxin antagonist hypaphorine blocked root hair elongation and caused moderate agravitropic root growth, also leading to PIN1 mislocalization. These results suggest that auxin imbalance leads to proximal and distal developmental defects in Arabidopsis root apex, associated with agravitropic root growth and root hair phenotype, respectively, providing evidence that these two auxin-regulated processes are coupled. Cell-specific subcellular localization of TRH1-YFP in stele and epidermis supports TRH1 engagement in auxin transport, and hence impaired function in trh1 causes dual defects of auxin imbalance. The interplay between intrinsic cues determining root epidermal cell fate through the TTG/GL2 pathway and environmental cues including abiotic stresses modulates root hair morphogenesis. As a consequence of auxin imbalance in Arabidopsis root apex, ectopic PIN1 mislocalization could be a risk aversion mechanism to trigger root developmental responses ensuring root growth plasticity.

EGF receptor (EGFR) signaling in human cancers elicits changes in protein-expression patterns that are crucial for potentiating tumor growth. Identifying those proteins with expression regulated by the EGFR and determining how they contribute to malignancy is fundamental for the development of more effective strategies to treat cancer. Here, we show that tissue transglutaminase (tTG) is one such protein. EGF up-regulates tTG expression in human breast-cancer cells, and knock-downs of tTG or the treatment of breast cancer cells with a tTG inhibitor blocks their EGF-stimulated anchorage-independent growth. We further show that the combined actions of Ras and Cdc42, leading to the activation of PI 3-kinase and NFkappaB, provide a mechanism by which EGF can up-regulate tTG in breast-cancer cells. Moreover, overexpression of wild-type tTG, but not its transamidation-defective counterpart, fully mimics the growth advantages afforded by EGF to these cancer cells. Surprisingly, the tTG-promoted growth of breast-cancer cells is dependent on its ability to activate the Src tyrosine kinase as an outcome of a complex formed between tTG and the breast-cancer marker and intermediate filament protein keratin-19. These findings identify tTG as a key participant in an EGFR/Src-signaling pathway in breast-cancer cells and a potential target for inhibiting EGFR-promoted tumor progression.

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.

To investigate knockdown resistance (kdr)-like mutations associated with pyrethroid resistance in Anopheles sinensis (Wiedemann, 1828), from Guangxi province, southwest China, a segment of a sodium channel gene was sequenced and genotyped using three new genotyping assays. Direct sequencing revealed the presence of TTG-to-TCG and TG-to-TTT mutations at allele position L1014, which led to L1014S and L1014F substitutions in a few individual and two novel substitutions of N1013S and L1014W in two DNA templates. A low frequency of the kdr allele mostly in the heterozygous state of L1014S and L1014F was observed in this mosquito population. In this study, the genotyping of An. sinensis using three polymerase chain reaction-based methods generated consistent results, which agreed with the results of DNA sequencing. In total, 52 mosquitoes were genotyped using a direct sequencing assay. The number of mosquitoes and their genotypes were as follows: L/L = 24, L/S = 19, L/F = 8, and F/W = 1. The allelic frequency of L1014, 1014S, and 1014F were 72, 18, and 9%, respectively.

BACKGROUND

Because the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor beta1 (TGF-beta1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain (NF-L), tissue transglutaminases (tTGs), beta-amyloid precursor proteins (AbetaPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT.

METHODS

BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi) to 8 weeks post-infection (wpi) by Western blotting and RT-PCR.

RESULTS

Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-beta1, S100B, NF-L, tTG, AbetaPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain.

CONCLUSIONS

Further studies are needed to determine whether there is an increased risk of CT progression into neurodegenerative disease because neurodegeneration-associated AbetaPP and phosphorylated tau emerged in the brain.

We previously reported that mice lacking the RARgamma gene and one or both alleles of the RARbeta gene (i.e., RARbeta+/-/RARgamma-/- and RARbeta-/-/RARgamma-/- mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme (Ghyselinck et al., 1997, Int. J. Dev. Biol. 41, 425-447). In the present study, these compound mutants were used to investigate the cellular and molecular mechanisms involved in retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease was not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues which normally attract these cells into the INZs were not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis was also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bax, and p53. In contrast, the mutant INZs displayed a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RARbeta gene from the RARgamma null genetic background. As retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes, we propose that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression in the INZs. Approximately 10% of the RARbeta-/- /RARgamma-/- mutants displayed a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype (Dudley et al., 1995, Genes Dev. 9, 2795-2807; Luo et al., 1995, Genes Dev. 9, 2808-2820). BMP-7 was globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression.

Tissue transglutaminase (tTG) catalyzes a Ca(2+)-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 +/- 10 microM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 +/- 35 microM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 microM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 microM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.

Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoH(I) and RpoH(II) using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoH(I) and RpoH(II) regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the -35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoH(I) promoter sequence logo, is critical for RpoH(I)-dependent transcription; and that several bases in the predicted -10 element were important for activity with either RpoH(II) or both RpoH homologs. Genes that are transcribed by both RpoH(I) and RpoH(II) are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoH(I) regulon are associated with a classic heat shock response, while those specific to RpoH(II) are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoH(I) and RpoH(II) allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.

Interferon-alpha (IFNalpha) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumors, and melanoma. IFNalpha, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumor cell growth is directly suppressed by IFNalpha is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will review the consolidate signal transducer and activator of transcription (STAT)-dependent mechanism of action of IFNalpha. We will discuss data obtained by us and others on the triggering of the stress-dependent kinase pathway induced by IFNalpha and its correlations with the apoptotic process. The regulation of the expression of proteins involved in apoptosis occurrence will be also described. In this regard, IFNalpha is emerging as a post-translational controller of the intracellular levels of the apoptosis-related protein tissue transglutaminase (tTG). This new way of regulation of tTG occurs through the modulation of their proteasome-dependent degradation induced by the cytokine. Until today, inconsistent data have been obtained regarding the clinical effectiveness of IFNalpha in the therapy of solid tumors. In fact, the benefit of IFNalpha treatment is limited to some neoplasms while others are completely or partially resistant. The mechanisms of tumor resistance to IFNalpha have been studied in vitro. The alteration of JAK-STAT components of the IFNalpha-induced signaling, can be indeed a mechanism of resistance to IFN. However, we have recently described a reactive mechanism of protection of tumor cells from the apoptosis induced by IFNalpha dependent on the epidermal growth factor (EGF)-mediated Ras/extracellular signal regulated kinase (Erk) signaling. The involvement of the Ras->>Erk pathway in the protection of tumor cells from the apoptosis induced by IFNalpha is further demonstrated by both Ras inactivation by RASN17 transfection and mitogen extracellular signal regulated kinase 1 (Mek-1) inhibition by exposure to PD098059. These data strongly suggest that the specific disruption of the latter could be a useful approach to potentiate the antitumour activity of IFNalpha against human tumors based on the new mechanistic insights achieved in the last years.

The nucleotide sequence of an endo-beta-1,4-glucanase gene of Clostridium acetobutylicum contained two putative extended promoter consensus sequences, a Shine-Dalgarno sequence and a TTG initiation codon. The nucleotide sequence of the gene coding for the C-terminal region of this enzyme was not required for activity. Extensive homology in the nucleotide and amino acid sequences of the endoglucanase genes from C. acetobutylicum and Bacillus spp. was demonstrated.

GnRH plays a pivotal role in regulating human reproductive functions. This hypothalamic peptide interacts with its receptor (GnRHR) on the pituitary gonadotropes to trigger the secretion of gonadotropins, which, in turn, regulates the release of sex steroids from the gonads. In light of the importance of GnRHR, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR (hGnRHR) gene become a key issue in understanding human reproduction. In this report, the possible involvement of steriodogenic factor-1 (SF-1) as a key cell-specific regulator for hGnRHR gene expression was examined. By the transient luciferase reporter gene assays, the wild-type promoter, containing 2.3 kb ofthe hGnRHR gene 5'-flanking region relative to the ATG codon, was able to drive a 3.6 +/- 0.2-fold (P < 0.05) increase in luciferase activity in the mouse alphaT3-1 gonadotropes. Subsequent deletion analysis indicated that the most proximal 173 bp within the first exon of the gene, although not a promoter itself, contains a critical regulatory element(s) essential for the basal expression of the hGnRHR gene. The functional roles of the putative gonadotrope-specific elements (GSE; consensus 5'-CTG(A)/(T)CCTTG-3') residing at positions -5, -134, and -396 were studied by site-directed mutagenesis, and it was found that only the mutation at position -134 significantly reduced the promoter activity (80% reduction; P < 0.05). The attenuation effect of this GSE mutant was cell specific, as it was restricted to alphaT3-1 cells, but not to COS-7 and human ovarian adenocarcinoma (SKOV-3) cells. Competitive mobility shift assays using either alphaT3-1 nuclear extract or recombinant SF-1 protein clearly indicated that SF-1 is able to interact specifically with this GSE element positioned at -134. Using a SF-1 antibody that completely abrogated complex formation in the gel shift assays, the involvement of endogenous nuclear SF-1 was further evidenced. By competitive gel shift assays using oligoprimers with 2-bp scanning mutations, the sequences essential for the interaction with SF-1 were identified (5'-TTG(A)/(T)CCCTG-3', underlined sequences were important). To study the in vivo function of SF-1, vector directing expression of sense or antisense SF-1 messenger RNA (mRNA) was cotransfected with the hGnRHR promoter-luciferase construct into alphaT3-1, SKOV-3, and COS-7 cells. Overexpression of the SF-1 mRNA was able to enhance promoter activities in all of the cells tested. On the contrary, expression of the antisense SF-1 mRNA reduced the hGnRHR promoter activity only in alphaT3-1 cells, not in COS-7 or SKOV-3 cells. In summary, the data reported here provide conclusive evidence that SF-1 interacts with the GSE motif at position -134 within the first exon of the hGnRHR gene to mediate its cell-specific expression.

An association between celiac disease (CD) and other autoimmune diseases such as connective tissue diseases (CTD), inflammatory bowel diseases (IBD), and primary biliary cirrhosis (PBC) has been reported in several studies. However, a high rate of false positives in autoantibody testing was noted, especially when tissue transglutaminase (tTG) from guinea pig liver was used. Thus, the real prevalence of CD in CTD, IBD, and PBC is unclear. In a case-control study, 400 patients with CTD, 170 with IBD, 48 with PBC, and 120 healthy subjects were investigated for CD by the analysis of IgA and IgG tTG antibodies using the more specific human recombinant tTG immunoenzymatic assay. Patients and controls with positive findings were further tested for antiendomysial antibodies by indirect immunofluorescence and HLA typing, and those found positive by either of these tests underwent duodenal biopsy to confirm a possible diagnosis of CD. Twelve patients were positive for IgA or IgG tTG antibodies, showing an overall prevalence of 1.9%. Only 1 healthy subject (0.8%) had a low level positive reaction for IgA anti-tTG. Among the 12 patients and the healthy subject, only 2 (1 SLE and 1 ulcerative colitis patient) were subsequently confirmed to be affected with CD by positive EMA, HLA, and small bowel biopsy findings. The highest rate of false positives was found in PBC patients (10.4%). For these reasons, serological screening testing for CD is not recommended in CTD patients or in subjects affected with IBD or PBC, unless there is a relevant clinical suspicion of CD.

We recently reported that activation of "tissue" transglutaminase (EC 2.3.2.13; tTG) in liver cells undergoing apoptosis determines extensive cross-linking of cellular proteins resulting in the formation of SDS-insoluble shells in the so-called "apoptotic bodies". In attempt to obtain further insight into the role played by tTG in apoptosis of liver cells, we investigated its expression in primary cultures of neonatal rat liver cells stimulated with epidermal growth factor (EGF). EGF-treatment of neonatal rat liver cells induces first hyperplasia of hepatocytes, followed by involution characterized by a high incidence of apoptosis. The proliferative phase of hepatocytes is paralleled by a 10-fold increase in tTG mRNA level, which is followed, during the phase of involution, by sequential increases in enzyme activity and levels of SDS-insoluble apoptotic bodies. tTG immunostaining at both the light- and electron-microscopic levels shows that the most intensive reaction is present in globular structures showing the typical morphological appearance of mature apoptotic bodies. In early apoptotic stages, tTG protein is localized in the perinuclear region of the cell. Intense immunostaining is also found in the apoptotic bodies present inside phagosomes within the cytoplasm of neighboring cells. This evidence confirms and extends our previous findings, indicating that tTG induction and activation specifically takes place in cells undergoing apoptosis, suggesting a key role for the enzyme in the apoptotic program.

We present a PCR procedure for identification of Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis. 16S and 23S ribosomal DNAs (rDNAs) of B. multivorans and B. vietnamiensis were sequenced and aligned with published sequences for definition of species-specific 18-mer oligonucleotide primers. Specific antisense 16S rDNA primers (for B. cepacia, 5'-AGC ACT CCC RCC TCT CAG-3'; for B. multivorans, 5'-AGC ACT CCC GAA TCT CTT-3') and 23S rDNA primers (for B. vietnamiensis, 5'-TCC TAC CAT GCG TGC AA-3') were paired with a general sense primer of 16S rDNAs (5'-AGR GTT YGA TYM TGG CTC AG-3') or with a sense primer of 23S rDNA (5'-CCT TTG GGT CAT CCT GGA-3'). PCR with these primers under optimized conditions is appropriate to specifically and rapidly identify B. multivorans, B. vietnamiensis, and B. cepacia (genomovars I, III, and IV are not discriminated). In comparison with the polyphasic taxonomic analyses presently necessary for species and genomovar identification within the B. cepacia complex, our procedure is more rapid and easier to perform and may contribute to clarifying the clinical significance of individual members of the complex in cystic fibrosis.

Basic biological processes in which tissue transglutaminase (TG2, tTG) is thought to be important including apoptosis, cell adhesion and migration, ECM homeostasis and angiogenesis are key stages in the multistage tumour progression cascade. Studies undertaken with primary tumours and experimental models suggest that TG2 expression and activity in the tumour body and surrounding matrix generally decreases with tumour progression, favouring matrix destabilisation, but supporting angiogenesis and tumour invasion. In contrast, in the secondary metastatic tumour TG2 is often highly expressed whereby its potential roles in cell survival both at the intra- and extracellular level become important. In the following review the underlying molecular basis for the selection of these different phenotypes in tumour types and the anomaly for the requirement of TG2 is discussed in relation to the complex events of tumour progression.

It was recently demonstrated that renal tissue transglutaminase (tTg) protein and its catalytic product the epsilon(gamma-glutamyl) lysine protein cross-link are significantly increased in the subtotal (5/6) nephrectomy model (SNx) of renal fibrosis in rats. It was proposed that the enzyme had two important physiologic functions in disease development; one of stabilizing the increased extracellular matrix (ECM) by protein cross-linking, the other in a novel form of tubular cell death. This study, using the same rat SNx model, demonstrates first by Northern blotting that expression of tTg mRNA when compared with controls is increased by day 15 (+70% increase, P < 0.05), then rises steadily, peaking at day 90 (+391%, P < 0.01), and remains elevated at 120 d (+205%, P < 0.05) when compared with controls. In situ hybridization histochemistry demonstrated that the tubular cells were the major site of the additional tTg synthesis. Immunohistochemistry on cryostat sections revealed a sixfold increase (P < 0.001) in ECM-bound tTg antigen at 90-d post-SNx, whereas in situ transglutaminase activity demonstrated by the incorporation of fluorescein cadaverine into cryostat sections indicated a 750% increase (P < 0.001) on day 90 in SNx animals. This increased activity was extracellular and predominantly found in the peritubular region. These results indicate that increased tTg gene transcription by tubular cells underlies the major changes in renal tTg protein reported previously in SNx rats, and that the presence of the epsilon(gamma-glutamyl) lysine cross-links in the extracellular environment is the result of the extracellular action of tTg. These changes may be in response to tubular cell injury during the scarring process and are likely to contribute to the progressive expansion of the ECM in renal fibrosis.

Alzheimer's disease, Parkinson's disease and Huntington's disease are neurodegenerative diseases, characterized by the accumulation and deposition of neurotoxic protein aggregates. The capacity of specific proteins to self-interact and form neurotoxic aggregates seems to be a common underlying mechanism leading to pathology in these neurodegenerative diseases. This process might be initiated and/or accelerated by proteins that interact with these aggregating proteins. The transglutaminase (TG) family of proteins are calcium-dependent enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine isopeptide bonds, which can result in both intra- and intermolecular cross-links. Intramolecular cross-links might modify self-interacting proteins, and make them more prone to aggregate. In addition, intermolecular cross-links could link self-aggregating proteins and thereby initiate and/or stimulate the aggregation process. So far, increased levels and activity of tissue transglutaminase (tTG), the best characterized member of the TG family, have been observed in many neurodegenerative diseases, and the self-interacting proteins, characteristic of Alzheimer's disease, Parkinson's disease and Huntington's disease, are known substrates of tTG. Here, we focus on the role of tTG in the initiation of the aggregation process of self-interacting proteins in these diseases, and promote the notion that tTG might be an attractive novel target for treatment of neurodegenerative diseases.

OBJECTIVE

Clinically diagnosed coeliac disease patients carry an increased risk of mortality. As coeliac disease is markedly underdiagnosed, we aimed to quantify the risk of mortality in subjects with unrecognized and thus untreated coeliac disease.

METHODS

Blood samples from 6,987 Finnish adults were drawn in 1978-80, and sera were tested for immunoglobulin A (IgA)-class tissue transglutaminase antibodies (Eu-tTG) in 2001. Positive sera were further analysed for endomysial (EMA) and tissue transglutaminase antibodies by another test (Celikey tTG). EMA- and Celikey tTG-positive cases were compared to negatives as regards mortality in up to 28 years of surveillance, yielding a total follow-up of 147,646 person years. Dates and causes of death were extracted from the nation-wide database.

RESULTS

Altogether 74 (1.1%) of the participants were EMA- and 204 (2.9%) Celikey tTG-positive. The age- and sex-adjusted relative risk of overall mortality was not increased in either EMA (0.78, 95% CI 0.52-1.18) or Celikey tTG (1.19, 95% CI 0.99-1.42) -positive subjects. However, antibody-positive cases evinced a tendency to die from lymphoma, stroke, and diseases of the respiratory system.

CONCLUSIONS

The prognosis of unrecognized coeliac disease was good as regards overall mortality, which does not support screening of asymptomatic coeliac disease cases.

Transgenic Arabidopsis expressing beta-glucuronidase (GUS) driven by a chalcone synthase gene (CHS) promoter were produced. GUS activity in the leaves increased with increasing fluence rates of white light in parallel with endogenous CHS transcript levels. An isogenic line homozygous for the transgene was obtained and mutagenized seedlings of this line were screened for altered light-induction of the transgene. Putative mutants with low GUS activity were not altered in the light-induction of endogenous CHS transcripts and are therefore not regulatory mutants. Two mutant lines (A12 and C10) with elevated levels of GUS activity in the light show a corresponding increase in CHS transcript levels. The A12 mutant was focussed upon and designated icx1 (increased chalcone synthase expression). This mutant has enhanced light-stimulation of CHS expression since CHS transcript levels in darkness in icx1 are very low, as in the wild-type. The transcript levels of two other genes involved in flavonoid biosynthesis are elevated in the light in icx1 as is anthocyanin formation. However, there is no alteration in LHCII chlorophyll a/b-binding protein gene (CAB) transcript levels under the same conditions. The altered gene expression phenotype of icx1 co-segregates with several other phenotypic characteristics, including fewer leaf trichomes and alterations to the seed coat. On the basis of these data and comparison with the Arabidopsis ttg (transparent testa glabra) mutant, it is suggested that the ICX1 gene product may be concerned both with the light-regulation of gene expression and with developmental processes occurring in the epidermis.

Neisseria gonorrhoeae is an obligate human pathogen that can escape immune surveillance through antigenic variation of surface structures such as pili. A G-quadruplex-forming (G4) sequence (5'-G(3)TG(3)TTG(3)TG(3)) located upstream of the N. gonorrhoeae pilin expression locus (pilE) is necessary for initiation of pilin antigenic variation, a recombination-based, high-frequency, diversity-generation system. We have determined NMR-based structures of the all parallel-stranded monomeric and 5' end-stacked dimeric pilE G-quadruplexes in monovalent cation-containing solutions. We demonstrate that the three-layered all parallel-stranded monomeric pilE G-quadruplex containing single-residue double-chain reversal loops, which can be modeled without steric clashes into the 3 nt DNA-binding site of RecA, binds and promotes E. coli RecA-mediated strand exchange in vitro. We discuss how interactions between RecA and monomeric pilE G-quadruplex could facilitate the specialized recombination reactions leading to pilin diversification.

OBJECTIVE

Positivity of both immunoglobulin A anti-tissue transglutaminase (TTG) and anti-endomysium antibodies (EMA) has a positive predictive value of nearly 100% for celiac disease (CD). The objective of the present study was to evaluate whether patients of any age, with high pretest probability of CD and high titre of anti-TTG and EMA positivity, have a high probability of intestinal damage and may not require the biopsy for final diagnosis.

METHODS

A retrospective analysis of 412 consecutively referred patients, age range 10 months to 72 years, who underwent small-bowel biopsy for suspicion of CD and positivity to both anti-TTG and EMA, was performed at 4 Italian centers. Biopsies were evaluated independently by 2 pathologists using Marsh modified classification; in cases of dissimilar results, a third pathologist examined the biopsy. The final histological finding diagnosis was expressed as the prevalent or highest score assigned by the pathologist board.

RESULTS

Three hundred ninety-six patients (96.1%) had histological findings consistent with CD (grade 2 and 3a, 3b, or 3c of modified Marsh classification). An anti-TTG ratio ≥ 7 was able to identify with the 3 assays used (Celikey, anti-TTG immunoglobulin A, EuTTG) all of the patients with significant mucosal damage (Marsh ≥ 2) independent of age and sex; specificity and positive predictive value were 100%. An anti-TTG ratio >20 was more specific (99.8%) for identification of patients with villous atrophy (Marsh 3 a, b, or c).

CONCLUSIONS

Patients with positivity of anti-TTG ≥ 7-fold cutoff, confirmed by positivity to EMA, have a high-degree probability of duodenal damage. In selected conditions, a duodenal biopsy may be avoided and a confirmed greatly positive anti-TTG result could be the basis to prescribe a gluten-free diet.

BACKGROUND

Knockdown resistance in insects resulting from mutation(s) in the voltage gated Na+ channel (VGSC) is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common kdr mutation in insects, was reported in Anopheles culicifacies-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an An. culicifacies population from Malkangiri district of Orissa, India.

METHODS

Anopheles culicifacies sensu lato (s.l.) samples, collected from a population of Malkangiri district of Orissa (India), were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR) was developed for the detection of the new mutation L1014S. The An. culicifacies population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS) and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing.

RESULTS

DNA sequencing of An. culicifacies individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA)-to-Phe (TTT) or -Ser (TCA) changes, respectively. A third and novel substitution, Val (GTG)-to-Leu (TTG or CTG), was identified at residue V1010 resulting from either of the two transversions-3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the identification of the new mutation L1014S was found specific as evident from DNA sequencing results of respective samples. Since L1014S was found tightly linked to V1010L, no separate assay was developed for the latter mutation. Screening of population using PIRA-PCR assays for 1014S and ARMS for 1014F alleles revealed the presence of all the three amino acid substitutions in low frequency.

CONCLUSIONS

This is the first report of the presence of L1014S (homologous to the kdr-e in An. gambiae) and a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC of An. culicifacies in addition to the previously described mutation L1014F. The V1010L substitution was tightly linked to L1014S substitution. A new PIRA-PCR strategy was developed for the detection of L1014S mutation and the linked V1010L mutation.

Gluten-sensitive enteropathy, otherwise known as celiac sprue, is characterized by an abnormal proximal small intestinal mucosa arising as a result of an inappropriate inflammatory response to ingested gluten antigens present in wheat in genetically susceptible individuals. This immune response is directed to a 33-mer peptide of the alpha gliadin component of gluten. The generation of an epitope for the recognition by CD4+ T cells requires deamination of the protein by tissue transglutaminase (tTG). Moreover, IgA anti tTG is highly sensitive and is specific serologic marker (95-99%) of celiac disease. They can be easily determined quantitatively, by ELISA of an accurate and relatively inexpensive technique. Therefore, tTG can be used as the first line diagnostic test in the work-up of celiac disease, as well as for screening purposes. Finally, tTG may contribute to future strategies in treating celiac disease either by producing nontoxic wheat or by generating oral vaccination that can prevent the disease.