Transglutaminase Levels Research Articles

SERUM TRANSGLUTAMINASE IN CHILDREN WITH INFLAMMATORY BOWEL DISEASE

21 Background: The clinical activity of inflammatory bowel diseases(IBD) is based on clinical and laboratory findings that do not always reflect the pathogenic processes taking place in the intestine. There is evidence that Factor XIIIa, a plasma born transglutaminase (TG), plays a key role in the extracellular matrix assembly in damaged tissue following various intestinal injuries. We previously found that TG levels are decreased in serum of adult patients with IBD and demonstrated in a rat model of chronic colitis that serum TG is closely related to the severity of intestinal mucosal damage.Aims: The aim of this study was to extend our findings to children and to examine whether serum TG levels are related to the endoscopic activity index in children with IBD. Patients/Methods: Serum was collected from 16 children with Crohn's disease (mean age, 12.2±3 years; range, 8-17); from 11 children with ulcerative colitis (mean age, 10.4±3.3 years; range, 9-15); and from 9 age-matched healthy controls (mean age, 8±3.4 years; range, 5-16). Besides scoring clinical parameters, the disease activity of each patient was endoscopically evaluated as inactive(0-3), milde/moderate (3-5) or severe (5-10) scoring on a 0-2 scale the erythema, friability, granularity erosions and ulcerations. Serum TG activity was assayed by a radioenzymatic method. Results: Serum TG was significantly lower in patients with active disease compared with inactive disease or normal subjects (0.66±0.17 vs 1.34±0.62, p<0.001, and 0.66±0.17 vs 1.36±0.40, p<0.001; respectively). Serum TG showed a negative correlation with endoscopic scores both in children with Crohn's disease (r = -0.77, p<0.001) and ulcerative colitis (r = -0.74, p<0.01). Conclusions: These data raise the possibility that TG assay may prove useful in management of IBD in children as a new serological non-invasive indicator of intestinal mucosal status.

Keratinocyte Differentiation is Stimulated by Activators of the Nuclear Hormone Receptor PPARα

Peroxisome proliferator activated receptors (PPAR) belong to the superfamily of nuclear hormone receptors that heterodimerize with the retinoid X receptor and regulate transcription of several genes involved in lipid metabolism and adipocyte differentiation. Because of the role of 1,25-dihydroxyvitamin D3 and retinoic acid working through similar receptors (the vitamin D receptor and retinoic acid receptor, respectively) on keratinocyte differentiation, we have examined the effects of activators of PPARalpha on keratinocyte differentiation. The rate of cornified envelope formation was increased 3-fold in keratinocytes maintained in low calcium (0.03 mM) and incubated in the presence of clofibric acid, a potent PPARalpha activator. Involucrin, a cornified envelope precursor, and the cross-linking enzyme transglutaminase, were increased at both the message level (2-7-fold) and the protein level (4-12-fold) by clofibric acid. Furthermore, physiologic doses of the fatty acids oleic acid, linoleic acid, and eicosatetraynoic acid, which are also activators of PPARalpha, also induced involucrin and transglutaminase protein and mRNA. In contrast, the PPARgammaligand prostaglandin J2 had no effect on protein or mRNA levels of involucrin or transglutaminase. Levels of involucrin and transglutaminase mRNA and protein were induced by clofibric acid in keratinocytes incubated in 1.2 mM calcium, a concentration which by itself induces keratinocyte differentiation. Finally, PPARalpha activators inhibit DNA synthesis. This study demonstrates that PPARalpha activators, including putative endogenous ligands such as fatty acids, induce differentiation and inhibit proliferation in keratinocytes, and suggests a regulatory role for the PPARalpha in epidermal homeostasis.

All-trans retinoic acid blocks the antiproliferative prodifferentiating actions of 1,25-dihydroxyvitamin D3 in normal human keratinocytes.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans retinoic acid (RA), the active metabolites of vitamins D and A respectively, regulate the proliferation and differentiation of keratinocytes. Both the vitamin D receptor (VDR) and the retinoic acid receptor family (RAR) bind to DNA response elements as heterodimers with the retinoic X receptor (RXR), suggesting that there are pathways of action that are shared by both compounds. Therefore, we examined the interactions of 1,25(OH)2D3 and RA upon the proliferation and differentiation of normal human keratinocytes (NHK) and of a squamous cell carcinoma cell line, SCC4. Although both 1,25(OH)2D3 and RA were each able to inhibit NHK proliferation in a dose-dependent manner, when they were administered in combination, proliferation was stimulated, suggesting mutual antagonism. In contrast, SCC4 cells proved insensitive in terms of proliferation to 1,25(OH)2D3 and to all but the highest concentration (10(-6) M) of RA. 1,25(OH)2D3 exerted a biphasic effect on transglutaminase (TGase) and involucrin (INV) mRNA levels, with maximal stimulation at 10(-9) M. RA inhibited TGase and INV mRNA levels and antagonized the stimulation by 1,25(OH)2D3. A similar pattern was observed for TGase protein, but, RA, which, by itself, reduced INV, markedly enhanced the ability of 1,25(OH)2D3 to raise INV levels, possibly by inhibiting 1,25(OH)2D3-stimulated TGase activity and cross-linking of soluble INV into the insoluble cornified envelope (CE). Thus, in NHK cells, RA antagonizes the antiproliferative prodifferentiating actions of 1,25(OH)2D3, but assessment of a single marker, such as INV protein, may be misleading.

Identification of Cytoplasmic Actin as an Abundant Glutaminyl Substrate for Tissue Transglutaminase in HL-60 and U937 Cells Undergoing Apoptosis

A lysine derivative, 3-[Nalpha[Nepsilon-[2', 4'-dinitrophenyl]-amino-n-hexanoyl-L-lysylamido]-propane-1-ol, a novel amine substrate of transglutaminases, was synthesized and delivered into intact HL-60 and U937 human leukemia cells to probe the function of the intracellular enzyme. The novel substrate compound was covalently incorporated into intracellular proteins in these cells expressing high levels of tissue transglutaminase and undergoing apoptosis following the induction of their differentiation with dimethyl sulfoxide and retinoic acid. Immunoaffinity purification and microsequencing of labeled proteins identified cytoplasmic actin as the main endogenous glutaminyl substrate in these cells. As shown by confocal image analysis, cells revealed distinct labeling of the microfilament meshwork structures by the novel compound as the result of the intracellular action of transglutaminase.

Transglutaminase activity is increased in Alzheimer's disease brain

Transglutaminase is a calcium-activated enzyme that crosslinks substrate proteins into insoluble, often filamentous aggregates resistant to proteases. Because the neurofibrillary tangles in Alzheimer's disease have similar characteristics, and because tau protein, the major component of these tangles is an excellent substrate of transglutaminase in vitro, transglutaminase activity and levels were measured in control and Alzheimer's disease brain. Frozen prefrontal cortex and cerebellum samples from Alzheimer's disease and control cases matched for age and postmortem interval were used in the analyses. Total transglutaminase activity was significantly higher in the Alzheimer's disease prefrontal cortex compared to control. In addition the levels of tissue transglutaminase, as determined by quantitative immunoblotting, were elevated approximately 3-fold in Alzheimer's disease prefrontal cortex compared to control. To our knowledge, this is the first demonstration that transglutaminase is increased in Alzheimer's disease brain. There were no significant differences in transglutaminase activity or levels in the cerebellum between control and Alzheimer's disease cases. Because the elevation of transglutaminase in the Alzheimer's disease samples occurred in the prefrontal cortex, where neurofibrillary pathology is usually abundant, and not in the cerebellum, which is usually spared in Alzheimer's disease, it can be suggested that transglutaminase could be a contributing factor in neurofibrillary tangle formation.

Cell-biomaterial interactions: role of transglutaminase enzyme

Cell interaction(s) with biomaterial undergo a multistep-step paradigm of adhesion, contact, attachment, and spreading. However, the molecular mechanism(s) underlying the cell anchorage on a material surface is not yet well understood. Transglutaminases, a family of calcium-dependent enzymes, has been implicated in the interactions between cells and extracellular matrix. To investigate the functions of this enzyme in cell-material interactions, Balb-C 3T3 fibroblasts were cultured, stably expressing high levels of tissue transglutaminase (TGase), on dishes precoated with thin layers of the hydrophilic hydrogel poly (2-hydroxyethyl methacrylate) (polyHEMA). Results suggest that in the presence of a relatively high poly (HEMA) dilution, the transfected cells showed a more complex morphologic pattern, characterized by many spread and flattened cells, in comparison with a control culture. These studies support the hypothesis that the overexpression of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness.

Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures.

Lesional psoriatic epidermis displays a number of phenotypic changes that are distinct from the differentiation program found in normal interfollicular epidermis. In psoriatic epidermis, keratinocytes are hyperproliferative and several differentiation-associated molecules are expressed that are absent in normal skin (e.g., cytokeratins (CK) 6, 16, and 17, and the epidermal proteinase inhibitor SKALP/ elafin). In addition, several molecules which are normally restricted to the stratum granulosum are strongly upregulated in the stratum spinosum (e.g., psoriasis-associated fatty acid binding protein (PA-FABP), psoriasin, involucrin, and transglutaminase). The aim of this study was to develop in vitro culture systems which (a) would allow to study the induction of normal and psoriatic differentiation pathways, and (b) would be amenable for screening of antipsoriatic drugs. Here we have investigated several models for induction of differentiation with respect to the expression of markers for the normal and psoriatic phenotype. Cell cycle parameters and expression levels of CK1, CK10, CK16, SKALP/elafin, transglutaminase, involucrin, psoriasin, and PA-FABP were assessed in these models using flow cytometry, immunocytochemistry, and Northern blot analysis. We observed that induction of differentiation with fetal calf serum resembled the psoriatic phenotype (sustained hyperproliferation; high levels of CK16, SKALP/elafin, transglutaminase, and involucrin; moderate psoriasin expression), whereas differentiation induced by growth factor depletion in a confluent culture resembled the normal differentiation phenotype (low proliferative rate; high expression levels of CK1 and CK10; moderate expression of involucrin and transglutaminase; low expression levels of SKALP/elafin and CK16; absence of psoriasin). We propose that these models can be used to study expression and pharmacological modulation of selected differentiation genes and the coordinated expression of sets of genes associated with epidermal differentiation programs.

Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures

Lesional psoriatic epidermis displays a number of phenotypic changes that are distinct from the differentiation program found in normal interfollicular epidermis. In psoriatic epidermis, keratinocytes are hyperproliferative and several differentiation-associated molecules are expressed that are absent in normal skin (e.g., cytokeratins (CK) 6, 16, and 17, and the epidermal proteinase inhibitor SKALP/elafin). In addition, several molecules which are normally restricted to the stratum granulosum are strongly upregulated in the stratum spinosum (e.g., psoriasis-associated fatty acid binding protein (PA-FABP), psoriasin, involucrin, and transglutaminase). The aim of this study was to develop in vitro culture systems which (a) would allow to study the induction of normal and psoriatic differentiation pathways, and (b) would be amenable for screening of antipsoriatic drugs. Here we have investigated several models for induction of differentiation with respect to the expression of markers for the normal and psoriatic phenotype. Cell cycle parameters and expression levels of CK1, CK10, CK16, SKALP/elafin, transglutaminase, involucrin, psoriasin, and PA-FABP were assessed in these models using flow cytometry, immunocytochemistry, and Northern blot analysis. We observed that induction of differentiation with fetal calf serum resembled the psoriatic phenotype (sustained hyperproliferation; high levels of CK16, SKALP/elafin, transglutaminase, and involucrin; moderate psoriasin expression), whereas differentiation induced by growth factor depletion in a confluent culture resembled the normal differentiation phenotype (low proliferative rate; high expression levels of CK1 and CK10; moderate expression of involucrin and transglutaminase; low expression levels of SKALP/elafin and CK16; absence of psoriasin). We propose that these models can be used to study expression and pharmacological modulation of selected differentiation genes and the coordinated expression of sets of genes associated with epidermal differentiation programs. © 1996 Wiley-Liss, Inc.

Butyrate enemas in experimental colitis and protection against large bowel cancer in a rat model

BACKGROUND & AIMS: Butyrate is effective in experimental colitis by increasing transglutaminase activity. Because ulcerative colitis increases the risk of colonic neoplasia, the aim of this study was to investigate whether butyrate treatment reduces mucosal sensitivity to colon cancer development in rats with experimental colitis. METHODS: Colon cancer was induced by azoxymethane injections in 10 rats with trinitrobenzensulfonic acid-induced colitis and 10 rats without colitis. Three additional groups of rats with colitis were treated with butyrate, mesalamine, and saline enemas, respectively, twice daily for 8 weeks; 1 week after colitis induction, tumors were induced. Biopsy specimens for assessment of proliferation pattern and transglutaminase activity were obtained during the latent period of cancer development. Characteristics of tumors were recorded 27 weeks after the first exposure to azoxymethane. RESULTS: Experimental colitis enhanced carcinogenesis; butyrate therapy reduced both incidence and size of tumors and also affected colonic proliferation pattern. Transglutaminase levels were restored by butyrate treatment in rats with colitis. CONCLUSIONS: The protective effect of butyrate against large bowel cancer in experimental colitis suggests its usefulness in long-term therapy to decrease disease relapses and to reduce colon cancer risk in ulcerative colitis. (Gastroenterology 1996 Jun;110(6):1727-34)

Evidence for Separate Control Mechanisms at the Message, Protein, and Enzyme Activation Levels for Transglutaminase During Calcium-Induced Differentiation of Normal and Transformed Human Keratinocytes

We analyzed the effects of three different calcium concentrations on the RNA and functional protein levels of transglutaminase (TGase) and involucrin (INV) over time in culture. We compared the results in normal human keratinocytes with those in a squamous cell carcinoma, SCC4. The highest calcium concentration (1.2 mM) induced the greatest levels of INV and TGase message, INV protein, and rates of CE formation, but not maximal levels of TGase protein. By examining cytosol and membrane fractions of keratinocytes, we found that after synthesis, TGase protein shifts, under the influence of calcium (both 0.1 mM and 1.2 mM), from the cytosol into the membrane in postconfluent cells. However, only 1.2 mM calcium induced significant amounts of TGase activity. These data indicate that elevated calcium (1.2 mM) achieves the expected induction in keratinocyte differentiation by regulation of not only INV and TGase message levels, but also the translation and activation of TGase protein. Our data suggest that this calcium-induced activation of TGase protein occurs while the protein is anchored in the membrane. In contrast, despite ample INV and TGase message levels within SCC4 cells, these RNA levels are not regulated by calcium or translated into protein, suggesting that the transformed phenotype of SCC4 cells results not only in a failure of calcium to regulate gene transcription, but also in a defect within the translation machinery of these differentiation-specific proteins.

Tissue-Type Transglutaminase Is Not a Tumor-Related Marker

The level of transglutaminase (TGase) expressed in various tumor cell lines was investigated. We found that each cell line could be categorized into three distinct groups, that is, (i) cell lines with low or negligible TGase activity, (ii) cell lines with significant or high TGase activity and immunoreactive to a monoclonal antibody against tissue type TGase, and (iii) cell lines with significant or high TGase activity but not immunoreactive to the antibody. Results reported here argue against the former proposal that tissue-type TGase is expressed at a lower level in malignant cells compared to the level in normal cells.

Involvement of transglutaminase in myofibril assembly of chick embryonic myoblasts in culture.

Involvement of transglutaminase in myofibrillogenesis of chick embryonic myoblasts has been investigated in vitro. Both the activity and protein level of transglutaminase initially decreased to a minimal level at the time of burst of myoblast fusion but gradually increased thereafter. The localization of transglutaminase underwent a dramatic change from the whole cytoplasm in a diffuse pattern to the cross-striated sarcomeric A band, being strictly colocalized with the myosin thick filaments. For a brief period prior to the appearance of cross-striation, transglutaminase was localized in nonstriated filamental structures that coincided with the stress fiber-like structures. When 12-o-tetradecanoyl phorbol acetate was added to muscle cell cultures to induce the sequential disassembly of thin and thick filaments, transglutaminase was strictly colocalized with the myosin thick filaments even in the myosacs, of which most of the thin filaments were disrupted. Moreover, monodansylcadaverine, a competitive inhibitor of transglutaminase, reversibly inhibited the myofibril maturation. In addition, myosin heavy chain behaved as one of the potential intracellular substrates for transglutaminase. The cross-linked myosin complex constituted approximately 5% of the total Triton X-100-insoluble pool of myosin molecules in developing muscle cells, and its level was reduced to below 1% upon treatment with monodansylcadaverine. These results suggest that transglutaminase plays a crucial role in myofibrillogenesis of developing chick skeletal muscle.

Transglutaminase in azoxymethane-induced colon cancer in the rat.

A widespread from of transglutaminase, tissue transglutaminase, has been identified in a number of mammalian cell types, both normal and transformed cells; its biological role is not well understood. We investigated the effect of experimentally induced colon cancer on transglutaminase activity in the rat. Azoxymethane (15 mg/kg for six weeks), given by a course of weekly intraperitoneal injections, produces tumors almost exclusively confined to the intestinal tract. Transglutaminase activity was assayed on tissue homogenates both during the period of treatment and, when the cancer had developed, on tumor tissue and on microscopically uninjured adjacent tissue. A transient proliferative phase was present in the intestine during azoxymethane treatment: in this phase we found a coincidentally increased transglutaminase levels. Transglutaminase activity in tumors of both small and large intestine was significantly higher than in adjacent tissue. Immunohistochemistry revealed higher levels of transglutaminase in tumors, mainly localized in the extracellular matrix, than in adjacent tissues, where it was widely distributed. The present study shows that transglutaminase, besides its potential role in intracellular process during early proliferative phase of carcinogenesis, may also play an important role in matrix processing during tumor growth and differentiation.

1,25-Dihydroxyvitamin D3 potentiates the keratinocyte response to calcium.

Extracellular calcium (Cao) stimulates the differentiation of keratinocytes; 1,25 dihydroxyvitamin D3 1,25(OH)2D3) does likewise. Since 1,25(OH)2D3 regulates calcium flux in other cells, we hypothesized that 1,25(OH)2D)3 acted through and promoted the effects of calcium on keratinocyte differentiation. To test this hypothesis, we evaluated the effects of calcium and 1,25(OH)2D3 alone and in combination on the mRNA and protein levels of involucrin and transglutaminase in neonatal human keratinocytes. Cao alone increased these mRNA levels in a dose-dependent fashion (0.03 to 1.2 mM) over a 24-h period. This increase in mRNA levels was associated with a stimulation by calcium of involucrin and transglutaminase gene transcription. However, by 72 h, the mRNA levels of involucrin and transglutaminase decreased. At 0.03 mM Cao, 1,25(OH)2D3 showed a dose-dependent stimulation of involucrin and transglutaminase mRNA for up to 48 h and potentiated the initial (4-h) stimulation by Cao of involucrin and transglutaminase mRNA. As for calcium alone, this increase in mRNA was associated with an increase in transcription of the involucrin and transglutaminase genes. However, by 24 h of exposure to both calcium and 1,25(OH)2D3, a dose-dependent fall in mRNA levels was seen. The mRNA levels of involucrin and transglutaminase were stable for 24 h when neonatal human keratinocytes were grown in serum-free keratinocyte growth medium containing 0.03 or 1.2 mM Cao alone. However, the mRNAs of both genes underwent rapid degradation when neonatal human keratinocytes were treated with 1,25(OH)2D3, especially in high Cao. 1,25(OH)2D3 and Cao increased the protein levels of involucrin and transglutaminase activity in a synergistic fashion throughout the 48-h time course. These data support the hypothesis that 1,25(OH)2D3 promotes calcium-induced differentiation at the level of both gene expression and mRNA stability.

Butyrate, mesalamine, and factor XIII in experimental colitis in the rat: Effects on transglutaminase activity

Background/Aims: Butyrate and factor XIII may improve ulcerative colitis; they also affect tissue and serum transglutaminase levels. We investigated the therapeutic potential of sodium butyrate and factor XIII and the role of transglutaminase during mucosal repair in experimental colitis. Methods: Rats with induced colitis were treated with sodium butyrate, mesalamine, sodium butyrate plus mesalamine, or saline enemas. Thromboxane B2 was monitored as index of inflammation. In a fifth group, the effectiveness of intravenous Factor XIII was assessed. Results: Sodium butyrate, alone or plus mesalamine, reduced histological activity from 13.7 ± 1.7 (saline) to 2.5 ± 1.3 and 2.3 ± 1.1 (P < 0.01), respectively. Transglutaminase, reduced in the colons of the saline group (783 ± 157 vs. normal 1800 ± 192 mU/g; P < 0.01), returned toward normal values in the sodium butyrate or sodium butyrate plus mesalamine groups (1390 ± 228 and 1226 ± 172 mU/g, respectively; P < 0.01 vs. saline). Furthermore, sodium butyrate plus mesalamine reduced thromboxane B2 levels by day 5 (0.92 ± 0.16 vs. saline 1.85 ± 0.34 ng/mL; P < 0.05). Factor XIII therapy improved the histological picture (2.7 ± 2.1 vs. saline 13.8 ± 1.7; P < 0.01) and increased transglutaminase levels both in serum (2.81 ± 0.11 vs. saline 1.45 ± 0.09 mu/mL; P < 0.01) and in colon (1503 ± 127 vs. saline 747 ± 103). Conclusions: Sodium butyrate and factor XIII improve colitis, sodium butyrate plus mesalamine reduce early thromboxane B2 synthesis, and transglutaminase(S) plays a role in ulcer healing.

Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis).

Proliferating primordial germ cells (PGCs) isolated from mouse embryos soon after their arrival in the genital ridges would only survive in vitro at temperature of less than 30 degrees C (De Felici, M. and McLaren, A. (1983). Exp. Cell. Res. 144, 417-427; Wabik-Sliz, B. and McLaren, A. (1984). Exp. Cell. Res. 154, 530-536) or when co-cultured on cell feeder layers (Donovan, P. J., Stott, D., Godin, I., Heasman, J. and Wylie, C. C. (1986). Cell 44, 831-838; De Felici, M. and Dolci, S. (1991). Dev. Biol. 147, 281-284). In the present paper we report that mouse PGC death in vitro occurs with all the hallmarks of programmed cell death or apoptosis. We found that after 4-5 hours in culture many PGCs isolated from 12.5 dpc fetal gonads assumed a nuclear morphology and produced membrane bound fragments (apoptotic bodies) typical of apoptotic cells. In addition, PGCs in culture accumulated high level of tissue transglutaminase (tTGase; an enzyme that is induced and activated during apoptosis) and showed extensive degradation of DNA to oligonucleosomal fragments, which is characteristic of apoptosis. The physiological relevance of this mechanism of PGC death is supported by the finding that some PGCs undergoing apoptosis, as revealed by the high level of tTGase expression, were detected in the embryo. Most importantly, we show that the addition of stem cell factor (SCF) or leukemia inhibitory factor (LIF) to the culture medium, two cytokines known to favour PGC survival and/or proliferation in vitro, markedly reduced the occurrence of apoptosis in PGCs during the first hours in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotype-specific "tissue" transglutaminase regulation in human neuroblastoma cells in response to retinoic acid: correlation with cell death by apoptosis.

Neuroblastomas in culture are characterized by the presence of 2 morphologically and biochemically distinct phenotypes (i.e., neural "N-type" and flat substrate-adherent "S-type") which undergo transdifferentiation. Human neuroblastoma SK-N-BE(2) cells differentiate toward a neural phenotype upon retinoic acid (RA) treatment. However, we recently showed that, during the RA treatment, a subset of SK-N-BE(2) cells undergo apoptosis; these cells specifically express a high "tissue" transglutaminase (tTG) level. This study was undertaken to investigate the cellular and molecular basis of the action of retinoic acid on apoptosis in human neuroblastoma cells. As a biochemical marker of the phenomenon we studied the tTG gene expression in the parental line SK-N-BE(2) and in 2 clones which stably express neuroblastic [BE(2)-M17] and substrate-adherent [BE(2)-C] features, respectively. Data showed a differential phenotype-specific regulation of tTG gene expression. In fact, RA treatment enhanced tTG expression and apoptotic index in the flat substrate-adherent variant, whereas, in cells expressing the neural phenotype, very low tTG expression and apoptosis were found. Northern-blotting analysis revealed that the substrate-adherent cells had a basal 3-fold higher level of tTG mRNA. An increase in tTG mRNA major transcript levels (3.7 kb) occurred within a few hours of exposure to RA in both the phenotypic variants. By contrast, tTG protein level was very low in the cell expressing the neuronal phenotype, even after prolonged exposure to RA. Immunohistochemical analysis indicated that tTG protein, in addition to mature apoptotic cells, was specifically localized in the flat substrate-adherent variant both in the wild-type and in the BE(2)-C clone. These findings suggest that the ability to undergo apoptosis in the neuroblastoma cells is associated with the expression of a non-neuronal neuroectodermal (substrate-adherent cells) immature phenotype.

Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.

Transglutaminase levels in Dupuytren's disease

By use of immunoperoxidase staining, tissue transglutaminase and aminopropeptide of collagen type III were localized to fibroblasts running within cords of Dupuytren's fascia. Quantitative analysis of transglutaminase revealed that activity levels were significantly higher in acutely contracting fascia than in chronic contractures. The results show that contractures in Dupuytren's fascia may be reflected by the level of transglutaminase activity in the tissue. Furthermore, it is suggested that isopeptide bond formation, involving collagen type III aminopropeptide moieties, may be the biochemical mechanism by which transglutaminase maintains the contracted state.

The existence of an inactive form of transglutaminase within metastasising tumours

Separation by anion exchange chromatography of detergent extracts from a poorly metastatic HSV-2-induced hamster fibrosarcoma, its highly metastatic variant and a highly metastatic rat fibrosarcoma indicated the presence of an inactive form of transglutaminase antigen, when eluent fractions were assayed for transglutaminase activity and antigen. This inactive antigenic transglutaminase was clearly separable from the particulate and cytosolic forms of the transglutaminase enzyme. Unlike tumours, its presence could not be demonstrated in extracts from normal rat liver. Measurement of activity levels during tumour growth indicated that the progression of the two highly metastatic tumours was accompanied by a decrease in cytosolic transglutaminase activity, whilst the activity of this enzyme form remained constant in the poorly metastatic tumour. Measurement of antigen levels indicated an inverse relationship between the level of inactive transglutaminase and the level of cytosolic transglutaminase activity, suggesting that the two forms are inter-related. Gel filtration indicated the molecular weight of the inactive form to be greater than both the particulate and cytosolic forms, and it was estimated to be 120 000. Partial proteolysis of the semi-purified inactive form, by either trypsin or thrombin, led to its activation and to the appearance of a transglutaminase similar in molecular weight and ionic mobility, both by anion-exchange chromatography and electrophoresis, to the cytosolic transglutaminase.