Dietary Gluten and Neurodegeneration: A Case for Preclinical Studies.

Although celiac disease (CD) is an autoimmune disease that primarily involves the intestinal tract, mounting evidence suggests that a sizeable number of patients exhibit neurological deficits. About 40% of the celiac patients with neurological manifestations have circulating antibodies against neural tissue transglutaminase-6 (tTG6). While early diagnosis and strict adherence to a gluten-free diet (GFD) have been recommended to prevent neurological dysfunction, better therapeutic strategies are needed to improve the overall quality of life. Dysregulation of the microbiota-gut-brain axis, presence of anti-tTG6 antibodies, and epigenetic mechanisms have been implicated in the pathogenesis. It is also possible that circulating or gut-derived extracellular structures and including biomolecular condensates and extracellular vesicles contribute to disease pathogenesis. There are several avenues for shaping the dysregulated gut homeostasis in individuals with CD, non-celiac gluten sensitivity (NCGS) and/or neurodegeneration. In addition to GFD and probiotics, nutraceuticals, such as phyto and synthetic cannabinoids, represent a new approach that could shape the host microbiome towards better prognostic outcomes. Finally, we provide a data-driven rationale for potential future pre-clinical research involving non-human primates (NHPs) to investigate the effect of nutraceuticals, such as phyto and synthetic cannabinoids, either alone or in combination with GFD to prevent/mitigate dietary gluten-induced neurodegeneration.

Bioengineering of microbial transglutaminase for biomedical applications.

Microbial transglutaminase (mTGase) is commonly known in the food industry as meat glue due to its incredible ability to "glue" meat proteins together. Aside from being widely exploited in the meat processing industries, mTGase is also widely applied in other food and textile industries by catalysing the formation of isopeptide bonds between peptides or protein substrates. The advancement of technology has opened up new avenues for mTGase in the field of biomedical engineering. Efforts have been made to study the structural properties of mTGase in order to gain an in-depth understanding of the structure-function relationship. This review highlights the developments in mTGase engineering together with its role in biomedical applications including biomaterial fabrication for tissue engineering and biotherapeutics.

Improvement of skin barrier dysfunction by Scutellaria baicalensis GEOGI extracts through lactic acid fermentation.

BACKGROUND: The development of an alternative medicine to treat atopic dermatitis (AD) from natural sources is necessary. AIMS: To improve skin barrier dysfunction by enhancing the differentiation of human keratinocytes with the fermented Scutellaria baicalensis. METHODS: Scutellaria baicalensis was fermented with Lactobacillus plantarum and extracted with 70% ethanol (FE). Antioxidant activities and the regulation of the gene expression related to keratinocyte differentiation were measured as well as its proliferation. RESULT: This work first proved that the FE had multiple activities, both increasing keratinocyte differentiation and proliferation: The FE greatly up-regulated expression of the genes of keratinocyte differentiation such as involucrin, keratin 10, and transglutaminase-1 (TG-1) up to 4.06-fold, which was 3 times higher than the 2 other extracts. The effect of baicalein on keratinocyte differentiation was also first found; however, its efficacy was lower than that of the fermented extract. The FE proved to effectively accelerate keratinocyte differentiation, rather than to initiate the differentiation, and also showed an ability of stimulating keratinocyte proliferation up to 2.8 × 106 viable cells/mL as well as 70.24 ng/mL of collagen production in fibroblasts. High efficacy of the FE was confirmed by synergistic effects of large amounts of various bioactive substances in the extracts as baicalein alone did not show remarkable effects and even positive controls had not much better activities than the FE. CONCLUSION: The fermented extract was able to improve skin barrier dysfunction, and the ointment with 1%-5% (v/v) of the extract be directly used for skin clinical trials to treat AD.

Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations.

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes.

The known two types of transglutaminases regulate immune and stress responses in white shrimp, Litopenaeus vannamei.

Transglutaminases (TGs) play critical roles in blood coagulation, immune responses, and other biochemical functions, which undergo post-translational remodeling such as acetylation, phosphorylation and fatty acylation. Two types of TG have been identified in white shrimp, Litopenaeus vannamei, and further investigation on their potential function was conducted by gene silencing in the present study. Total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory bursts (release of superoxide anion), superoxide dismutase activity, transglutaminase (TG) activity, haemolymph clotting time, and phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when shrimps were individually injected with diethyl pyrocarbonate-water (DEPC-H2O) or TG dsRNAs. In addition, haemolymph glucose and lactate, and haemocytes crustin, lysozyme, crustacean hyperglycemic hormone (CHH), transglutaminaseI (TGI), transglutaminaseII (TGII) and clotting protein (CP) mRNA expression were determined in the dsRNA injected shrimp under hypothermal stress. Results showed that TG activity, phagocytic activity and clearance efficiency were significantly decreased, but THC, hyaline cells (HCs) and haemolymph clotting time were significantly increased in the shrimp which received LvTGI dsRNA and LvTGI + LvTGII dsRNA after 3 days. However, respiratory burst per haemocyte was significantly decreased in only LvTGI + LvTGII silenced shrimp. In hypothermal stress studies, elevation of haemolymph glucose and lactate was observed in all treated groups, and were advanced in LvTGI and LvTGI + LvTGII silenced shrimp following exposure to 22 °C. LvCHH mRNA expression was significantly up-regulated, but crustin and lysozyme mRNA expressions were significantly down-regulated in LvTGI and LvTGI + LvTGII silenced shrimp; moreover, LvTGII was significantly increased, but LvTGI was significantly decreased in LvTGI silenced shrimp following exposure to 28 and 22 °C. Knockdown of LvTGI and LvTGI + LvTGII also significantly increased the mortality of L. vannamei challenged with the pathogen V. alginolyticus. The same consequences have been confirmed in LvTGII silenced shrimp in our previous study. These results indicate that LvTGI and LvTGII not only reveal a complementary effect in gene expression levels but also play a key function in the immune defence mechanism of shrimp, by regulating the haemolymph coagulation, immune parameters and immune related gene expression, and in the regulation of carbohydrate metabolism.

P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2.

Transglutaminases (denoted TG or TGM) are externalized from cells via an unknown unconventional secretory pathway. Here, we show for the first time that purinergic signaling regulates active secretion of TG2 (also known as TGM2), an enzyme with a pivotal role in stabilizing extracellular matrices and modulating cell-matrix interactions in tissue repair. Extracellular ATP promotes TG2 secretion by macrophages, and this can be blocked by a selective antagonist against the purinergic receptor P2X7 (P2X7R, also known as P2RX7). Introduction of functional P2X7R into HEK293 cells is sufficient to confer rapid, regulated TG2 export. By employing pharmacological agents, TG2 release could be separated from P2X7R-mediated microvesicle shedding. Neither Ca(2+) signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement. A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity. These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions.

Immunological and protective effects of Bordetella bronchiseptica subunit vaccines based on the recombinant N-terminal domain of dermonecrotic toxin.

Dermonecrotic toxin (DNT) produced by Bordetella bronchiseptica (B. bronchiseptica) can cause clinical turbinate atrophy in swine and induce dermonecrotic lesions in model mice. We know that the N-terminal of DNT molecule contains the receptor-binding domain, which facilitates binding to the target cells. However, we do not know whether this domain has sufficient immunogenicity to resist B. bronchiseptica damage and thereby to develop a subunit vaccine for the swine industry. In this study, we prokaryotically expressed the recombinant N-terminal of DNT from B. bronchiseptica (named DNT-N) and prepared it for the subunit vaccine to evaluate its immunogenicity. Taishan Pinus massoniana pollen polysaccharide (TPPPS), a known immunomodulator, was used as the adjuvant to examine its immune-conditioning effects. At 49 d after inoculation, 10 mice from each group were challenged with B. bronchiseptica, and another 10 mice were intradermally challenged with native DNT, to examine the protection imparted by the vaccines. The immune parameters (T-lymphocyte counts, cytokine secretions, serum antibody titers, and survival rates) and skin lesions were determined. The results showed that pure DNT-N vaccine significantly induced immune responses and had limited ability to resist the B. bronchiseptica and DNT challenge, whereas the mice administered with TPPPS or Freund's incomplete adjuvant vaccine could induce higher levels of the above immune parameters. Remarkably, the DNT-N vaccine combined with TPPPS adjuvant protected the mice effectively to prevent B. bronchiseptica infection. Our findings indicated that DNT-N has potential for development as an effective subunit vaccine to counteract the damage of B. bronchiseptica infection, especially when used conjointly with TPPPS.

The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: Activity and gene transcription.

The non-specific immune parameters, disease resistance and immune genes expressions in Macrobrachium rosenbergii were evaluated at 120 days of post feeding the diets containing the extracts of banana, Musa acuminate, fruit's peel (banana peels extract, BPE) at 0, 1.0, 3.0 and 6.0 g kg(-1). Results showed that prawns fed with a diet containing BPE at the level of 1.0, 3.0 and 6.0 g kg(-1) for 120 days had a significantly higher survival rate (30.0%, 40.0% and 56.7%, respectively) than those fed with the control diet after challenge with Lactococcus garvieae for 144 h, and the respective relative survival percentages were 22.2%, 33.3%, and 51.9%, respectively. Dietary BPE supplementation at 3.0 and/or 6.0 g kg(-1) for 120 days showed a significant increase total haemocyte count (THC), granular cell (GC), superoxide dismutase (SOD) activity, phenoloxidase (PO) activity, transglutaminase (TG) activity, and phagocytic activity and clearance efficiency to L. garvieae infection, and meanwhile, the significant decrease in haemolymph clotting times and respiratory bursts (RBs) per haemocyte of prawns were revealed. Furthermore, the mRNA expressions of prophenoloxidase (proPO), lipopolysaccharide and ß-1,3-glucan binding protein (LGBP), peroxinectin (PE), transglutaminase (TG), and crustin (CT) were significantly increased. We therefore recommend that BPE can be used as an immunomodulator for prawns through dietary administration at 6.0 g kg(-1) for a long term (over 120 days) to modify immune responses and genes expression following the enhanced resistance against pathogens.

Antioxidant properties of Berberis aetnensis C. Presl (Berberidaceae) roots extract and protective effects on astroglial cell cultures.

Berberis aetnensis C. Presl (Berberidaceae) is a bushy-spiny shrub common on Mount Etna (Sicily). We demonstrated that the alkaloid extract of roots of B. aetnensis C. Presl contains prevalently berberine and berbamine, possesses antimicrobial properties, and was able to counteract the upregulation evoked by glutamate of tissue transglutaminase in primary rat astroglial cell cultures. Until now, there are no reports regarding antioxidant properties of B. aetnensis C. Presl collected in Sicily. Air-dried, powdered roots of B. aetnensis C. Presl were extracted, identified, and quantified by HPLC. We assessed in cellular free system its effect on superoxide anion, radicals scavenging activity of antioxidants against free radicals like the 1,1-diphenyl-2-picrylhydrazyl radical, and the inhibition of xanthine oxidase activity. In primary rat astroglial cell cultures, exposed to glutamate, we evaluated the effect of the extract on glutathione levels and on intracellular production of reactive oxygen species generated by glutamate. The alkaloid extract of B. aetnensis C. Presl inhibited superoxide anion, restored to control values, the decrease of GSH levels, and the production of reactive oxygen species. Potent antioxidant activities of the alkaloid extract of roots of B. aetnensis C. Presl may be one of the mechanisms by which the extract is effective against health disorders associated to oxidative stress.

Novel anti-nociceptive effects of cardamonin via blocking expression of cyclooxygenase-2 and transglutaminase-2.

Recently, we reported that Alpinia katsumadai (AK) has anti-nociceptive activity in vivo and that cardamonin (CDN) from AK suppresses the activity and expression of transglutaminase-2 (Tgase-2). However, it remains unknown whether CDN contributes to the anti-nociceptive activities of AK in vivo. We examined the anti-inflammatory effects of CDN in MG63 osteoblast-like cells and Raw264.7 macrophage-like cells treated with interleukin-1ß treatment. CDN suppressed the expression of Tgase-2, cyclooxygenase-2 (COX-2), and p65 (nuclear factor-κB) in a concentration-dependent manner, and restored the expression of IκB in MG63 and Raw264.7 cells. However, CDN did not inhibit the activity of COX-2. Gene silencing of Tgase-2 reduced the COX-2 expression in MG63 cells. Phenylbenzoquinone (PBQ)-induced writhing, carrageenan-induced hyperalgesia, and rota-rod test were used to evaluate the anti-nociceptive activity in vivo. CDN (3-30 mg/kg, orally administered) significantly inhibited PBQ-induced writhing. CDN also produced a significant, dose-dependent increase in the withdrawal response latencies in carrageenan-induced hyperalgesia. The effects of CDN on PBQ-induced writhing were not caused by impaired motor functions. These results suggest that CDN might be helpful in controlling the pain from inflammatory diseases.

Identification and cloning of the second type transglutaminase from Litopenaeus vannamei, and its transcription following pathogen infection and in relation to the haemolymph coagulation.

Complementary (c)DNA encoding transglutaminaseII (TGII) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134), tiger shrimp, Penaeus monodon (AAV49005; AAO33455), kuruma shrimp, Marsupenaeus japonicus (BAD36808) and Pacifastacus leniusculus (AAK69205) TG. The 2405-bp cDNA contained an open reading frame (ORF) of 2292 bp, a 31-bp 5'-untranslated region (UTR), and an 82-bp 3'-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (764 aa) was 85.9 kDa with an estimated pI of 5.32. The L. vannamei TGII (abbreviated LvTGII) contains a typical TG-like homologue, two putative integrin binding motif (RGD and KGD), and five calcium-binding sites; three catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two groups, STGI and STGII, and LvTGII is more closely related to STGII than to STGI. LvTGII mRNA was detected in all tested tissues of L. vannamei, and was highly expressed in haemocytes. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant increase of LvTGI and LvTGII mRNA expression at 6 h followed by a notable decrease at 24 h in LvTGI and a continually increase in LvTGII indicating a complementary effect, which implied that both LvTGs involved in the immune response of shrimp, and LvTGII was more important in the later defense response. The gene silencing of LvTGII in shrimp significantly decreased LvTGII expression and TG activity of haemocytes, and significantly increased clotting time of haemolymph, suggests that the cloned LvTGII is a clotting enzyme involved in haemolymph coagulation of L. vannamei. In conclusion, the cloned LvTGII is a clotting enzyme involved in coagulation of haemolymp and immune response of white shrimp, L. vannamei.

The antioxidant property of chitosan green tea polyphenols complex induces transglutaminase activation in wound healing.

The present study examined, for the first time, the in vitro wound healing potential of chitosan green tea polyphenols (CGP) complex based on the activation of transglutaminase (TGM) genes in epidermal morphogenesis. Response surface methodology was applied to determine the optimal processing condition that gave maximum extraction of green tea polyphenols. The antioxidant activity, scavenging ability, and chelating ability were studied and expressed as average EC50 values of CGP and other treatments. In silico analysis and gene coexpression network was subjected to the TGM sequences analysis. The temporal expressions of TGMs were profiled by semi-quantitative reverse transcription (RT)-PCR technology within 10 days after wounding and 2 days postwounding. CGP showed the effectiveness of antioxidant properties, and the observations of histopathological photography showed advanced tissue granulation and epithelialization formation by CGP treatment. In silico and coexpression analysis confirmed the regulation via TGM gene family in dermatological tissues. RT-PCR demonstrated increased levels of TGM1-3 expression induced by CGP treatment. The efficacy of CGP in wound healing based on these results may be ascribed to its antioxidant properties and activation of the expression of TGMs, and is, thus, essential for the facilitated repair of skin injury.

Transglutaminase inhibitors attenuate vascular calcification in a preclinical model.

OBJECTIVE: In vitro, transglutaminase-2 (TG2)-mediated activation of the ß-catenin signaling pathway is central in warfarin-induced calcification, warranting inquiry into the importance of this signaling axis as a target for preventive therapy of vascular calcification in vivo. METHODS AND RESULTS: The adverse effects of warfarin-induced elastocalcinosis in a rat model include calcification of the aortic media, loss of the cellular component in the vessel wall, and isolated systolic hypertension, associated with accumulation and activation of TG2 and activation of ß-catenin signaling. These effects of warfarin can be completely reversed by intraperitoneal administration of the TG2-specific inhibitor KCC-009 or dietary supplementation with the bioflavonoid quercetin, known to inhibit ß-catenin signaling. Our study also uncovers a previously uncharacterized ability of quercetin to inhibit TG2. Quercetin reversed the warfarin-induced increase in systolic pressure, underlying the functional consequence of this treatment. Molecular analysis shows that quercetin diet stabilizes the phenotype of smooth muscle and prevents its transformation into osteoblastic cells. CONCLUSIONS: Inhibition of the TG2/ß-catenin signaling axis seems to prevent warfarin-induced elastocalcinosis and to control isolated systolic hypertension.

Enhancement of transglutaminase production in Streptomyces mobaraensis as achieved by treatment with excessive MgCl2.

In this study, we first tested the capacity for eight different salts as stress-mediated bioprocesses in the production of transglutaminase (TGase). A significant effect on the cell growth and TGase production was obtained with the highest yield of TGase being observed at 96 h of incubation (4.3 U/ml) when the basic medium was supplemented 0.10 M MgCl(2), as opposed to that observed with the basic medium control (2.1 U/ml at 120 h). Data from Western blot assays showed that transformation of pro-TGase to its mature enzyme occurred more rapidly in MgCl(2) medium. Furthermore, total protease, metalloprotease, and serine protease were also synthesized at a faster rate in the medium containing MgCl(2). The results demonstrate that MgCl(2) enhanced the production of key proteases involved in the activation of TGase biosynthesis. To explore the mechanism, viability assay was performed. The results show that MgCl(2) induced the mycelia differentiation, decreased cell growth rate, and stimulated cell death. We argue that TGase production was promoted by the stimulation of mycelium differentiation induced by MgCl(2) stress.

TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2.

The decision between survival and death in cells exposed to TNF relies on a highly regulated equilibrium between proapoptotic and antiapoptotic factors. The TNF-activated antiapoptotic response depends on several transcription factors, including NF-κB and its RelA/p65 subunit, that are activated through phosphorylation-mediated degradation of IκB inhibitors, a process controlled by the IκB kinase complex. Genetic studies in mice have identified the IκB kinase-related kinase TANK-binding kinase 1 (TBK1; also called NAK or T2K) as an additional regulatory molecule that promotes survival downstream of TNF, but the mechanism through which TBK1 exerts its survival function has remained elusive. Here we show that TBK1 triggers an antiapoptotic response by controlling a specific RelA/p65 phosphorylation event. TBK1-induced RelA phosphorylation results in inducible expression of plasminogen activator inhibitor-2 (PAI-2), a member of the serpin family with known antiapoptotic activity. PAI-2 limits caspase-3 activation through stabilization of transglutaminase 2 (TG2), which cross-links and inactivates procaspase-3. Importantly, Tg2(-/-) mice were found to be more susceptible to apoptotic cell death in two models of TNF-dependent acute liver injury. Our results establish PAI-2 and TG2 as downstream mediators in the antiapoptotic response triggered upon TBK1 activation.

A unique role for heat shock protein 70 and its binding partner tissue transglutaminase in cancer cell migration.

Cell migration is essential for several important biological outcomes and is involved in various developmental disorders and disease states including cancer cell invasiveness and metastasis. A fundamental step in cell migration is the development of a leading edge. By using HeLa carcinoma cells as an initial model system, we uncovered a surprising role for the heat shock protein 70 (Hsp70) and its ability to bind the protein cross-linking enzyme, tissue transglutaminase (tTG), in cancer cell migration. Treatment of HeLa cells with EGF results in the activation of a plasma membrane-associated pool of tTG and its redistribution to the leading edges of these cells, which are essential events for EGF-stimulated HeLa cell migration. However, we then found that the ability of tTG to be localized to the leading edge is dependent on Hsp70. Similarly, the localization of tTG to the leading edges of MDAMB231 breast carcinoma cells, where it also plays an essential role in their migration, has a strict requirement for Hsp70. Treatment of these different cell lines with inhibitors against the ATP hydrolytic activity of Hsp70 prevented tTG from localizing to their leading edges and thereby blocked EGF-stimulated HeLa cell migration, as well as the constitutive migration normally exhibited by MDAMB231 cells. These findings highlight a new and unconventional role for the chaperonin activity of Hsp70 in the localization of a key regulatory protein (tTG) at the leading edges of cancer cells and the important consequences that this holds for their ability to migrate.

Identification and cloning of a transglutaminase from giant freshwater prawn, Macrobrachium rosenbergii, and its transcription during pathogen infection and moulting.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5'-untranslated region (UTR), and a 316-bp 3'-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii.

Differential cytotoxic responses to low- and high-dose photodynamic therapy in human gastric and bladder cancer cells.

Here, we present differential cytotoxic responses to two different doses of photodynamic therapies (PDTs; low-dose PDT [LDP] and high-dose PDT [HDP]) using a chlorin-based photosensitizer, DH-II-24, in human gastric and bladder cancer cells. Fluorescence-activated cell sorting analysis using Annexin V and propidium iodide (PI) showed that LDP induced apoptotic cell death, whereas HDP predominantly caused necrotic cell death. The differential cytotoxic responses to the two PDTs were further confirmed by a DiOC(6) and PI double-staining assay via confocal microscopy. LDP, but not HDP, activated caspase-3, which was inhibited by Z-VAD, Trolox, and BAPTA-AM. LDP and HDP demonstrated opposite effects on intracellular reactive oxygen species (ROS)/Ca(2+) signals; LDP stimulated intracellular ROS production, contributing to a transient increase of intracellular Ca(2+) , whereas HDP induced a massive and prolonged elevation of intracellular Ca(2+) responsible for the transient production of intracellular ROS. In addition, the two PDTs also increased in situ transglutaminase 2 (TG2) activity, with a higher stimulation by HDP, and this increase in activity was prevented by Trolox, BAPTA-AM, and TG2-siRNA. LDP-induced apoptotic cell death was strongly inhibited by Trolox and TG2-siRNA and moderately suppressed by BAPTA-AM. However, HDP-mediated necrotic cell death was partially inhibited by BAPTA-AM but not by TG2-siRNA. Thus, these results demonstrate that LDP and HDP induced apoptotic and necrotic cell death by differential signaling mechanisms involving intracellular Ca(2+) , ROS, and TG2.

Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvß3 integrin-induced Src tyrosine kinase signaling.

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFα production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the IκBα. Low basal levels of IκBα explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-κB. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.