MicroRNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβRII

Atrial fibrosis serves as an important contributor to atrial fibrillation (AF). Recent data have suggested that microRNA‐30c (miR‐30c) is involved in fibrotic remodelling and cancer development, but the specific role of miR‐30c in atrial fibrosis remains unclear. The purpose of this study was to investigate the role of miR‐30c in atrial fibrosis and its underlying mechanisms through in vivo and in vitro experiments. Our results indicate that miR‐30c is significantly down‐regulated in the rat abdominal aortic constriction (AAC) model and in the cellular model of fibrosis induced by transforming growth factor‐β1 (TGF‐β1). Overexpression of miR‐30c in cardiac fibroblasts (CFs) markedly inhibits CF proliferation, differentiation, migration and collagen production, whereas decrease in miR‐30c leads to the opposite results. Moreover, we identified TGFβRII as a target of miR‐30c. Finally, transferring adeno‐associated virus 9 (AAV9)‐miR‐30c into the inferior vena cava of rats attenuated fibrosis in the left atrium following AAC. These data indicate that miR‐30c attenuates atrial fibrosis via inhibition of CF proliferation, differentiation, migration and collagen production by targeting TGFβRII, suggesting that miR‐30c might be a novel potential therapeutic target for preventing atrial fibrosis.     

Pivotal role for TGF-beta in infectious heart disease: The case of Trypanosoma cruzi infection and consequent Chagasic myocardiopathy

This paper summarizes recent data from the literature suggesting that transforming growth factor-β (TGF-β) participates at least in four different processes influencing development of myocardiopathy in Chagas disease, a major parasitic illness caused by Trypanosoma cruzi infection: (a) invasion of cardiac fibroblasts and myocytes; (b) intracellular parasite cycle; (c) regulation of inflammation and immune response; (d) fibrosis and heart remodeling during acute and chronic disease. All these effects point to an important role of TGF-β in Chagas disease myocardiopathy and suggest that monitoring the circulating levels of this cytokine could be of help in clinical prognosis and management of patients. Moreover, TGF-β-interfering therapies appear as interesting adjuvant interventions during acute and chronic phases of T. cruzi infection.     

Two callose synthases,GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility

Callose, a β-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1–12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.     

Islet neogenesis-associated protein pentadecapeptide (INGAP-PP): Mechanisms involved in its effect upon β-cell mass and function

The effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) administration to normal male hamsters upon serum glucose and triglyceride levels, β-cell mass and function was studied. INGAP-PP (500 μg) or saline was injected twice daily during 10 days. Both groups showed comparable body weight, serum glucose and triglyceride levels. INGAP-PP treated animals had significantly higher HOMA-IR and HOMA-β and their islets released more insulin in response to glucose; they had lower islet DNA content, significantly increased number of islets/unit area, β-cell replication rate and mass, cells co-expressing Pdx-1/INGAP and islets in contact with ducts, and decreased β-cell apoptosis rate. The percentage of cells expressing Pdx-1 alone or together with INGAP or insulin increased significantly in ducts. These animals also showed a significantly higher concentration of Pdx-1 and Ngn-3 mRNA and a lower number of INGAP-positive cells. In conclusion, INGAP-PP promoted a controlled and functionally active increase of β-cell mass; our data demonstrate for the first time the mechanism responsible for such changes; that Ngn-3 would be involved in INGAP-PP-induced neogenesis; and the existence of a negative feedback loop with endogenous INGAP-producing cells. Accordingly, INGAP-PP could be used to induce these effects in people with or at risk of developing diabetes.     

Efficient expression and purification of human interferon alpha2b in the methylotrophic yeast, Pichia pastoris

The human interferon alpha2b (hIFN-alpha2b) is the most widely used member of IFNalpha family, and it exerts many biological actions including broad-spectrum antiviral effects, inhibition of tumor cell proliferation and enhancement of immune functions. Herein, the cDNA coding for hIFN-alpha2b has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of hIFN-alpha2b has been achieved. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hIFN-alpha2b, a 18.8 kDa protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using Source Q ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 298 mg of the protein was obtained in high purity from 1l of the supernatant and its identity to hIFN-alpha2b was confirmed by NH(2)-terminal amino acid sequence analysis. The bioassay of the recombinant protein gave a specific activity of 1.9 x 10(9)IU/mg. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hIFN-alpha2b for both research and industrial purpose.     

Chemotaxonomic discrimination among the fungal genera Tolypocladium, beauveria and Paecilomyces

Fungi of the genera Beauveria and Paecilomyces were found to produce cyclotetradepsipeptides, beauverolides. Production of beauverolides was not detected at the genus Tolypocladium. Analysis of beauverolides therefore provides a very simple chemotaxonomic test which seems to be suitable for fast discrimination between the genera Beauveria vs Tolypocladium and complementing morphological examination. A GC-MS study of β-hydroxy acid distribution in the beauverolide hydrolyzates revealed that all strains prdouce γ-methyl-β-hydroxy acids only. Their occurrence thus cannot be used as a taxonomic marker of different species within the genera Beauveria and Paecilomyces.     

Molecular Systematics of Zopfiella and allied genera: evidence from multi-gene sequence analyses

This study aims to reveal the phylogenetic relationships of Zopfiella and allied genera in the Sordariales. Multiple gene sequences (partial 28 S rDNA, ITS/5.8 S rDNA and partial β-tubulin) were analysed using MP and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to infer phylogenies. Phylogenetic analyses show that currently recognised Zopfiella species are polyphyletic. Based on sequence analyses and morphology, it appears that Zopfiella should be restricted to species having ascospores with a septum in the dark cell. Our molecular analysis also shows that Zopfiella should be placed in Lasiosphaeriaceae rather than Chaetomiaceae. Cercophora and Podospora are also polyphyletic, which is in agreement with previous studies. Our analyses show that species possessing a Cladorrhinum anamorph are phylogenetically closely related. In addition, there are several strongly supported clades, characterised by species possessing divergent morphological characters. It is difficult to predict which characters are phylogenetically informative for delimiting these clades.     

Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway

Transforming growth factor-beta (TGF-beta) and its family are potent and multi-functional cytokines that affect various fundamental biological events. TGF-beta has a unique signaling pathway that is carried by Smad family, and many recent studies showed the extensive crosstalk between Smad pathway and other signaling pathway. There were also clear evidences for the involvement of oxidative events in TGF-beta signaling pathway. To elucidate the role of oxidative events in carrying TGF-beta signals, we examined the effect of various antioxidants on TGF-beta activities in osteoblastic cell line. Among the examined compounds, we found nordihydroguaiaretic acid (NDGA) has a unique and strong inhibitory effect on various TGF-beta activities. Since the majority of TGF-beta activities are mediated by Smad, we questioned whether NDGA blocks the Smad signaling pathway. The result showed that NDGA inhibits the translocation of Smad2 to the nucleus. Further study revealed the strong inhibitory effect of NDGA on the phosphorylation of Smad2. This result may be important for designing chemical modulators of TGF-beta and its family related events and may provide new insights into the action mechanism of antioxidant.     

The differential effect of Eriobotrya japonica hydrophilic leaf extract on cytokines production and modulation

Stimulating or modulating the release of cytokines by immunomodulators or immunostimulating agents is an attractive mode for treating several diseases such as viral infections. For instance, patients with viral infections may be in need of increasing or inducing T helper 1 (Th1) or proinflammatory cytokines, which ultimately activate T cytotoxic and Natural killer lymphocytes to kill virally infected cells. Of these agents, we found that Eriobotrya japonica hydrophilic leaf extract (EJHE) can induce and modulate cytokines in dose-dependent manner. Twenty-four hour exposure of increasing concentrations of EJHE increased significantly (p < 0.001) the production of IFN-γ and TNF-α, from PHA+LPS-stimulated whole blood. However, the production of IFN-γ and TNF-α plateaued at high EJHE concentrations (10–100 μg/ml). No significant changes in the production of IL-10 were seen. In addition, EJHE at 1 and 10 μg/ml reversed significantly (p < 0.01) the inhibitory effect of hydrocortisone on the IL-12 p70, IFN-γ and TNF-α production from PHAS+LPS stimulated whole blood. Without PHA and LPS, EJHE was found to induce significantly (p < 0.001) IFN-γ, IL-12 p70, TNF-α, and IL-10 from whole blood culture in concentration dependent manner. The maximum induction of IFN-γ, IL-12 p70, and TNF-α by EJHE was at 1 and 10 μg/ml. On the other hand, IL-10 induction kept increasing even at the highest concentration used (100 μg/ml) of EJHE. Furthermore, intra-peritoneal injection of EJHE into mice increased significantly serum cytokines level mainly at 10 and 100 μg/ml. Two-hour post i.p. injection, EJHE increased serum IFN-γ, TNF-α, and IL-10 to 750, 1000, and 250 pg/ml, respectively. However, 24 h post i.p. injection, the levels of TNF-α, and IL-10 were similar to basal levels but IFN-γ levels were 200 pg/ml. These results indicate that EJHE induces proinflammatory and Th1 cytokines in concentration dependent manner and the effect of this induction should be studied further in viral models to check the efficacy of such cytokine induction.     

Characterization of the oligosaccharide component of alpha3beta1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin alpha3beta1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, alpha3beta1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the alpha3 and beta1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In alpha3beta1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of alpha3beta1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex7HexNAc6FucSia4 was present. In a direct ligand binding assay, desialylated alpha3beta1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, alpha3beta1 integrin was shown to take part in T24 cell migration on fibronectin: anti-alpha3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of beta1,6 branched complex type glycans in this process. Our data show that alpha3beta1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.     

Tepidimonas thermarum sp. nov., a new slightly thermophilic betaproteobacterium isolated from the Elisenquelle in Aachen and emended description of the genus Tepidimonas

Several non-pigmented bacterial isolates, with an optimum growth temperature of about 50-55 degrees C, were recovered from the Elisenquelle at Aachen, Germany. Phylogenetic analysis of the 16S rRNA gene sequence of strains AA-1(T) and AA-2 indicated that these organisms represent a new species of the genus Tepidimonas. The major fatty acids of strains AA-1(T) and AA-2 are 16:0 and 16:1 omega7c. Ubiquinone 8 is the major respiratory quinone, the major polar lipids are phosphotidylethanolamine and phosphotidylglycerol. The new isolates are aerobic; thiosulfate is oxidized to sulfate in the presence of a metabolizable carbon source. The organism assimilated organic acids and amino acids, but did not assimilate carbohydrates or polyols. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strains AA-1(T) (=LMG 23094(T); =CIP 108777(T)) and AA-2 (=LMG 23095; =CIP 108778) represents a new species for which we recommend the name Tepidimonas thermarum.     

Intrinsic ligand binding properties of the human and bovine α,-interferon receptors

The Type I interferon receptor (IFN-αR) interacts with all IFN-αs, IFN-β and IFN-ω, and seems to be a multisubunit receptor. To investigate the role of a cloned receptor subunit (IFN-αR1), we have examined the intrinsic ligand binding properties of the bovine and human IFN-αR1 polypeptides expressed in Xenopus laevis oocytes. Albeit with different efficiencies, Xenopus oocytes expressing either the human or bovine IFN-αR1 polypeptide exhibit significant binding and formation of crosslinked complexes with human IFN-αA and IFN-αB. Thus, the IFN-αR1 polypeptide most likely plays a direct role in ligand binding.     

Alternative pathway for the role of furin in tumor cell invasion process. Enhanced MMP-2 levels through bioactive TGFbeta

The mammalian convertase furin plays a significant role in tumorigenesis and its overexpression was observed in a number of different cancer types. To date, however, few mechanisms of action have been described. Most of the information available concerns the invasion step and designates MT1-MMP, through the activation of MMP-2, as the bona fide substrate mediating furin activity. However, recent reports indicate furin-independent pathways for MT1-MMP activation. To gain further insights into the role of furin in the invasion process, we studied the in vitro invasive capacity of LoVo cells, a furin-deficient adenocarcinoma cell line transfected with wild-type furin. Furin complementation resulted in an increased cell invasiveness that correlated with their capacity to produce MMP-2. Chemical blockage of MMPs activity with BB-3103 or MMP-2-specific antibodies revealed that the increased invasive capacity of furin-complemented cells was mediated by MMP-2. Unexpectedly, furin complementation did not change the status of MT1-MMP expression or activation, but instead resulted in the production of mature and bioactive TGFbeta1. Western blot-analysis of TGFbeta1 fragmentation species indicated that TGFbeta maturation step required furin activity, whereas results from TGFbeta-inducible reporter assays in the presence of MMP inhibitors or exogenous MMP-2 suggested that the activation step was under MMP influence. In addition, blockage with TGFbeta neutralizing antibodies revealed that furin-induced invasiveness was mediated by endogenous production of TGFbeta. Taken together, our findings established the existence of a novel alternative/complementary pathway by which furin increases tumor cell invasion through an amplification/activation loop between MMP-2 and TGFbeta.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.