Diabetic peripheral neuropathy (DPN) is one of the most common diabetic chronic complications. The pathogenesis of DPN is complex and involves an intertwined array of mechanisms. The purposes of this study were to evaluate the association of oxidative stress and vascular risk factors with the prevalence of DPN and to determine the role of these biochemical parameters in the prognosis of DPN. One hundred patients with type 2 diabetes mellitus and 40 clinically healthy individuals were evaluated. The patients were divided into two groups. Group 1 included 40 diabetic patients without peripheral neuropathy, and group 2 consisted of 60 patients with DPN. Erythrocytes glutathione (GSH) level, plasma malondialdehyde (MDA), nitrite/nitrate (NOx) and homocysteine (Hcy) levels as well as serum ceruloplasmin (Cp), total antioxidants (TAO), endothelin-1 (ET-1) levels and γ-glutamyl transferase (GGT) activity were estimated. A significant decrease of erythrocyte GSH was observed in groups 1 and 2 relative to the controls. An increase in glycosylated haemoglobin (HbA1c), MDA, NOx, GGT, Cp, TAO, Hcy and ET-1 was noted in patients with DPN. In conclusion, oxidative stress biomarkers and vascular risk factors could be important in the pathogenesis of DPN. The measurement of serum GGT and Hcy in addition to HbA1c and disease duration could facilitate the early detection of neuropathy in diabetic patients. Copyright © 2012 John Wiley & Sons, Ltd.

It is well established that the development of insulin resistance shows a temporal sequence in different organs and tissues. Moreover, considering that the main aspect of insulin resistance in liver is a process of glucose overproduction from gluconeogenesis, we investigated if this metabolic change also shows temporal sequence. For this purpose, a well-established experimental model of insulin resistance induced by high-fat diet (HFD) was used. The mice received HFD (HFD group) or standard diet (COG group) for 1, 7, 14 or 56 days. The HFD group showed increased (P < 0.05 versus COG) epididymal, retroperitoneal and inguinal fat weight from days 1 to 56. In agreement with these results, the HFD group also showed higher body weight (P < 0.05 versus COG) from days 7 to 56. Moreover, the changes induced by HFD on liver gluconeogenesis were progressive because the increment (P < 0.05 versus COG) in glucose production from l-lactate, glycerol, l-alanine and l-glutamine occurred 7, 14, 56 and 56 days after the introduction of the HFD schedule, respectively. Furthermore, glycaemia and cholesterolemia increased (P < 0.05 versus COG) 14 days after starting the HFD schedule. Taken together, the results suggest that the intensification of liver gluconeogenesis induced by an HFD is not a synchronous ‘all-or-nothing process’ but is specific for each gluconeogenic substrate and is integrated in a temporal manner with the progressive augmentation of fasting glycaemia. Copyright © 2012 John Wiley & Sons, Ltd.

Triptolide is a diterpenoid triepoxide derived from the traditional Chinese medical herb Tripterygium wilfordii. In the present study, we demonstrated that this phytochemical attenuated colon cancer growth in vitro and in vivo. Using a proteomic approach, we found that 14-3-3 epsilon, a cell cycle- and apoptosis-related protein, was altered in colon cancer cells treated with triptolide. In this regard, triptolide induced cleavage and perinuclear translocation of 14-3-3 epsilon. Taken together, our findings suggest that triptolide may merit investigation as a potential therapeutic agent for colon cancer, and its anticancer action may be associated with alteration of 14-3-3 epsilon. Copyright © 2012 John Wiley & Sons, Ltd.

The signal transducers and activators of transcription 3 (Stat3) has been detected in many types of cancer and plays an important role in tumour-cell survival, proliferation, self-renewal and invasion. To address the possibility that Stat3 may be involved in the metastasis and prognosis in lingual squamous cell carcinoma, we examined whether Stat3 expression associates with metastasis or survival rate in human clinical samples. We found that there was a significant correlation between Stat3 expression and lymph node metastasis (P = 0.009), stages (P = 0.029), recurrence (P = 0.0032) and death (P = 0.0356). The specific knockdown of Stat3 by RNA interference strongly inhibited the motile and invasion activity of tumour cells. Our results suggest that Stat3 is involved in the motility, metastasis and progression in human lingual squamous cell carcinoma, and thus, it may be a therapeutic target for human lingual squamous cell carcinoma. Copyright © 2012 John Wiley & Sons, Ltd.

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS–sGC–cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague–Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight-matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle + septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α₁ mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension. Copyright © 2012 John Wiley & Sons, Ltd.

The aim of this study was to investigate the effects of exercise in multiple organs of rats treated with doxorubicin. Male adult Wistar rats were distributed into the following groups: sedentary + NaCl; exercise + NaCl; sedentary + doxorubicin; and exercise + doxorubicin. Animals were sacrificed 2 days following injections. Central fragments from heart, liver, and kidney were collected and minced in 0.9% NaCl being cellular suspensions used for the single-cell gel (comet) assay. The results showed that exercise was able to prevent genotoxicity induced by doxorubicin in heart cells. By contrast, exercise was not able to prevent genotoxicity induced by doxorubicin in liver cells. The same occurred to kidney cells, i.e. no statistically significant differences (p > 0.05) were found when compared with groups not exposed to doxorubicin. Taken together, our results support the idea that exercise could contribute to the protective effect against genotoxicity induced by doxorubicin in heart cells. Copyright © 2012 John Wiley & Sons, Ltd.

During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D_9k, claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca²⁺-ATPase (PMCA)-1b and Na⁺/Ca²⁺ exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells. Copyright © 2012 John Wiley & Sons, Ltd.

Organochalcogens are extensively produced and employed by industry and agriculture, and the risk of occupational and environmental toxicity to them has been poorly understood. Here, we investigated the acute effect of a new organochalcogen 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one on biochemical and hematological parameters in male Wistar rats. The animals were treated with a single intraperitoneal injection of the organochalcogen at doses of 125, 250 or 500 µg·kg^–1. After 60 min, the animals were sacrificed by decapitation, and the trunk blood was collected for determination of glucose, triglycerides, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase, lactate dehydrogenase, urea, creatinine, C-reactive protein, red blood cells, hematocrit, hemoglobin and white blood cells (WBC). Our results showed a reduction in cholesterol levels in all treated groups, an increase in ALT activity at doses of 250 and 500 µg·kg^–1, a decrease of hemoglobin and an increase in WBC in animals that received 250 and 500 µg·kg^–1 of the organoselenium. In addition, we observed an increase in neutrophil counts at 125 µg·kg^–1 dose and a decrease at 500 µg·kg^–1 dose. We also verified an increase in lymphocyte counts at the dose of 500 µg·kg^–1. Thus, the present study shows that the acute treatment with this new organochalcogen causes biochemical changes and hematological disorders in male rats. Copyright © 2012 John Wiley & Sons, Ltd.

JMJD3, a Jumonji C family histone demethylase, is induced by transcription factor, nuclear factor-kappa B (NF-κB), in response to various stimuli. JMJD3 is crucial for erasing histone-3 lysine-27 trimethylation (H3K27me3), a modification associated with transcriptional repression and is responsible for the activation of a diverse set of genes. Here, we identify the genes in human leukaemia monocyte (THP-1) human monocytic cells that are significantly affected by the stable knockdown (kd) of JMJD3. Global gene expression levels were detected in stable JMJD3 knockdown THP-1 cells and in tumor necrosis factor-alpha (TNF-α)-stimulated JMJD3-kd THP-1 cells by using a 12-plex NimbleGen human whole genome array. In addition, datasets were analysed by using Ingenuity Pathway Analysis. Stable knockdown of JMJD3 in THP-1 cells affected particularly in expression levels and in downstream effects on inflammatory signalling pathways. JMJD3 attenuation down-regulates various key genes in NF-κB, chemokine and CD40 signalling, and mostly affects inflammatory disease response molecules. In addition, chromatin immunoprecipitation revealed that JMJD3-kd could inhibit several NF-κB-regulated inflammatory genes by recruiting repressive histone-3 lysine-27 trimethylation to their promoters. Moreover, this study significantly highlights the connexion of NF-κB with JMJD3, which suggests an epigenetic regulation in different signalling pathways. Finally, this study establishes novel JMJD3 targets through Ingenuity Pathway Analysis. Copyright © 2012 John Wiley & Sons, Ltd.

Insulin and insulin-like growth factor 1 (IGF-1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF-1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF-1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF-1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF-1 promote osteoblast differentiation and mineralization in vitro, IGF-1, but not insulin, can induce osteoblast proliferation. The IGF-1-induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF-1-mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3-kinase inhibitor. Osteocalcin, an osteoblast-specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose- and time-dependent manner after insulin treatment, whereas it was decreased with IGF-1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF-1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.

Indoleamine 2,3-dioxygenase (IDO) converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, to investigate the effects of IDO in carbon tetrachloride (CCl₄)–induced hepatitis model, the levels of IDO enzymic activities in the mock group, the control group and the 1-methyl- d-tryptophan (1-MT)–treated group were confirmed by determination of l-kynurenine concentrations. Serum alanine aminotransferase levels in 1-MT-treated rats after CCl₄ injection significantly increased compared with those in mock and control groups. In CCl₄-induced hepatitis models, tumour necrosis factor-α (TNF-α) is critical in the development of liver injury. The mRNA expression and secretion levels of TNF-α in the liver from 1-MT-treated rats were more enhanced compared with those in the mock and the control groups. Moreover, the levels of cytokine and chemokine from mock, control group and 1-MT-treated rats after treated with CCl₄ were analyzed by ELISA, and the level of interleukin-6 was found to increase in 1-MT-treated rats. It was concluded that the deficiency of IDO exacerbated liver injury in CCl₄-induced hepatitis and its effect may be connected with TNF-α and interleukin-6. Copyright © 2012 John Wiley & Sons, Ltd.

P-bodies (processing bodies) are observed in different organisms such as yeast, Caenorhabditis elegans and mammals. A typical eukaryotic cell contains several types of spatially formed granules, such as P-bodies, stress granules and a variety of ribonucleoprotein bodies. These microdomains play important role in mRNA processing, including RNA interference, repression of translation and mRNA decay. The P-bodies components as well as stress granules may play an important role in host defense against viral infection. The complete set of P-bodies protein elements is still poor known. They contain conserved protein core limited to different organisms or to stress status of the cell. P-bodies are related also to some neuronal mRNA granules as well as to maternal RNA granules or male germ cell granules. In this mini-review, we focus on the structure of P-bodies and their function in the mRNA utilization and processing because of the high mRNA's dynamics between different cellular compartments and its key role in modulation of gene expression. Copyright © 2012 John Wiley & Sons, Ltd.

This study was designed to investigate whether the short-term extracorporeal shockwave lithotripsy (ESWL) exposure to kidney produces an oxidative stress and a change in some trace element levels in liver and diaphragm muscles of rats. Twelve male Wistar albino rats were divided randomly into two groups, each consisting of six rats. The animals in the first group did not receive any treatment and served as control group. The right-side kidneys of animals in group 2 were treated with two-thousand 18 kV shock waves while anesthetized with 50 mg kg⁻¹ ketamine. The localization of the right kidney was achieved after contrast medium injection through a tail vein under fluoroscopy control. The animals were killed 72 h after the ESWL treatment, and liver and diaphragm muscles were harvested for the determination of tissue oxidative stress and trace element levels. Although the malondialdehyde level increased, superoxide dismutase and glutathione peroxidase enzyme activities decreased in the livers and diaphragm muscles of ESWL-treated rats. Although glutathione level increased in liver, it decreased in diaphragm muscles of ESWL-treated animals. Fe, Mg and Mn levels decreased, and Cu and Pb levels increased in the livers of ESWL-treated animals. Fe and Cu levels increased, and Mg, Pb, Mn and Zn levels decreased in the diaphragm muscles of ESWL-treated animals. It also causes a decrease or increase in many mineral levels in liver and diaphragm muscles, which is an undesirable condition for the normal physiological function of tissues. Copyright © 2012 John Wiley & Sons, Ltd.

α-Tocopherol (α-Toc) is involved in various physiologic processes, which present antioxidant and neuroprotective properties. High-fat diets have an important role in neurodegenerative diseases and neurological disturbances. This study aimed to investigate the effects of treatment with α-Toc and the consumption of high-fat diets on ectonucleotidase activities in synaptosomes of cerebral cortex, hippocampus and striatum of rats. Animals were divided into four different groups, which received standard diet (control), high-fat saturated diet (HF), α-Toc and high-fat saturated diet plus α-Toc (α-Toc + HF). High-fat saturated diet was administered ad libitum and α-Toc by gavage using a dose of 50 mg·kg^–1. After 3 months of treatment, animals were submitted to euthanasia, and cerebral cortex, hippocampus and striatum were collected for biochemical assays. Results showed that adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) hydrolysis in the cerebral cortex, hippocampus and striatum were decreased in HF in comparison to the other groups (P < 0·05). When rats that received HF were treated with α-Toc, the activity of the ectonucleotidases was similar to the control. ATP, ADP and AMP hydrolysis in the cerebral cortex, hippocampus and striatum were increased in the α-Toc group when compared with the other groups (P < 0·05). These findings demonstrated that the HF alters the purinergic signaling in the nervous system and that the treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition. Copyright © 2012 John Wiley & Sons, Ltd.

Hemichannels, which are one half of the gap junction channels, have independent physiological roles. Although hemichannels consisting of connexins are more widely documented, hemichannels of pannexins, proteins homologous to invertebrate gap junction proteins also have been studied. There are at least 21 different connexin and three pannexin isotypes. This variety in isotypes results in tissue-specific hemichannels, which have been implicated in varied events ranging from development, cell survival, to cell death. Hemichannel function varies with its spatio-temporal opening, thus demanding a refined degree of regulation. This review discusses the activity of hemichannels and the molecules released in different physiological states and their impact on tissue functioning. Copyright © 2012 John Wiley & Sons, Ltd.

Based on the fact that vitamin A in clinical doses is a potent pro-oxidant agent to the lungs, we investigated here the role of nitric oxide (NO^•) in the disturbances affecting the lung redox environment in vitamin A-treated rats (retinol palmitate, doses of 1000–9000 IU·kg^–1·day^–1) for 28 days. Lung mitochondrial function and redox parameters, such as lipid peroxidation, protein carbonylation and the level of 3-nytrotyrosine, were quantified. We observed, for the first time, that vitamin A supplementation increases the levels of 3-nytrotyrosine in rat lung mitochondria. To determine whether nitric oxide (NO •) or its derivatives such as peroxynitrite (ONOO-) was involved in this damage, animals were co-treated with the nitric oxide synthase inhibitor L-NAME (30 mg·kg^–1, four times a week), and we analysed if this treatment prevented (or minimized) the biochemical disturbances resulting from vitamin A supplementation. We observed that L-NAME inhibited some effects caused by vitamin A supplementation. Nonetheless, L-NAME was not able to reverse completely the negative effects triggered by vitamin A supplementation, indicating that other factors rather than only NO• or ONOO- exert a prominent role in mediating the redox effects in the lung of rats that received vitamin A supplementation. Copyright © 2011 John Wiley & Sons, Ltd.

Blood metabolic parameters of Walker-256 tumour-bearing rats, on days 5, 8, 11 and 14 after implantation of tumour, were compared with those of rats without tumour fed ad libitum (free-fed control) or with reduced feeding (pair-fed control), similar to the anorexic tumour-bearing rats. Cachexia parameters and tumour mass also were investigated. In general, especially on day 14 after implantation of tumour, there was reduction of body mass, gastrocnemius muscle mass, food intake and glycemia and increase of blood triacylglycerol, free fatty acids, lactate and urea, compared with free-fed controls rats. These changes did not occur in pair-fed control, except a slight reduction of glycemia. Pair-fed control showed no significant changes in blood cholesterol and glycerol in comparison with free-fed control, although there was reduction of cholesterol and increase of blood glycerol on day 14 after tumour implantation compared with pair-fed control. The results demonstrate that, besides the characteristic signs of the cachexia syndrome such as anorexia, weight loss and muscle catabolism, Walker-256 tumour-bearing rats show several blood metabolic alterations, some of which begin as early as day 5 after implantation of tumour, and are accentuated during the development of cachexia. Evidence that the alterations of blood metabolic parameters of tumour-bearing rats were not found in pair-fed control indicate that they were not caused by decreased food intake. These changes were probably mediated by factors produced by tumour or host tissue in response to the presence of tumour. Copyright © 2011 John Wiley & Sons, Ltd.

The process of placental iron transfer is an important physiological process during pregnancy. However, the molecular mechanism of placental iron transport has not been completely elucidated until now. Ferroportin 1 (FPN1) and hephaestin (Heph) have been identified as the important molecules involved in duodenal iron export. However, whether they participate in the placental iron efflux has been undefined until now. In this study, the BeWo cells were treated with desferrioxamine and Holo-transferrin human in different concentrations and harvested at 48 and 72 h. The mRNA expression of FPN1 and Heph was detected with quantitative real-time polymerase chain reaction, and the protein expression was detected with western blots. The results showed an up-regulated FPN1 expression with desferrioxamine treatment and down-regulated expression with Holo-transferrin human supplementation. However, the change of FPN1 expression at protein level was limited. Heph expression enhanced when cells were treated with desferrioxamine although the quantity of Heph expression was low. Heph expression showed no significant change with Holo-transferrin human supplementation. It indicates that FPN1 may participate in placental iron transport, and placental FPN1 expression is obviously not dependent on the iron regular element/iron regular protein regulation. An alternatively spliced FPN1 isoform that lacks an iron regular element may be the predominant expression in BeWo cells. It also demonstrates that Heph is active in placenta but may not play a key role in placental iron transport because it is not the main part of placental copper oxidase. Copyright © 2011 John Wiley & Sons, Ltd.

Silibinin, an effective anti-cancer and chemopreventive agent in various epithelial cancer models, has been reported to inhibit cancer cell growth through mitogenic signalling pathways including cervical cancer. However, the underlying mechanisms are still not well elucidated. Here, we assessed the effect of silibinin on human cervical carcinoma cell cycle modulation, apoptosis induction and associated molecular alterations by employing HeLa cell line. Silibinin treatment of HeLa cells resulted in a G2 arrest and induced a decrease in cyclin-dependent kinases involved in both G1 and G2 progression. In addition, silibinin showed a dose-dependent and a time-dependent apoptotic death in HeLa cells in both the mitochondrial pathway and the death receptor-mediated pathway, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in cervical cancer pre-clinical models. Copyright © 2011 John Wiley & Sons, Ltd.

Alzheimer's disease (AD) is accompanied by oxidative stress in the brain. Because the brain tissue is rich in polyunsaturated fatty acids, it is prone to the free radical attack resulting in lipid peroxidation. Intermediates of lipid peroxidation may diffuse from the primary site, cross the blood–brain barrier and modify erythrocyte membranes in the bloodstream. We exposed isolated erythrocyte membranes from patients with AD and the control group to in vitro free radical damage and monitored the accumulation of the end products of lipid peroxidation, lipofuscin-like pigments (LFPs), by fluorescence spectroscopy. LFPs were analyzed by means of tridimensional and synchronous fluorescence spectroscopy. The levels of LFP formed during in vitro peroxidation were significantly higher in erythrocyte membranes from patients with AD compared with the control group. Furthermore, the chemical composition of LFP in AD was different from the control group. The analysis of the specific modifications of erythrocyte membranes in AD is of great medical importance regarding the need of a diagnostic blood biomarker. Copyright © 2011 John Wiley & Sons, Ltd.

Studies revealed that Nijmegen Breakage Syndrome protein 1 (NBS1) plays an important role in maintaining genome stability, but the underlying mechanism is controversial and elusive. Our results using clinical samples showed that NBS1 was involved in ataxia-telangiectasia mutated (ATM)-dependent pathway. NBS1 deficiency severely affected the phosphorylation of ATM as well as its downstream targets. BrdU proliferation assay revealed a delay of NBS cells in inhibiting DNA synthesis after Doxorubicin (Dox) treatment. In addition, under higher concentrations of Dox, NBS cells exhibited a much lower level of apoptosis compared to their normal counterparts, indicating a resistance to Dox treatment. Accelerated telomere shortening was also observed in NBS fibroblasts, consistent with an early onset of cellular replicative senescence in vitro. This abnormality may be due to the shelterin protein telomeric binding factor 2 (TRF2) which was found to be upregulated in NBS fibroblasts. The dysregulation of telomere shortening rate and of TRF2 expression level leads to telomere fusions and cellular aneuploidy in NBS cells. Collectively, our results suggest a possible mechanism that NBS1 deficiency simultaneously affects ATM-dependent DNA damage signaling and TRF2-regulated telomere maintenance, which synergistically lead to genomic abnormalities. Copyright © 2011 John Wiley & Sons, Ltd.

In this study, the activity of the antioxidant enzyme network was assessed spectrophotometrically in samples of dental pulp and dental papilla taken from third-molar gem extracts. The production of nitric oxide by the conversion of l-(2,3,4,5)-[3H] arginine to l-(3H) citrulline, the activity of haem oxygenase 1 (HO-1) through bilirubin synthesis and the expression of inducible nitric oxide synthase (iNOS), HO-1 proteins and messenger RNA by Western blot and reverse-transcribed polymerase chain reaction were also tested. The objective of this study was to evaluate the role of two proteins, iNOS and HO-1, which are upregulated by a condition of oxidative stress present during dental tissue differentiation and development. This is fundamental for guaranteeing proper homeostasis favouring a physiological tissue growth. The results revealed an over-expression of iNOS and HO-1 in the papilla, compared with that in the pulp, mediated by the nuclear factor kappa B transcription factor activated by the reactive oxygen species that acts as scavengers for the superoxide radicals. HO-1, a metabolically active enzyme in the papilla, but not in the pulp, seems to inhibit the iNOS enzyme by a crosstalk between the two proteins. We suggest that the probable mechanism through which this happens is the interaction of HO-1 with haem, a cofactor dimer indispensible for iNOS, and the subsequent suppression of its metabolic activity. Copyright © 2011 John Wiley & Sons, Ltd.

The possibility of differentiating bone marrow-derived mesenchymal stem cells (BMSCs) into tubular epithelial-like cells is explored in vitro. Purified BMSCs from Sprague–Dawley rats were obtained by density gradient centrifugation. Third generation BMSCs were divided into six groups and were cultured under different conditions. The expression of alkaline phosphatase and cytokeratin (CK)-18 protein was detected through staining and immunocytochemistry, respectively, and the expression of E-cadherin proteins was recorded through immunofluorescence. Some cells in ischemia/reperfusion (I/R), all-trans retinoic acid (ATRA), epidermal growth factor (EGF) and bone morphogenetic protein-7 (BMP-7) groups turned positive, whereas the positive cells in the combined group significantly increased compared with the other groups. Compared with the control group, the positive expression rates of CK-18 in the I/R, ATRA, EGF, BMP-7 and the combined group were 11·50% ± 3·84%, 27·40% ± 2·70%, 29·60% ± 4·51%, 26·80% ± 5·00% and 44·00% ± 3·16%, respectively, and CK-18 mRNA expression in the combined group was obviously higher than that in the other groups (P < 0·01). Immunofluorescence detection showed that E-cadherin expression was not detectable in the control group, whereas the positive expression rates of E-cadherin in the I/R, ATRA, EGF, BMP-7 and the combined group were 6·75% ± 2·13%, 16·40% ± 2·69%, 18·25% ± 3·50%, 16·06% ± 2·00% and 30·26% ± 5·16%, respectively. The addition of ATRA, EGF and BMP-7 induces BMSCs differentiation into tubular epithelial-like cells in stimulated acute renal failure microenvironment in vitro. Copyright © 2011 John Wiley & Sons, Ltd.

Total parenteral nutrition (TPN) is essential for patients with postoperative impairing gastrointestinal function who are unable to receive and absorb oral/enteral feeding for at least 7 days. Oxidative stress plays a major role in the ethiopathogenesis of cancers. In this study, total antioxidant status (TAS), glutathione peroxidase (GPx), superoxide dismutase, malondialdehyde and ascorbic acid were studied in patients operated because of small intestine, colorectal or pancreatic cancer and subsequently receiving TPN in comparison with patients receiving standard nutrition after the operation. TAS level and GPx activity were decreased in patients with small intestine cancer but did not differ in patients with colorectal and pancreatic cancer before and after surgery. In all patient groups receiving TPN, superoxide dismutase activity after the surgery was kept at the same level as before. On the fifth day after the surgery, malondialdehyde concentration in each group was restored to the value observed before surgery. On the fifth day of TPN treatment, ascorbic acid concentration was increased in every group of patients.

During the life span, phenotypic and structural modifications on skeletal muscle contribute to a reduction on glucose uptake either in basal state or triggered by insulin, but the underlying mechanisms for this decline are not entirely identified. A reduction in the expression of skeletal muscle glucose transporters (GLUTs), glucose transporter type 1 (GLUT1) and glucose transporter type 4 (GLUT4), has been associated to such phenomena, but unlike the case of insulin, only few studies have addressed the effect of age on muscle-contraction-induced glucose uptake. The aim of the study was to investigate the influence of age on GLUT1 and GLUT4 expression in skeletal muscle and its relation to the glucose uptake induced by muscle contraction. For this purpose, soleus muscle from Wistar rats aged 4, 10, 22 and 42 weeks were isolated and electrically stimulated (30 min, 10 Hz, 20 V, 0.2 ms). After stimulation, glucose uptake and GLUT1 and GLUT4 expression and localisation were evaluated. Muscle contraction caused an increase in glucose uptake in all studied groups. In addition, the absolute rates of glucose uptake were negatively correlated with age. The expression of GLUT4 was lower in older animals, whereas no relation between age and GLUT1 expression was found. Immunohistochemistry confirmed the ontogenic effect on GLUT4 expression and suggested an age-related modification on GLUT1 distribution within the muscle fibres; for instance, this protein seems to be present mainly out of the sarcoplasm. The present findings demonstrate that the ability of muscle contraction to increase glucose uptake is not influenced by age, whereas glucose uptake under basal conditions decreases with age. Copyright © 2011 John Wiley & Sons, Ltd.

The objectives of this study were to examine whether the methanolic and aqueous extracts from the haulm and flower of Gentiana asclepiadea exhibited free radical scavenging and protective (antigenotoxic) effect against DNA oxidation induced by H₂O₂ in human lymphocytes and human embryonic kidney cells (HEK 293). All four extracts exhibited high scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at concentrations 2.5 and 25 mg ml⁻¹. The level of DNA damage was measured using the alkaline version of single-cell gel electrophoresis (comet assay). Challenge with H₂O₂ shows that the pre-treatment of the cells with non-genotoxic doses of Gentiana extracts protected human DNA—either eliminated or significantly reduced H₂O₂ induced DNA damage. The genotoxic activity of H₂O₂ was most effectively decreased after 30 min of pre-incubation with 0.05 mg ml⁻¹ (range, 93.5%–96.3% of reduction in lymphocytes) and 0.25 mg ml⁻¹ (range, 59.5%–71.4% and 52.7%–66.4% of reduction in lymphocytes and HEK 293 cells, respectively) of G. asclepiadea extracts. These results suggest that the tested G. asclepiadea extracts could be considered as an effective natural antioxidant source. Copyright © 2011 John Wiley & Sons, Ltd.

The aim of this study was to investigate the levels of the oxidant and antioxidant changes in orthodontic tooth movement and the effects of vitamin E on these parameters. For this purpose, 50 orthodontic patients (aged 13–18 years) required non-extracted treatment were divided randomly into the following groups: Control and Vitamin E. Same pre-adjusted appliances were applied to all patients, and vitamin E (300 mg day⁻¹) was given during 1 month in vitamin E group. Gingival crevicular fluid was collected and periodontal indexes were recorded at the baseline and after 1 month. Lipid peroxidation (LP) levels as malonyldialdehyde, reduced glutathione (GSH) and glutathione peroxidase (GSH-Px), vitamin C and E levels were measured in the anterior and posterior regions of the dentition. After 1 month, orthodontic treatment LP levels increased in control group in both anterior and posterior regions in vitamin E group. LP levels also increased in vitamin E group in only posterior region. The level of GSH and vitamin C did not change statistically in control and vitamin E groups. Periodontal indexes did not show any differences in comparison with the groups.

Nephrotoxic serum nephritis (NSN) is a well-established animal model of glomerulonephritis, a frequent clinical condition with a high mortality rate owing to the ineffectiveness of current therapies. Mesenchymal stem cells (MSCs) are adult stem cells with potential as novel therapies in regenerative medicine owing to the absence of allogenic rejection. Glial cell-derived neurotrophic factor (GDNF) acts as a morphogen in kidney development. The therapeutic effectiveness of bone marrow MSCs overexpressing GDNF (GDNF-MSCs) was evaluated in an NSN rat model. An adenoviral vector was used to transduce MSCs with GDNF and a green fluorescent protein reporter gene. Then, GDNF-MSCs were injected into NSN rats via the renal artery. The influence of GDNF on renal injury was assessed. The location of GDNF-MSCs in kidneys was detected using fluorescence microscopy, cells were counted, and kidney function was measured. Infusion of GNDF-MSCs enhanced the recovery of renal function in NSN rats. MSCs were detected in the kidney cortex after injection. Compared with control MSCs, GDNF-MSCs led to significantly better renal function and injury recovery in NSN rats. GDNF has a positive effect on MSC differentiation in renal tissue. Owing to their highly renoprotective capacity, GDNF-MSCs represent a possible novel cell-based paradigm for treatment of glomerulonephritis. Copyright © 2011 John Wiley & Sons, Ltd.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of 17β-estradiol. In this study, we determined the antitumour activities of 2-ME on the well-differentiated EC9706 esophageal carcinoma cells in vitro. 2-ME had a strong antiproliferative effect on EC9706 cells and caused an increase in the population of apoptotic cells, detected by flow cytometry. A significant number of cells were blocked in the G₂/M phase of the cell cycle. 2-ME-treated cells demonstrated an increase in cyclin B1 and c-Myc protein levels, as well as an increase in the percentage of G₂/M phase. Their up-regulation may be involved in 2-ME-induced apoptosis and G₂/M cell cycle arrest of the EC9706 cells, and it precedes the onset of apoptosis. Copyright © 2011 John Wiley & Sons, Ltd.

Activation of protein kinase G (PKG) by cyclic guanosine 3,5-monophosphate (cGMP) has become of considerable interest as a novel molecular approach for the induction of apoptosis in cancer cells. This study was conducted to investigate the role of PKG isoforms in the regulation of cell growth in human breast cancer cell lines MCF-7 and MDA-MB468. The expression levels of PKG isoforms were also examined using real-time reverse transcriptase polymerase chain reaction. No differences in the gene expression of PKG isoforms were observed between MCF-7 and MDA-MB-468 cells. To investigate the effects of PKG isoforms on the regulation of cell growth, the cGMP analogues 8-APT-cGMP (PKGIα activator), 8-Br-PET-cGMP (PKGIβ activator) and 8-pCPT-cGMP (PKGII activator) were employed. Apoptosis was assessed with the Annexin-V–propidium iodide (PI) staining, cell cycle analysis and caspase-3/9 activity assay. Treatment of MCF-7 and MDA-MB-468 cells with 8-Br-PET-cGMP resulted in a concentration-dependent cell growth inhibition and apoptosis, whereas neither PKGIα nor PKGII activators had any effect on the cell growth. The role of PKGIβ in the inhibition of cell growth was confirmed using PKGI and PKGII inhibitors. The present study is the first to demonstrate the involvement of PKGIβ in the inhibition of cell growth and induction of apoptosis in breast cancer cells. Copyright © 2011 John Wiley & Sons, Ltd.

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H₂O₂) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H₂O₂ formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H₂O₂ generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis. Copyright © 2011 John Wiley & Sons, Ltd.

Vascular endothelial growth factor (VEGF) is an endothelium-specific mitogen and a promising inducer of angiogenesis and lymphangiogenesis. The VEGF receptors on endothelial cell membrane include the tyrosine kinases VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and VEGFR-3 (Flt-4). KDR is a major mediator of mitogenic, angiogenic and permeability-enhancing effects of VEGF. KDR is upregulated in response to hypoxia, a major inducer of VEGF gene transcription. A HEK293 cell line overexpressing KDR was established under cell hypoxic stress to explore the function of KDR. A hypoxia-inducing agent, cobalt chloride (CoCl₂) was applied to detect whether KDR was able to prevent against chemical hypoxic toxicity. The results indicate that KDR attenuated CoCl₂-induced cell injury in HEK293 cells. Furthermore, the underlying mechanisms may be explained by the increased expression of Bcl-2, AKT1 and phosphorylated AKT, key members of cell survival pathway, and decreased expression of pro-apoptosis protein Bax. Copyright © 2011 John Wiley & Sons, Ltd.

Many small mammals survive the winter by hibernating, entering long periods of cold torpor that are interspersed with brief periods of arousal back to euthermia. This cycling is accompanied by wide changes in oxygen consumption, perfusion of tissues and ATP turnover, and the arousal period in particular is challenging because of oxidative stress associated with the huge increase in oxygen consumption needed to support thermogenesis by brown adipose tissue and skeletal muscle. Well-developed antioxidant defences are needed. The present study analyses responses of the redox-sensitive transcription factor, NF-κB, in skeletal muscle over six points on the torpor–arousal cycle to gain insight into its regulation and role during hibernation. Immunoblotting was used to analyse NF-κB p50 and p65 subunit levels, nuclear versus cytoplasmic localization and DNA-binding activity as well as levels and phosphorylation state of the IκBα inhibitor and the kinase IKK that phosphorylates IκBα to trigger its dissociation from NF-κB. The data were generally consistent with an activation of NF-κB during the entrance into torpor with responses including an auto-up-regulation of p50 subunits seen during early torpor and elevated IκBα protein during arousal. Protein levels of two downstream antioxidant targets showed differential regulation, Mn-superoxide dismutase (MnSOD) rising during early torpor versus heme oxygenase 1 (HO-1) increasing during early arousal. The mRNA transcript levels of p50, p65, HO-1 and MnSOD also showed differential expression over the torpor–arousal cycle. The results suggest that antioxidant defences are up-regulated at specific phases of the torpor–arousal cycle and that NF-κB mediates such protective responses. Copyright © 2011 John Wiley & Sons, Ltd.

Myostation (MSTN), which is primarily expressed in muscle, plays an important role in myogenic and adipogenic cells. However, there is little information about whether MSTN displays different roles between adipose-derived stem cells (ADSCs) and muscle satellite cells (MSCs). The two kinds of cells can both exist in the muscle and differentiate into adiposities. In this research, we isolated ADSCs and MSCs from porcine fat tissues and semitendinosus muscle, respectively, to investigate the effect of MSTN on the adipogenesis of those cells. ADSCs and MSCs were treated with recombinant human MSTN during the induction of adipogenesis or before the induction of differentiation. Then, we evaluated adipogenesis by Oil Red O staining and assessed the expression patterns of adipocyte-specific fatty acid binding protein (aP2) and peroxisome proliferator-activated receptor (PPAR) γ using real-time polymerase chain reaction methods. Our results indicated that the treatment with MSTN before or during the induction of differentiation in MSCs could both inhibit the adipogenesis. However, the treatment with MSTN only during the induction of differentiation in ADSCs could suppress the adipogenesis. Those results showed that MSTN had different roles in the adipogenesis of ADSCs and MSCs. It can shed new light on the origin of adipocyte located in muscle. Copyright © 2011 John Wiley & Sons, Ltd.

Organophosphorous pesticides, commonly used in agriculture for achieving better quality products, are toxic substances that have harmful effects on human health. Recent research on pesticides, especially pesticide mixtures, has shown that they are one of the key environmental health issues. The aim of the present study was to investigate whether dichlorvos, acephate, dimethoate and phorate, either used separately or in combination, can induce oxidative damage in rat livers. The levels of superoxide dismutase, glutathione peroxidase, catalase and lipid peroxidation products (malondialdehyde) were used as criteria. Low, middle and high doses of pesticides in drinking water were continuously administered orally to rats ad libitum for 24 weeks. Results show that the antioxidative defense mechanisms and lipid peroxidation in the rat livers display different responses, depending on the pesticide treatments and doses. The parameters for acephate, dichlorvos, phorate and dimethoate in the low-dose group, and the corresponding low-dose co-treated group were not altered. The oxidative damage in rat livers showed different responses with increasing pesticide dose according to the different pesticide treatments. The combination group of dichlorvos, acephate, dimethoate and phorate displayed different responses compared with the single pesticide-treated group. However, these responses did not constitute the sum of the response produced by each pesticide in the liver. Copyright © 2011 John Wiley & Sons, Ltd.

In pancreatic islets, glucose metabolism is a key process for insulin secretion, and pregnancy requires an increase in insulin secretion to compensate for the typical insulin resistance at the end of this period. Because a low-protein diet decreases insulin secretion, this type of diet could impair glucose homeostasis, leading to gestational diabetes. In pancreatic islets, we investigated GLUT2, glucokinase and hexokinase expression patterns as well as glucose uptake, utilization and oxidation rates. Adult control non-pregnant (CNP) and control pregnant (CP) rats were fed a normal protein diet (17%), whereas low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) rats were fed a low-protein diet (6%) from days 1 to 15 of pregnancy. The insulin secretion in 2.8 mmol l⁻¹ of glucose was higher in islets from LPP rats than that in islets from CP, CNP and LPNP rats. Maximal insulin release was obtained at 8.3 and 16.7 mmol l⁻¹ of glucose in LPP and CP groups, respectively. The glucose dose–response curve from LPNP group was shifted to the right in relation to the CNP group. In the CP group, the concentration–response curve to glucose was shifted to the left compared with the CNP group. The LPP groups exhibited an “inverted U-shape” dose–response curve. The alterations in the GLUT2, glucokinase and hexokinase expression patterns neither impaired glucose metabolism nor correlated with glucose islet sensitivity, suggesting that β-cell sensitivity to glucose requires secondary events other than the observed metabolic/molecular events. Copyright © 2011 John Wiley & Sons, Ltd.

The genetic basis for the phenotypic switching of vascular smooth muscle cells (VSMCs) is unclear in atherosclerosis. Recent studies showed that the 21-base pair deletion mutation (Δ21) in myocyte enhancer factor 2A (MEF2A) gene could be an inherited marker for coronary artery disease. MEF2A mutation may affect the phenotypic switching of VSMCs. Human aortic VSMCs were used. Four groups of VSMCs transfected with green fluorescent protein plasmid (control group), MEF2A wild-type (WT) plasmid (WT group), MEF2A Δ21 plasmid (Δ21 group) or MEF2A siRNA (siRNA group) were studied. The proliferation of VSMCs was determined by methylthiazolyldiphenyl-tetrazolium bromide, and the migration of VSMCs was measured by Millicell chamber. The protein expressions of MEF2A, smooth muscle α-actin, SM22α, osteopontin and p38 mitogen-activated protein kinase signaling pathway were detected by Western blotting. MEF2A protein expression was knockdown by siRNA transfection. MEF2A protein was overexpressed in WT and Δ21 groups. Δ21 and siRNA groups obviously showed more proliferation (methylthiazolyldiphenyl-tetrazolium bromide, 0.63 vs 0.66 vs 0.31, P < 0.01) and migration (52.6 vs 58.0 vs 21.2, P < 0.01) of VSMCs as compared with the WT group. In addition, the transfection of Δ21 and siRNA could induce the down-regulation of smooth muscle α-actin and SM22α (P < 0.01) and the up-regulation of osteopontin (P < 0.01) in VSMCs. The phosphorylated p38 signaling pathway expression was significantly enhanced in the Δ21 and siRNA groups as compared with that of the WT group (P < 0.01). These results suggest that MEF2A dominant negative mutation and RNA silence could induce the phenotypic switching of VSMCs, leading to its increased proliferation and migration, and p38 mitogen-activated protein kinase signaling pathway may participate in it. Copyright © 2011 John Wiley & Sons, Ltd.

No abstract is available for this article.

Propolis has been highlighted for its antioxidant, anti-inflammatory and antiviral properties. The purpose of this study was to investigate if brown Brazilian hydroalcoholic propolis extract (HPE) protects against vaginal lesions caused by herpes simplex virus type 2 (HSV-2) in female BALB/c mice. The treatment was divided in 5 days of pre-treatment with HPE [50 mg·kg^–1, once a day, intragastric (i.g.)], HSV-2 infection [10 µl of a solution 1 × 10² plaque-forming unit (PFU·ml^–1 HSV-2), intravaginal inoculation at day 6] and post-treatment with HPE (50 mg·kg^–1) for 5 days more. At day 11, the animals were killed, and the in vivo analysis (score of lesions) and ex vivo analysis [haematological and histological evaluation; superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) activities; reactive species (RS), tyrosine nitration levels, non-protein thiols (NPSH) and ascorbic acid (AA) levels] were carried out. HPE treatment reduced extravaginal lesions and the histological damage caused by HSV-2 infection in vaginal tissues of animals. HPE was able to decrease RS, tyrosine nitration, AA levels and MPO activity. Also, it protected against the inhibition of CAT activity in vaginal tissues of mice. HPE promoted protective effect on HSV-2 infected animals by acting on inflammatory and oxidative processes, and this effect probably is caused by its antioxidant and anti-inflammatory properties. Copyright © 2011 John Wiley & Sons, Ltd.

High-mobility group box 1 (HMGB1) is a multifunctional protein with intranuclear and extracellular functions. Although HMGB1 is overexpressed in approximately 85% of gastric cancers, the role of HMGB1 in gastric cancer biology remains unclear. In this study, we investigate the effect of downregulation of HMGB1 on the biological behavior of gastric cancer cells. MGC-803 gastric cancer cells were transduced with HMGB1-specific RNAi lentiviral vectors. Real-time polymerase chain reaction and Western blot analysis of HMGB1 mRNA and protein, respectively, validated the silencing effects. HMGB1-specific silencing significantly decreased cell proliferation. The impact on proliferation was observed at the cell cycle level—the number of cells in the G0/G1 phase increased, whereas that in S and G2/M phases decreased. Cell cycle changes were accompanied by decreases in cyclin D1 expression. Furthermore, HMGB1 silencing sensitized cells to apoptosis that was induced by oxaliplatin and mediated by the caspase-3 pathway. Finally, silencing of HMGB1 expression significantly reduced cellular metastatic ability and MMP-9 expression in MGC-803 cells. In summary, HMGB1 not only plays an essential role in the proliferation and invasion of MGC-803 cells but also represents a potential target for the therapeutic intervention of gastric cancer. Copyright © 2011 John Wiley & Sons, Ltd.

The pathogenesis of many diseases and different pathological conditions, including inflammation, is associated with excess production of reactive oxygen species (ROS). The present study aimed to investigate the effects of the antidepressant desipramine (DES) on carrageenan (CG)-induced inflammation, as well as on the endogenous levels of cell enzyme and non-enzyme antioxidants in rat liver and spleen, 4 and 24 h after CG injection. The intra-plantar CG injection into the right hind paw resulted in a time-dependent increase in the paw volume; the maximum of CG-induced edema peak was in 2–4 h. A single DES dose of 20 mg·kg⁻¹, administered 30 min before CG, had no effect on paw edema, whereas the higher drug dose used (50 mg·kg⁻¹) suppressed the edematous response to CG. The latter drug dose protected CG-induced decrease of glutathione (non-enzyme antioxidant) in the liver; it did not affect CG-unchanged activities of superoxide dismutase, glutathione peroxidase (enzyme antioxidants) and glucose-6-phosphate dehydrogenase (enzyme, important for the activity of glutathione-conjugated antioxidant enzymes) in both liver and spleen. The drug showed an efficient antioxidant capacity in ROS-generating chemical systems; it was higher than that of fluoxetine (another type of antidepressant). The present results suggest that the good antioxidant activity of DES might contribute to its beneficial effects in liver injuries. Copyright © 2011 John Wiley & Sons, Ltd.

Adenovirus-mediated expression of hypoxia-inducible factor 1α double mutant (pAd-HIF-1α-Ala564-Ala803) can be effectively transfected into bone marrow stem cells (MSCs) in the MSCs and cardiomyocytes co-culture system at normoxia to regulate the expression of downstream target genes of hypoxia-inducible factor 1α (HIF-1α), which in turn can promote MSC differentiation into cardiomyocytes. Fibroblasts share common characteristics with MSCs such as the morphology, phenotype and differentiation potential. Therefore, we further studied whether the pAd-HIF-1α-Ala564-Ala803 also can convert neonatal rat cardiac fibroblasts (NCFs) into (cardio)myocyte phenotype via regulating the downstream target genes of HIF-1α at normoxia. The immunostaining analysis showed that NCFs treated with pAd-HIF-1α-Ala564-Ala803 exhibited higher protein expression levels of smooth muscle α-actin (SMA, myocyte marker) and cardiac troponin T (cTnT, cardiomyocyte marker), compared with phosphate-buffered saline and pAd-LacZ treatments. The reverse transcription-polymerase chain reaction results showed that NCFs transfected with pAd-HIF-1α-Ala564-Ala803 augmented messenger RNA (mRNA) expression of transforming growth factor-β1 (TGF-β1), Smad4, NKx2.5, GATA4, myocardin, SMA and cTnT. The effects of HIF-1α-Ala564-Ala803 on NCFs were attenuated by pre-transfection of TGF-β1 or myocardin small interference RNAs. Adult CFs transfected with pAd-HIF-1α-Ala564-Ala803 showed a lower protein expression of SMA but not cTnT without any change in the mRNA expression level of NKx2.5, myocardin. Therefore, NCFs but not adult CFs possess a similar differentiation potential to MSCs as evidenced by the fact that pAd-HIF-1α-Ala564-Ala803 can convert NCFs into (cardio)myocyte phenotype via regulating its downstream target genes. Copyright © 2011 John Wiley & Sons, Ltd.

We have shown that SU6656, a potent Src family kinase inhibitor, has the ability to induce multinucleation at a high frequency in diverse cells: rat skin fibroblasts, bone marrow adherent cells, 5F9A mesenchymal stem cell-like clones, 2C5 tracheal epithelial cells and MDCK epithelial cells from dog kidney. To gain insight into the mechanism of multinucleation, we observed the process by time-lapse and confocal microscopy. These multinuclei generally seem to exist independently in one cell without any connections with each other. By time-lapse microscopy, multinucleated cells were found to be formed through the mechanism of plasmodium: karyokinesis without cytokinesis. The observation of EGFP-actin transfected cells by time-lapse confocal laser scanning microscopy suggested that plasmodium occurred with deficient contractile ring formation. Although we examined the differentiation of these cells, the multinucleated cells could not be categorized into any type of cell in vivo known to exhibit multinuclei. Copyright © 2011 John Wiley & Sons, Ltd.

Na⁺/H⁺ exchanger 1 (NHE1), acting as an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been known to play a key role in the metastasis of many solid tumours. However, the exact mechanism underlying these processes, especially in cervical cancer, is still poorly understood. In the current study, we first showed that the inhibition of NHE1 activity by the specific inhibitor cariporide could suppress migration and invasion of HeLa cells in vitro. Moreover, cariporide also reversed the enhanced migration and invasion in HeLa cells by overexpressed membrane-type 1 matrix metalloproteinase (MT1-MMP). Subsequently, our results showed that NHE1 regulated the expression of MT1-MMP at both messenger RNA and protein levels as well as its localization. Meanwhile, we observed slight modification in the morphology of HeLa cell after treating with cariporide. The present work indicates that NHE1 mediates HeLa cell metastasis via regulating the expression and localization of MT1-MMP and provides a theoretical basis for the development of novel therapeutic strategies targeting cervical cancer. Copyright © 2011 John Wiley & Sons, Ltd.

The placenta is a glucocorticoid target organ, and glucocorticoids (GCs) are essential for the development and maturation of fetal organs. They are widely used for treatment of a variety of diseases during pregnancy. In various tissues, GCs have regulated by glucose transport systems; however, their effects on glucose transporters in the human placental endothelial cells (HPECs) are unknown.

The transcription factors DP1 and DP2 have been implicated in crucial gene regulation as heterodimer partners of E2F; however, the functional differences between DP1 and DP2 remain poorly understood. To gain insight into DPs in human somatic cells, we first suppressed endogenous DP1 and DP2 using RNA interference and examined the effect of their loss on gene expression changes in HeLa cervical cancer cells. A DNA microarray and gene pathway analysis revealed that the suppression of well-known E2F/DP-regulated pathways, including the G1 to S phase transition of the cell cycle and DNA replication, was manifested in accordance with the acute loss of DP1 and DP2. On the other hand, the acute loss of DP1 and DP2 increased the p21Waf1/Cip1 mRNA level compared with the control RNA treatment. We further showed that the inactivation of DP1, but not DP2, resulted in mRNA induction for p53, an upstream regulator of p21Waf1/Cip1. Furthermore, in A549 lung cancer cells as well as HeLa cells, the mRNA and protein levels of p53 and p21Waf1/Cip1 were stabilized specifically upon DP1 depletion, whereas p53-regulated apoptotic factor BAX mRNA was unchanged. Finally, the impairment of DP1, but not DP2, increased senescence in HeLa, A549 and WI-38 diploid fibroblasts but not in p53 null Saos-2 osteosarcoma cells. Taken together, these results suggest that DP1, but not DP2, is uniquely involved in the regulation of the p53 and p21Waf1/Cip1 pathway, thereby augmenting senescence in human somatic cells. Copyright © 2011 John Wiley & Sons, Ltd.

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34⁺-haemopoietic stem cells (CD34⁺-HSCs), thus mimicking the Tay-Sachs (HEXA−/−) and Sandhoff (HEXB−/−) cells.

Two different α-glucosidase-producing thermophilic E134 strains were isolated from a hot spring in Kozakli, Turkey. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, the strain was proposed to be a species of G. toebii. Its thermostable exo-α-1,4-glucosidases also were characterized and compared, which were purified from the intracellular and extracellular fractions with estimated molecular weights of 65 and 45 kDa. The intracellular and extracellular α-glucosidases showed optimal activity at 65 °C, pH 7·0, and at 70 °C, pH 6·8, with 3·65 and 0·83 K_m values for the pNPG substrate, respectively. Both enzymes remained active over temperature and pH ranges of 35–70 °C and 4·5–11·0. They retained 82 and 84% of their activities when incubated at 60 °C for 5 h. Their relative activities were 45–75% and 45–60% at pH 4·5 and 11·0 values for 15 h at 35 °C. They could hydrolyse the α-1,3 and α-1,4 bonds on substrates in addition to a high transglycosylation activity, although the intracellular enzyme had more affinity to the substrates both in hydrolysis and transglycosylation reactions. Furthermore, although sodium dodecyl sulfate behaved as an activator for both of them at 60 °C, urea and ethanol only increased the activity of the extracellular α-glucosidase. By this study, G. toebii E134 strain was introduced, which might have a potential in biotechnological processes when the conformational stability of its enzymes to heat, pH and denaturants were considered. Copyright © 2011 John Wiley & Sons, Ltd.

The purposes of the present study were to elucidate the influences of the deficiency of teeth on masticatory muscles, such as the masseter, temporalis and digastric muscles and compare the influence among masticatory muscles. We analysed the expressions of myosin heavy chain (MyHC) isoform messenger RNA (mRNA) and protein in these muscles in the microphthalmic (mi/mi) mouse, whose teeth cannot erupt because of a mutation in the mitf gene locus. The expression levels of MyHC mRNA and protein in the masseter, temporalis, digastric, tibialis anterior and gastrocnemius muscles of +/+ and mi/mi mice were analysed with real-time polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The mi/mi masseter muscle at 8 weeks of age expressed 4·1-fold (p < 0·05) and 3.3-fold (p < 0·01) more MyHC neonatal mRNA and protein than that in the +/+, respectively; the expression level of MyHC neonatal protein was 19% of the total MyHC protein in the masseter muscle of mi/mi mice. In the digastric muscle, the expression levels of MyHC I mRNA and protein in the mi/mi mice were 4·7-fold (p < 0·05) and 5-fold (p < 0·01) higher than those in the +/+ mice. In the temporalis, tibialis anterior and gastrocnemius muscles, there was no significant difference in the expression levels of any MyHC isoform mRNA and protein between +/+ and mi/mi mice. These results indicate associations between the lack of teeth and the expression of MyHC in the masseter and digastric muscles but not such associations in the temporalis muscle, suggesting that the influence of tooth deficiency varies among the masticatory muscles. Copyright © 2011 John Wiley & Sons, Ltd.