Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Effects of 5-aza-2′deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2′deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECKgene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.

Plasma levels and placental expression of vaspin in pregnant women with diabetes mellitus

The present study aimed to investigate visceral adipose tissue-specific serpin (vaspin) concentrations in serum and term placentas and relate these values to insulin resistance and lipid parameters in women with gestational diabetes mellitus (GDM). A total of 30 GDM subjects and 27 age-matched pregnant women with normal glucose tolerance (NGT, control) were included. Serum glucose, glycated hemoglobin (HbA1c), lipid profile, insulin, and vaspin were measured at the end of pregnancy, and homeostasis model of assessment-insulin resistance (HOMA-IR) values were calculated. Vaspin mRNA and protein levels in placentas were measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Serum vaspin levels were significantly lower in the GDM group than in controls (0.49±0.24 vs 0.83±0.27 ng/mL, respectively; P<0.01). Three days after delivery, serum vaspin levels were significantly decreased in subjects with GDM (0.36±0.13 vs0.49±0.24 ng/mL, P<0.01). However, in the GDM group, serum vaspin levels were not correlated with the parameters evaluated. In contrast, in the control group, serum vaspin levels were positively correlated with triglycerides (TG; r=0.45, P=0.02) and very low-density lipoprotein cholesterol (VLDL-C; r=0.42, P=0.03). Placental mRNA vaspin (0.60±0.32 vs0.68±0.32, P=0.46) and protein (0.30±0.08 vs0.39±0.26; P=0.33) levels in the GDM group did not differ significantly from those in the control group, but were negatively correlated with neonatal birth weight in the GDM group (r=-0.48, P=0.03; r=-0.88; P<0.01). Our findings indicated that vaspin may be an important adipokine involved in carbohydrate and lipid metabolism and may also play a role in fetal development.

Abundant immunohistochemical expression of dopamine D2 receptor and p53 protein in meningiomas: follow-up, relation to gender, age, tumor grade, and recurrence

Meningiomas are common, usually benign tumors, with a high postoperative recurrence rate. However, the genesis and development of these tumors remain controversial. We aimed to investigate the presence and implications of a mutated p53 protein and dopamine D2 receptor in a representative series of meningiomas and to correlate these findings with age, gender, tumor grade, and recurrence. Tumor tissue samples of 157 patients diagnosed with meningioma (37 males and 120 females, mean age 53.6±14.3 years) who underwent surgical resection between 2003 and 2012 at our institution were immunohistochemically evaluated for the presence of p53 protein and dopamine D2 receptor and were followed-up to analyze tumor recurrence or regrowth. Tumors were classified as grades I (n=141, 89.8%), II (n=13, 8.3%), or grade III (n=3, 1.9%). Dopamine D2 receptor and p53 protein expression were positive in 93.6% and 49.7% of the cases, respectively. Neither of the markers showed significant expression differences among different tumor grades or recurrence or regrowth statuses. Our findings highlight the potential role of p53 protein in meningioma development and/or progression. The high positivity of dopamine D2 receptor observed in this study warrants further investigation of the therapeutic potential of dopamine agonists in the evolution of meningiomas.

Treatment with 1,25(OH)2D3induced HDAC2 expression and reduced NF-κB p65 expression in a rat model of OVA-induced asthma

Recent evidence indicates that a deficiency of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) may influence asthma pathogenesis; however, its roles in regulating specific molecular transcription mechanisms remain unclear. We aimed to investigate the effect of 1,25(OH)2D3 on the expression and enzyme activity of histone deacetylase 2 (HDAC2) and its synergistic effects with dexamethasone (Dx) in the inhibition of inflammatory cytokine secretion in a rat asthma model. Healthy Wistar rats were randomly divided into 6 groups: control, asthma, 1,25(OH)2D3 pretreatment, 1,25(OH)2D3 treatment, Dx treatment, and Dx and 1,25(OH)2D3 treatment. Pulmonary inflammation was induced by ovalbumin (OVA) sensitization and challenge (OVA/OVA). Inflammatory cells and cytokines in the bronchoalveolar lavage (BAL) fluid and histological changes in lung tissue were examined. Nuclear factor kappa B (NF-κB) p65 and HDAC2 expression levels were assessed with Western blot analyses and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Enzyme activity measurements and immunohistochemical detection of HDAC2 were also performed. Our data demonstrated that 1,25(OH)2D3 reduced the airway inflammatory response and the level of inflammatory cytokines in BAL. Although NF-κB p65 expression was attenuated in the pretreatment and treatment groups, the expression and enzyme activity of HDAC2 were increased. In addition, 1,25(OH)2D3 and Dx had synergistic effects on the suppression of total cell infusion, cytokine release, and NF-κB p65 expression, and they also increased HDAC2 expression and activity in OVA/OVA rats. Collectively, our results indicated that 1,25(OH)2D3might be useful as a novel HDAC2 activator in the treatment of asthma.

Biological significance of miR-126 expression in atrial fibrillation and heart failure

We investigated the biological significance of microRNA-126 (miR-126) expression in patients with atrial fibrillation (AF) and/or heart failure (HF) to examine the possible mechanism of miR-126-dependent AF and development of HF. A total of 103 patients were divided into three groups: AF group (18 men and 17 women, mean age: 65.62±12.72 years), HF group (17 men and 15 women, mean age: 63.95±19.71 years), and HF-AF group (20 men and 16 women, mean age: 66.56±14.37 years). Quantitative real-time PCR was used to measure relative miR-126 expression as calculated by the 2−ΔΔCt method. miR-126 was frequently downregulated in the 3 patient groups compared with controls. This reduction was significantly lower in permanent and persistent AF patients than in those with paroxysmal AF (P<0.05, t-test). Moreover, miR-126 expression was markedly lower in the HF-AF group compared with the AF and HF groups. The 3 patient groups had higher N-terminal prohormone brain natriuretic peptide (NT-proBNP) levels, lower left ventricular ejection fraction (LVEF), larger left atrial diameter, and higher cardiothoracic ratio compared with controls. There were significant differences in NT-proBNP levels and LVEF among the AF, HF, and HF-AF groups. Pearson correlation analysis showed that relative miR-126 expression was positively associated with LVEF, logarithm of NT-proBNP, left atrial diameter, cardiothoracic ratio, and age in HF-AF patients. Multiple linear regression analysis showed that miR-126 expression was positively correlated with LVEF, but negatively correlated with the logarithm of NT-pro BNP and the cardiothoracic ratio (all P<0.05). Serum miR-126 levels could serve as a potential candidate biomarker for evaluating the severity of AF and HF. However, to confirm these results, future studies with a larger and diverse patient population are necessary.

Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing

Transforming growth factor beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) are important regulators of bone repair and regeneration. In this study, we examined whether TGF-β1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-β1 and BMP2 in the fractured tibias were measured by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, weekly for the first 5 weeks post-fracture. Mechanical parameters (bending rigidity, torsional rigidity, destruction torque) of the healing bones were also assessed at 3, 4, and 5 weeks post-fracture, after the rats were sacrificed. The bending rigidity, torsional rigidity and destruction torque of the two groups increased continuously during the healing process. The diabetes group had lower mean values for bending rigidity, torsional rigidity and destruction torque compared with the control group (P<0.05). TGF-β1 and BMP-2 expression were significantly lower (P<0.05) in the control group than in the diabetes group at postoperative weeks 1, 2, and 3. Peak levels of TGF-β1 and BMP-2 expression were delayed by 1 week in the diabetes group compared with the control group. Our results demonstrate that there was a delayed recovery in the biomechanical function of the fractured bones in diabetic rats. This delay may be associated with a delayed expression of the growth factors TGF-β1 and BMP-2.

Expression of merlin, NDRG2, ERBB2, and c-MYC in meningiomas: relationship with tumor grade and recurrence

Meningiomas are common, usually benign tumors of the central nervous system that have a high rate of post-surgical recurrence or regrowth. We determined expression of the proteins merlin, NDRG2, ERBB2, and c-MYC in meningiomas using immunohistochemistry and assessed relationships between protein expression and gender, age, tumor grade, and recurrence or regrowth. The study sample comprised 60 patients, (44 women and 16 men) with a mean age of 53.2±12.7 years. Tumors were classified as grade I (n=48) or grades II and III (n=12). Expression of merlin, NDRG2, ERBB2, and c-MYC was not significantly different statistically with relation to gender, age, or meningioma recurrence or regrowth. Merlin was expressed in 100% of the cases. No statistically significant difference between tumor grade and recurrence or regrowth was identified. Statistically significant differences were identified between the mean age of patients with grade I (54.83±11.60) and grades II and III (46.58±15.08) meningiomas (P=0.043), between strong c-MYC expression and grades II and III (P<0.001), and between partial surgical resection and tumor recurrence or regrowth (P<0.001). These findings reveal the lower mean age among grades II and III meningioma patients than grade I patients, the influence of the protein merlin on tumorigenesis, the association of c-MYC with aggressive meningiomas, and that partial surgical resection is associated with tumor recurrence or regrowth.

Exercise improved lipid metabolism and insulin sensitivity in rats fed a high-fat diet by regulating glucose transporter 4 (GLUT4) and musclin expression

This study aimed to evaluate the effects of exercise training on triglyceride deposition and the expression of musclin and glucose transporter 4 (GLUT4) in a rat model of insulin resistance. Thirty male Sprague-Dawley rats (8 weeks old, weight 160±10 g) were fed a high-fat diet (40% calories from fat) and randomly divided into high-fat control group and swimming intervention group. Rats fed with standard food served as normal control. We found that 8-week swimming intervention significantly decreased body weight (from 516.23±46.27 to 455.43±32.55 g) and visceral fat content (from 39.36±2.50 to 33.02±2.24 g) but increased insulin sensitivity index of the rats fed with a high-fat diet. Moreover, swimming intervention improved serum levels of TG (from 1.40±0.83 to 0.58±0.26 mmol/L) and free fatty acids (from 837.80±164.25 to 556.38±144.77 μEq/L) as well as muscle triglycerides deposition (from 0.55±0.06 to 0.45±0.02 mmol/g) in rats fed a high-fat diet. Compared with rats fed a standard food, musclin expression was significantly elevated, while GLUT4 expression was decreased in the muscles of rats fed a high-fat diet. In sharp contrast, swimming intervention significantly reduced the expression of musclin and increased the expression of GLUT4 in the muscles of rats fed a high-fat diet. In conclusion, increased musclin expression may be associated with insulin resistance in skeletal muscle, and exercise training improves lipid metabolism and insulin sensitivity probably by upregulating GLUT4 and downregulating musclin.

Timing of di-ammonium phosphate addition to fermenting chardonnay must : effect on nitrogen utilization, ethyl-carbamate formation, and CAR1 expression by yeast /

The maximum amount of ethyl carbamate (EC), a known animal carcinogen produced by the reaction of urea and ethanol, allowed in alcoholic beverages is regulated by legislation in many countries. Wine yeast produce urea by the metabolism of arginine, the predominant assimilable amino acid in must. This action is due to arginase (encoded by CARl). Regulation of CARl, and other genes in this pathway, is often attributed to a well-documented phenomenon known as nitrogen catabolite repression. The effect of the timing of di-ammonium phosphate (DAP) additions on the nitrogen utilization, regulation of CARl, and EC production was investigated. A correlation was found between the timing of DAP addition and the utilization of nitrogen. When DAP was added earlier in the fermentations, less amino nitrogen and more ammonia nitrogen was sequestered from the media by the cells. It was also seen that early DAP addition led to more total nitrogen being used, with a maximal difference of ~25% between fermentations where no DAP was added versus addition at the start of the fermentation. The effect of the timing ofDAP addition on the expression of CARJ during fermentation was analyzed via northern transfer and the relative levels of CARl expression were determined. The trends in expression can be correlated to the nitrogen data and be used to partially explain differences in EC formation between the treatments. EC was quantified at the end of fermentation by GC/MS. In Montrachet yeast, a significant positive correlation was found between the timing of DAP addition, from early to late, and the final EC concentration m the wine (r = 0.9226). In one of the fermentations, EC levels of 30.5 ppb was foimd when DAP was added at the onset of fermentation. A twofold increase (69.5 ppb) was observed when DAP was added after 75% of the sugars were metabolized. When no DAP was added, the ethyl carbamate levels are comparable at a value of 38 ppb. In contrast, the timing of DAP additions do not affect the level EC produced by the yeast ECU 18 in this manner. The study of additional yeast strains shows that the effect of DAP addition to fermentations is strain dependent. Our results reveal the potential importance of the timing of DAP addition to grape must with respect to EC production, and the regulatory effect of DAP additions on the expression of genes in the pathway for arginine metabolism in certain wine yeast strains.

Expression and function of endothelin and its receptors in vascular adventitial fibroblasts

Objective: The adventitia has been recognized to play important roles in vascular oxidative stress, remodelling and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (ANG II). However, the mechanisms by which ANG II induces ET-1 expression are unknown. It is also unclear whether the ET-1 receptors are expressed in the adventitia. We therefore examined the role of oxidative stress in the regulation of ET-1. We also investigated the expression of both the ETA and ETB receptors and the roles of these two types of receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Methods and Results: Adventitial fibroblasts were isolated and cultured from the thoracic mouse aorta. Cells were treated with ANG II (lOOnM), ET-1 (lOpM), NADPH oxidase inhibitor apocynin (lOOfiM), the superoxide anion scavenger tempol (lOOfiM), the ANG II receptor antagonists (100[aM), losartan (AT| receptor) and PD 1233 19 (AT2 receptor), the ET-1 receptor antagonists (lOOuM), BQ123 (ETA receptor) and BQ788 (ETB receptor), and the ETB receptor agonist (lOOnM) Sarafotoxin 6C. ET-1 peptide levels were determined by ELISA, while ETA ,ETB and collagen levels were determined by Western blot. ANG II increased ET-1 peptide levels in a time-dependent manner reaching significance when incubated for 24 hours. NAD(P)H oxidase inhibitor, apocynin, as well as the superoxide scanverger, tempol, significantly reduced ANG Il-induced ET-1 peptide levels while over-expression of SOD1 (endogenous antioxidant enzyme) significantly decreased ANG Il-induced collagen I expression, therefore implicating reactive oxygen species in the mediation of ET-1. ANG II increased ETA receptor protein as well as collagen in a similar fashion, reaching significance after 4, 6, and 24 hours treatment. ANG II induced collagen was reduced while in the presence of the ETA receptor antagonist suggesting the role of the ETa receptor in the regulation of the extracellular matrix. ANG II treatment also increased ETB receptor protein levels in a time-dependent manner. ANG II treatment in the presence of the ETB receptor antagonist significantly increased ET-1 peptide levels. On another hand, the ETB receptor agonist, Sarafotoxin 6C, significantly decreased ET-1 peptide levels. These data implicate the role of the ETb receptor in the clearance of the ET-1 peptide. Conclusion: ANG II-induced increases of ET-1 peptide appears to be mediated by reactive oxygen species derived from NAD(P)H oxidase. Both the ETA and ETB receptors are expressed in adventitial fibroblasts. The ETA receptor subtype mediates collagen I expression, while the ETB receptor may play a protective role through increasing the clearance of the ET- 1 peptide.

Primary characterization of genes involved in the colony development of the insect pathogenic fungus Metarhizium anisopliae /

Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

The cumate gene-switch: a system for regulated expression in mammalian cells

Affiliation: Sophie Broussau, Amelie Pilotte & Bernard Massie : Départment de microbiologie et immunologie, Faculté de médecine, Université de Montréal