Influence of progesterone on GAD65 and GAD67 mRNA expression in the dorsolateral striatum and prefrontal cortex of female rats repeatedly treated with cocaine

Female rats are intensely affected by cocaine, with estrogen probably playing an important role in this effect. Progesterone modulates the GABA system and attenuates the effects of cocaine; however, there is no information about its relevance in changing GABA synthesis pathways after cocaine administration to female rats. Our objective was to investigate the influence of progesterone on the effects of repeated cocaine administration on the isoenzymes of glutamic acid decarboxylase (GAD65 and GAD67) mRNA in brain areas involved in the addiction circuitry. Ovariectomized, intact and progesterone replacement-treated female rats received saline or cocaine (30 mg/kg, ip) acutely or repeatedly. GAD isoenzyme mRNA levels were determined in the dorsolateral striatum (dSTR) and prefrontal cortex (PFC) by RT-PCR, showing that repeated, but not acute, cocaine decreased GADs/β-actin mRNA ratio in the dSTR irrespective of the hormonal condition (GAD65: P < 0.001; and GAD67: P = 0.004). In the PFC, repeated cocaine decreased GAD65 and increased GAD67 mRNA ratio (P < 0.05). Progesterone replacement decreased both GAD isoenzymes mRNA ratio after acute cocaine in the PFC (P < 0.001) and repeated cocaine treatment reversed this decrease (P < 0.001). These results suggest that cocaine does not immediately affect GAD mRNA expression, while repeated cocaine decreases both GAD65 and GAD67 mRNA in the dSTR of female rats, independently of their hormonal conditions. In the PFC, repeated cocaine increases the expression of GAD isoenzymes, which were decreased due to progesterone replacement.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70%) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62%) and left (about 81%) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%), DBH (about 37%) and PNMT (about 60%) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-β1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-β1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-β1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-β1. However, ED5 has no effect on VSMC apoptosis.

Mechanism of the transforming growth factor-β induction of fibronectin expression in hepatic stem-like cells

Transforming growth factor-β1 (TGF-β1) plays an important role in the fibrogenic process in the liver. The aim of the present study was to explore the action of TGF-β1 on fibronectin expression in rat hepatic stem-like cells and the underlying mechanisms. The level of fibronectin expression was determined in hepatic stem-like cells (WB cells) before and after TGF-β1 stimulation by RT-PCR and Western blot methods. Using immunogold transmission electron microscopy and the Western blot method, we observed the result of the expression and the distribution of cAMP, phosphorylated Smad3 and Smad7 before and after TGF-β1 treatment. The levels of fibronectin expression in both mRNA and protein increased 4- to 5-fold after TGF-β1 stimulation, reaching an optimum level after 8 h and then gradually falling back. Similarly, TGF-β1 stimulation resulted in an increase of cAMP in WB cells, peaking at 8 h. After treatment with TGF-β1 for 24 h, the expression of cAMP gradually decreased. In addition, we found that TGF-β1 treatment also contributed to the increased expression and to changes in cellular distribution of phosphorylated Smad3 (translocation from the cytoplasm to the nucleus) and Smad7 (translocation from the nucleus to the cytoplasm) in WB cells. The present study demonstrates that TGF-β is involved in the fibrogenic process in hepatic stem cells through up-regulation of fibronectin expression, and the mechanisms underlying this process may be associated with the activation of cAMP and Smad pathways.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancerpatients

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1,MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1based trios, with NOTCH1 or NUP210. Both trios correctly separated 86% of tumors (87% sensitivity and 80% specificity for predicting response), according to their response to chemotherapy (82% in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71% samples from the biological validation set were also correctly classified by both trios (72% sensitivity; 66% specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93% of samples from the technical validation group (95% sensitivity and 80% specificity; 86% accuracy by the cross-validation method) and 79% from the biological validation group (72% sensitivity and 100% specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

The effect of down-regulation of Smad3 by RNAi on hepatic stellate cells and a carbon tetrachloride-induced rat model of hepatic fibrosis

Searching for effective Smad3 gene-based gene therapies for hepatic fibrosis, we constructed siRNA expression plasmids targeting the rat Smad3 gene and then delivered these plasmids into hepatic stellate cells (HSCs). The effect of siRNAs on the mRNA levels of Smad2, Smad3, Smad4, and collagens I-α1, III-α1 and IV-α1 (Colα1, Col3α1, Col4α1, respectively) was determined by RT-PCR. Eighty adult male Sprague-Dawley rats were randomly divided into three groups. Twice a week for 8 weeks, the untreated hepatic fibrosis model (N = 30) and the treated group (N = 20) were injected subcutaneously with 40% (v/v) carbon tetrachloride (CCl4)-olive oil (3 mL/kg), and the normal control group (N = 30) was injected with olive oil (3 mL/kg). In the 4th week, the treated rats were injected subcutaneously with liposome-encapsulated plasmids (150 µg/kg) into the right liver lobe under general anesthesia once every 2 weeks, and the untreated rats were injected with the same volume of buffer. At the end of the 6th and 8th weeks, liver tissue and sera were collected. Pathological changes were assessed by a semi-quantitative scoring system (SSS), and a radioimmunoassay was used to establish a serum liver fibrosis index (type III procollagen, type IV collagen, laminin, and hyaluronic acid). The mRNA expression levels of the above cited genes were reduced in the HSCs transfected with the siRNA expression plasmids. Moreover, in the treated group, fibrosis evaluated by the SSS was significantly reduced (P < 0.05) and the serum indices were greatly improved (P < 0.01). These results suggest that Smad3 siRNA expression plasmids have an anti-fibrotic effect.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Effect of silybin on high-fat-induced fatty liver in rats

Silybin, a natural antioxidant, has been traditionally used against a variety of liver ailments. To investigate its effect and the underlying mechanisms of action on non-alcoholic fatty liver in rats, we used 60 4-6-week-old male Sprague-Dawley rats to establish fatty liver models by feeding a high-fat diet for 6 weeks. Hepatic enzyme, serum lipid levels, oxidative production, mitochondrial membrane fluidity, homeostasis model assessment-insulin resistance index (HOMA-IR), gene and protein expression of adiponectin, and resistin were evaluated by biochemical, reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Compared with the model group, silybin treatment (26.25 mg·kg-1·day-1, started at the beginning of the protocol) significantly protected against high-fat-induced fatty liver by stabilizing mitochondrial membrane fluidity, reducing serum content of alanine aminotransferase (ALT) from 450 to 304 U/L, decreasing hepatic malondialdehyde (MDA) from 1.24 to 0.93 nmol/mg protein, but increasing superoxide dismutase (SOD) and glutathione (GSH) levels from 8.03 to 9.31 U/mg protein and from 3.65 to 4.52 nmol/mg protein, respectively. Moreover, silybin enhanced the gene and protein expression of adiponectin from 215.95 to 552.40, but inhibited that of resistin from 0.118 to 0.018. Compared to rosiglitazone (0.5 mg·kg-1·day-1, started at the beginning of the protocol), silybin was effective in stabilizing mitochondrial membrane fluidity, reducing SOD as well as ALT, and regulating gene and protein expression of adiponectin (P < 0.05). These results suggest that mitochondrial membrane stabilization, oxidative stress inhibition, as well as improved insulin resistance, may be the essential mechanisms for the hepatoprotective effect of silybin on non-alcoholic fatty liver disease in rats. Silybin was more effective than rosiglitazone in terms of maintaining mitochondrial membrane fluidity and reducing oxidative stress.

Polymorphism of the aryl-hydrocarbon receptor gene in intron 10 of human cancers

Polychlorinated dibenzo-p-dioxins (PCDDs) and related halogenated aromatic hydrocarbons (e.g., PCDFs), often called "dioxins", are ubiquitously present environmental contaminants. Some of them, notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are among the most toxic synthetic compounds known. The biological effects of dioxins are mediated via the aryl hydrocarbon receptor (AhR). Mutations in the AhR transactivation domain are linked to sensitivity to the acute lethality of TCDD. We present here a study of AhR gene polymorphism in normal and cancer human tissues affecting pre-mRNA splicing in the AhR gene-coding transactivation domain region (exon 10, intron 10, exon 11 region), previously shown to be associated with AhR dysfunction. We tested 126 pairs of normal and cancer tissue samples from liver, lung, stomach, kidney, mucous, breast, and pancreas of 49 males and 77 females (45-70 years of age). We used in vitro splicing assay, RT-PCR and sequencing methods. Our results showed that in an in vitro system it is possible to reconstitute cellular pre-mRNA splicing events. Tested cancer tissues did not contain mutations in the AhR transactivation domain region when the DNA sequences were compared with those from normal tissues. There were also no differences in AhR mRNA splice variants between normal and malignant breast tissues and no polymorphisms in the studied regions or cDNA.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.