Semiquantitative Reverse Transcriptase-polymerase Chain Reaction Research Articles

The physiological impact of bisphenol A on the developmental and reproductive processes of Sesamia nonagrioides (Lepidoptera: Noctuidae) under LD and SD photoperiods

In this study, we examined the physiological impact of bisphenol A (BPA) on the developmental and reproductive processes of Sesamia nonagrioides (Lepidoptera: Noctuidae) under 16L:8D (long day, LD) and 10L:14D (short day, SD) photoperiods. Larvae were continuously exposed to different concentrations of BPA (1, 10, and 100 μg/L) applied in their artificial diets, under LD and SD conditions. During LD photoperiod, increased pupal weights were observed in insects grown in culture media with the higher dose of 100 μg/L BPA. The morphological effects of BPA on the reproductive system of S. nonagrioides were also evaluated. Testis size was significantly decreased, while ovaries’ size was increased in BPA-treated animals. The sex ratio shifted in favor of females when the larvae were exposed to BPA. During SD photoperiod, the BPA exposure significantly decreased larval weight. Additionally, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were used to identify the effects of BPA in the transcriptional regulation of heat shock protein genes SnoHsp20.8, SnoHsc70, and SnoHsp83 during SD conditions. During diapause conditions, the expression levels of SnoHsp20.8 and SnoHsp83 were not affected by BPA, while SnoHsc70 mRNA levels were decreased. Here, we observed a differential impact of BPA during non-diapausing and diapausing conditions. Our results revealed the physiological impact of environmentally relevant concentrations of BPA in S. nonagrioides.

Upregulated hepatic expression of mitochondrial PEPCK triggers initial gluconeogenic reactions inthe HCV-3 patients.

To identify the differential expression of candidate gluconeogenic genes which may initiate hepatitis C virus (HCV) related metabolic disorder during early stages of disease. Patients of diverse age and sex, with positive HCV genotype 3 (HCV-3) RNA in serum and with no history of other related infections, co-infections, alcoholism, diabetes or chemotherapeutic treatments were considered for this study. Semi-quantitative reverse transcriptase PCR analysis and quantitative fold change analysis of the fresh liver biopsies of eight chronically infected HCV-3 patients and six healthy individuals were evaluated for three potential biomarkers involved in glucose homeostasis induction, namely mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2), glucose-6-phosphatase catalytic subunit (G6PC) and associated forkhead box protein 01 (FOXO1). Symptomatic evaluation, clinical history and blood test were conducted according to general disease prognosis procedures and reported here. Significantly upregulated expression of PCK2 independent of age, sex and viral infectivity levels in all HCV patients was observed, whereas no significant changes in the expression of G6PC and FOXO1 were found. PCK2 triggers initial gluconeogenic reactions which ultimately result in the accumulation of glycogen in the liver hepatocytes. We therefore suggest that the overproduction of PCK2 has important physiological role in the onset of metabolic disorder in the HCV-3 patients.

Correlation of High Mobility Group Box-1 Protein (HMGB1) with Clinicopathological Parameters in Primary Retinoblastoma.

HMGB1 is considered to be DNA chaperone as it binds without any specificity. It is the structural protein which alters nuclear homeostasis and genomic stability of chromatin. Its role in retinoblastoma (Rb) remains unclear. The aim of the present study was to evaluate the expression of HMGB1 protein in primary enucleated retinoblastomas. Expression of HMGB1 in 69 prospective cases of primary retinoblastoma were assessed by immunohistochemistry and reverse transcriptase PCR (RT-PCR) technique and correlated with clinicopathological parameters. Immunohistochemical staining revealed expression of HMGB1 in 55.07 % (38/69) cases. Semi-quantitative RT-PCR was performed on 31 fresh tumor tissues. mRNA expression was observed in 77.41 % (24/31) cases. Expression of HMGB1 was statistically significant with poor tumor differentiation (p = 0.0440) & optic nerve invasion (p = 0.0128). HMGB1 expression was frequently seen in poorly differentiated tumors and those with histopathological high risk factors. Therefore, HMGB1 may contribute to tumor invasiveness and could serve as a poor prognostic marker in Rb.

Corticotropin-Releasing Hormone (CRH) Promotes Macrophage Foam Cell Formation via Reduced Expression of ATP Binding Cassette Transporter-1 (ABCA1).

Atherosclerosis, the major pathology of cardiovascular disease, is caused by multiple factors involving psychological stress. Corticotropin-releasing hormone (CRH), which is released by neurosecretory cells in the hypothalamus, peripheral nerve terminals and epithelial cells, regulates various stress-related responses. Our current study aimed to verify the role of CRH in macrophage foam cell formation, the initial critical stage of atherosclerosis. Our quantitative real-time reverse transcriptase PCR (qRT-PCR), semi-quantitative reverse transcriptase PCR, and Western blot results indicate that CRH down-regulates ATP-binding cassette transporter-1 (ABCA1) and liver X receptor (LXR)-α, a transcription factor for ABCA1, in murine peritoneal macrophages and human monocyte-derived macrophages. Oil-red O (ORO) staining and intracellular cholesterol measurement of macrophages treated with or without oxidized LDL (oxLDL) and with or without CRH (10 nM) in the presence of apolipoprotein A1 (apoA1) revealed that CRH treatment promotes macrophage foam cell formation. The boron-dipyrromethene (BODIPY)-conjugated cholesterol efflux assay showed that CRH treatment reduces macrophage cholesterol efflux. Western blot analysis showed that CRH-induced down-regulation of ABCA1 is dependent on phosphorylation of Akt (Ser473) induced by interaction between CRH and CRH receptor 1(CRHR1). We conclude that activation of this pathway by CRH accelerates macrophage foam cell formation and may promote stress-related atherosclerosis.

Propofol Inhibits Lipopolysaccharide-Induced Inflammatory Responses in Spinal Astrocytes via the Toll-Like Receptor 4/MyD88-Dependent Nuclear Factor-κB, Extracellular Signal-Regulated Protein Kinases1/2, and p38 Mitogen-Activated Protein Kinase Pathways.

In this study, we investigated the effect of propofol, a commonly used IV anesthetic, on lipopolysaccharide (LPS)-induced inflammatory responses in astrocytes and explored the molecular mechanisms by which it occurs. Astrocytes were stimulated with LPS (1.0 μg/mL) in the absence and presence of different concentrations of propofol. The expression of astrocyte marker glial fibrillary acidic protein (GFAP) in astrocytes was detected using immunofluorescence staining and Western blot analysis. The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were measured using an enzyme-linked immunosorbent assay. The mRNA level of Toll-like receptor 4 (TLR4) was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The protein expressions of TLR4, myeloid differentiation factor 88 (MyD88), p- extracellular signal-regulated protein kinases (ERK)1/2, p-c-Jun N-terminal kinase, p-p38 mitogen-activated protein kinase (MAPK), p-I-κBα, I-κBα, and p-nuclear factor-κB (NF-κB)p65 were detected by Western blot. Our results show that after stimulation with LPS, the levels of IL-1β, IL-6, and tumor necrosis factor-α and the expression of GFAP in astrocytes were up-regulated significantly. In addition, the expression of TLR4, MyD88, p-ERK1/2, p-c-Jun N-terminal kinase, p-p38 MAPK, and p-NF-κBp65 increased, whereas the expression of total I-κBα decreased upon stimulation with LPS. Propofol (10 μM) reduced the secretion of proinflammatory cytokines, inhibited the expressions of GFAP, TLR4, MyD88, p-ERK1/2, p-p38 MAPK, and p-NF-κBp65 in astrocytes challenged with LPS. In the present study, propofol 10 μM but not lower clinically relevant or higher supra-clinical concentrations attenuated LPS-induced astrocyte activation and subsequent inflammatory responses by inhibiting the TLR4/MyD88-dependent NF-κB, ERK1/2, and p38 MAPK pathways.

마우스 수컷 생식세포에서 비스페놀 A에 대한 인삼 에탄올 추출물의 보호 효과

Department of Herbal Crop Research, NIHHS, RDA, Eumsung 369-873, Korea.ABSTRACT : This study was carried out to evaluate the preventive effect of three forms of Korean ginseng roots (fresh,white and red) against bisphenol A (BPA) toxicity in mouse male germ cells (GC-2spd, TM3, TM4). ROS (reactive oxygenspecies) generation were measured by DCF-DA (2’,7’-dichlorohydrofluorescein diacetate) assay. Also, semi-quantitativereverse transcriptase polymerase chain reaction (RT-PCR) was performed to quantify the mRNA expression levels of apop-tosis-related genes, Bax (pro-apoptotic gene) and Bcl2 (anti-apoptotic gene). ROS generation was increased by 50µM BPA,but definitely decreased by treatment with Korean ginseng extracts (fresh, white and red) in mouse male germ cells. In espe-cial, Korean fresh ginseng extract reduced significantly ROS production to normal control. In addition, Korean fresh andwhite ginseng extracts suppressed the apoptosis of mouse male germ cells by fine-tuning mRNA levels of apoptotic geneschanged by BPA. In general, Korean fresh ginseng extract was more effective than white ginseng extract for reducing BPA-induced oxidative stress and apoptosis in mouse male germ cells. Therefore, Korean fresh and white ginseng may help toalleviate biphenol A toxicity in mouse male germ cells.Key Words : Panax ginseng, Bisphenol A, Germ Cell, Apoptosis, Reproductive Toxicity

Methylation of Integrin α4 and E-Cadherin Genes in Human Prostate Cancer.

Prostate cancer is the second most common malignancy in men worldwide. Abnormal epigenetic alterations such as DNA methylation and histone modification play an important role in tumor initiation, progression and regulation of cancer-related genes such as integrin α4 and E-cadherin. Expression of these genes was determined by semi-quantitative reverse transcriptase-PCR in prostate cancer cell lines, DU145 and PC3, before and after treatment with 5-aza-2-deoxycytidine and trichostatin A. Laser capture microdissection microscopy was used to obtain exclusively affected epithelial cells from prostate gland biopsies of 30 patients with prostate cancer and 40 with benign prostate hyperplasia. DNA bisulfite modifications followed by methylation-specific PCR were used to evaluate the promoter methylation status of E-cadherin and α4 integrin genes in extracted DNA from patients and aforementioned cell lines. The integrin α4 promoter in DU145 was fully methylated, whereas in PC3 cells, partial methylation was detected. E-cadherin was expressed in both cell lines; trichostatin A and 5-aza-2-deoxycytidine treatment had no effect on E-cadherin expression, however the combined treatment of both drugs or 5-aza-2-deoxycytidine alone increased integrin α4 expression. Integrin α4 and E-cadherin were hypermethylated in 66.6 % and 6.6 % of prostate cancer cases, respectively; no hypermethylation was observed in patients with benign prostate hyperplasia. These results together suggest that aberrant DNA methylation is one of the mechanisms involved in integrin α4 expression and may play an important role in human prostate carcinogenesis. In addition, the higher rate of integrin α4 gene methylation in prostate cancer patients elects it as a potential molecular tumor marker.

High SIPA-1 expression in proximal tubules of human kidneys under pathological conditions.

Systemic lupus erythematosus (SLE) and clear cell renal cell carcinoma (CC-RCC) are serious disorders and usually fatal, and always accompanied with pathological changes in the kidney. Signal-induced proliferation-associated protein 1 (SIPA-1) is a Rap1GTPase activating protein (Rap1GAP) expressed in the normal distal and collecting tubules of the murine kidney. Lupus-like autoimmune disease and leukemia have been observed in SIPA-1 deficient mice, suggesting a pathological relevance of SIPA-1 to SLE and carcinoma in human being. The expression pattern of SIPA-1 is as yet undefined and the pathogenesis of these diseases in humans remains elusive. In this study, we used both immunohistochemistry and quantum dot (QD)-based immunofluorescence staining to investigate the expression of SIPA-1 in renal specimens from SLE and CC-RCC patients. MTT assay and Western blotting were employed to evaluate the effects of SIPA-1 overexpression on the proliferation and apoptosis of renal cell lines. Semi-quantitative reverse transcriptase-PCR (RT-PCR) was applied to examine the changes of hypoxia-inducible factor-1α (HIF-1α) mRNA level. Results showed that SIPA-1 was highly expressed in the proximal and collecting tubules of nephrons in SLE patients compared to normal ones, and similar results were obtained in the specimens of CC-RCC patients. Although SIPA-1 overexpression did not affect cellular proliferation and apoptosis of both human 786-O renal cell carcinoma cells and rat NRK-52E renal epithelial cell lines, RT-PCR results showed that HIF-1α mRNA level was down-regulated by SIPA-1 overexpression in 786-O cells. These findings suggest that SIPA-1 may play critical roles in the pathological changes in kidney, and might provide a new biomarker to aid in the diagnosis of SLE and CC-RCC.

RBMS3 is a tumor suppressor gene that acts as a favorable prognostic marker in lung squamous cell carcinoma.

Recent research indicates that RBMS3 may act as a tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) and esophageal squamous cell carcinoma (ESCC). It has been reported that RBMS3 directly binds to the promoter region of c-Myc in ESCC and that β-catenin from both whole cell extracts and nuclear fractionation was significantly downregulated in RBMS3-transfected NPC cells compared to control cells. The aim of this study was to evaluate the clinical significance of the RBMS3 gene expression in relation to the expression of Wnt pathway components in patients with lung squamous cell carcinoma (LSCC). RBMS3, c-Myc and cytoplasmic β-catenin were detected in 39.76, 56.63 and 89.16% of 83 LSCC samples by immunohistochemistry, respectively, in 83 primary LSCC samples. Semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting demonstrated decreased RBMS3 mRNA and expression in 33.33% (10/30) and 36.67% (11/30) tumor tissues, respectively. Statistical correlation analysis showed RBMS3 to be negatively correlated with c-Myc (r=-0.384, p<0.001) and not correlated with cytoplasmic β-catenin in the LSCC samples. Multivariate Cox proportional hazards model analysis showed that the combined marker RBMS3/c-Myc was an independent prognostic indicator of overall survival (p=0.001; HR 3.470; IC 95%, 1.652-7.290), and c-Myc was a prognostic indicator of disease-free survival (p<0.001; HR 3.182; IC 95%, 1.961-8.920). RBMS3 is a novel TSG in LSCC, and its downregulation facilitates development and progression of LSCC. Therefore, it is suggested that Rbms3 as a tumor marker may play an important role in diagnosis of LSCC.

Effect of Solanum surattense on mitochondrial enzymes in diabetic rats and in vitro glucose uptake activity in L6 myotubes.

Background:S. surattense is widely used in Siddha medicine for various ailments.Objective:The aim was to evaluate the impact of alcoholic leaf-extract of S. surattense on mitochondrial enzymes in streptozotocin (STZ) induced diabetic rats and to study the in vitro muscle glucose uptake activity on L6 myotubes.Materials and Methods:The male albino Wistar rats were randomly divided into five groups of six animals each. Diabetes was induced by intraperitoneal injection of STZ (40 mg/kg body weight). After being confirmed the diabetic rats were treated with alcoholic leaf-extract of S. surattense (100 mg/kg body weight) for 45 days. The biochemical estimations (liver mitochondrial enzymes, antioxidants, thiobarbituric acid reactive substances [TBARS]) and histopathological studies were performed. Further, the in vitro muscle glucose uptake activity in L6 myotubes and messenger RNA (mRNA) expression of glucose transporter-4 (GLUT-4) was performed.Results:In diabetic rats, the activities of liver mitochondrial enzymes were found to be significantly lowered. The mitochondrial TBARS level increased, whereas the activities/level of enzymatic and non-enzymatic antioxidants decreased in diabetic rats. Administration of S. surattense to diabetic rats significantly reversed the above parameters toward normalcy. Furthermore in diabetic rats, the histopathological studies showed growth of adipose tissue and shrinkage of islets in the pancreas, liver showed fatty change with mild inflammation of portal triad, and kidney showed messangial capillary proliferation of glomeruli and fatty infiltration of tubules. Treatment with S. surattense brought back these changes to near normalcy. The extract was analyzed for in vitro muscle glucose uptake activity in L6 myotubes and mRNA expression of GLUT-4 by semi-quantitative reverse transcriptase-polymerase chain reaction. One nano gram per millilitre of S. surattense leaf-extract gave 115% glucose uptake on L6 myotubes. It also showed elevated levels of GLUT-4 mRNA transcripts, when compared with control cells.Conclusion:These studies strongly support the anti-diabetic nature of S. surattense.

Overexpression of granulocyte macrophage colony stimulating factor in breast cancer cells leads towards drug sensitization.

This report describes the effect of overexpressing granulocyte macrophage colony stimulating factor (GMCSF) in breast cancer cells, which otherwise is involved in proliferation and differentiation of granulocyte and macrophage lineages. The purified recombinant GMCSF cytokine is known to exert dose-dependent proliferative response on various cancer cells, but its effect during overexpression is yet to be evaluated. In our present study, we have generated MCF-7 (breast cancer) cells overexpressing GMCSF. Interestingly, cell viability studies showed pronounced sensitivity of GMCSF overexpressing MCF-7 cells towards anticancer drugs, such as, doxorubicin, 5FU and cisplatin. These findings were substantiated by cell cycle analysis of the drug-treated GMCSF overexpressing MCF-7 cells. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results revealed differential expressions of cyclins, and the carboxyfluorescein succinimidyl ester (CFSE)-based assay established decrease in doubling time of GMCSF overexpressed cells with respect to the control populations. Thus, overexpressing of proliferative GMCSF cytokine in breast cancer cells may increase susceptibility to anticancer drugs.

Oleanolic acid from Fragaria ananassa calyx leads to inhibition of α-MSH-induced melanogenesis in B16-F10 melanoma cells

Natural products with non-toxic and environmentally friendly properties are good sources for skin-whitening and brightening cosmetic agents. Strawberries (Fragaria ananassa), and their parts are used as cosmetic agents, because they contain high levels of bioactive substances. We isolated and identified compounds from F. ananassa calyx. Oleanolic acid has multiple biological activities, including anti-tumor, anti-angiogenic, antiinflammatory, anti-oxidant, and pro-apoptotic effects. However, no study has investigated the influence of oleanolic acid on melanin synthesis in B16-F10 melanoma cells. In the present study, we investigated the effect of oleanolic acid on melanin biosynthesis in B16-F10 melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH). Oleanolic acid-mediated melanogenesis inhibition was studied by measuring intracellular and secreted melanin levels and by using Western blot and semiquantitative reverse transcriptase-polymerase chain reaction analyses. Oleanolic acid suppressed melanin release and expression, resulting in a significant dose-dependent decrease in secreted and intracellular melanin levels and cellular tyrosinase activity. Furthermore, it inhibited the expression of melanogenesis-associated factors, including tyrosinase, tyrosinase-related proteins-1 and -2, and microphthalmia-associated transcription factor, in α-MSH-stimulated B16-F10 melanoma cells. The results of the present study can contribute to the development of cosmetic agents utilizing the skin whitening and brightening effect of oleanolic acid, which will likely have a wide range of applications in the cosmetic industry and/or clinical practice in the future.

The mannoprotein TIR3 (CAGL0C03872g) is required for sterol uptake in Candida glabrata

Sterol uptake in the pathogenic fungus, Candida glabrata, occurs via the sterol transporter, CgAus1p. Azole inhibition of sterol biosynthesis can under certain circumstances be reversed by adding exogenously sterol. Here we demonstrate that the CgTIR3 (CAGL0C03872g) gene product is also required for sterol uptake, since Cgtir3Δ strains fail to take up sterol both aerobically and under hypoxic conditions. Western analysis using an HA-tagged TIR3 strain showed that CgTir3p localizes to the cell wall, and its expression is induced by serum. Semi-quantitative reverse transcriptase-PCR also showed that two transcription regulatory genes, CgUPC2A and CgUPC2B, control CgTIR3 as well as CgAUS1 gene expression. Interestingly, complementation studies using Cgtir3Δ showed that ScDAN1, a mannoprotein required for sterol uptake in Saccharomyces cerevisiae, could not complement the C. glabrata TIR3 function. Furthermore, sterol analyses, in which both the CgAUS1 and CgTIR3 genes were constitutively expressed, resulted in aerobic sterol uptake although the amount of uptake was considerably less than that of cells cultured aerobically with serum. These results suggest that additional factors other than CgAUS1 and CgTIR3 are required for sterol uptake in C. glabrata.

Systemic delivery of human bone marrow embryonic-like stem cells improves motor function of severely affected dystrophin/utrophin–deficient mice

Background aimsEmbryonic-like stem cells (ELSCs) express embryonic stem cell–specific marker genes, such as SSEA-4, Oct-4 and Nanog, and can be induced to differentiate into cells of all 3 germ layers. Our preliminary data showed that ELSCs isolated from human bone marrow express multipotent antigen markers and differentiate into multinucleated myotube-like cells more efficiently than do mesenchymal stromal cells (MSCs) isolated from the same source. We investigated the therapeutic effect of ELSCs in dystrophin/utrophin double knock-out (dko) mice, one of the Duchenne muscular dystrophy animal models, by systemically transplanting them through tail-vein injection. MethodsELSCs and MSCs were both isolated from human bone marrow. Two months after equal amounts of ELSCs or MSCs were injected through tail-vein injection, we evaluated skeletal muscle motor function and serum creatine kinase activity and measured dystrophin expression by means of immunostaining, Western blotting and semi-quantitative reverse transcriptase–polymerase chain reaction. ResultsELSCs positive for Oct-4 and Nanog-3 expressed higher levels of SSEA-4, FZD-9 and CD105 and were induced to differentiate into myotube-like cells more efficiently than did MSCs in vitro. Transplantation of ELSCs through the tail vein improved motor function and decreased serum creatine kinase activity at 2 months after cell transplantation. In addition, dystrophin protein and messenger RNA were upregulated and the skeletal muscle histology was improved in these dko mice transplanted with ELSCs. ConclusionsELSCs could be more efficiently induced to differentiate into myotubes than were MSCs in vitro, and systematically transplanting ELSCs improved muscle motor function and muscle histology in dko mice.

Molecular characterization of chicken class II transactivator gene.

Class II transactivator (CIITA) is an effective transcriptional factor regulating various genes in the immune system. Since the detection of CIITA in 1993, there has been considerable progress toward understanding its role as an activator of MHC II genes in human and mouse; however, there is little knowledge of this gene in other animals such as chicken. Molecular characterization of the chicken CIITA gene transcript was performed to determine its sequence and expression in different tissues. The CIITA cDNA was first generated through reverse transcriptase-polymerase chain reaction (RT-PCR) from Cobb chicken spleen cell RNA, using oligonucleotide primers based on the predicted cDNA sequence. The effect of the immune system stimulation on the CIITA gene expression in kidney, liver, thymus, and spleen were assessed by semi-quantitative RT-PCR analysis. A partial cDNA sequence (1,688bp) encoding part of the NACHT domain followed by seven of the transactivator and one of the NLS domains were obtained. Comparison of the deduced amino acid sequence with other CIITAs reveals high level of similarities in amino acid composition, secondary structure and phosphorylation sites. Furthermore, in comparison to the Red Jungle Fowl (RJF) sequence, we found 17 single nucleotide polymorphisms in Cobb broiler chicken, ten of which were reported for the first time. Gene expression analysis indicated that CIITA RNA amounts increased in all the examined tissues following stimulation with Brucella antigen. This investigation may indicate that CIITA molecule has an important role in the chicken immune responses as well as human and other animals.

Overexpression of CD133 enhances chemoresistance to 5-fluorouracil by activating the PI3K/Akt/p70S6K pathway in gastric cancer cells.

CD133 has been reported to be associated with chemoresistance in various cancer cells. The efficacy of 5-fluorouracil (5-FU), an important chemotherapeutic agent for advanced gastric cancer (GC), is limited by 5-FU resistance. Hence, the present study investigated the function of CD133 in 5-FU resistance in human GC cells. We isolated CD133+ GC cells by immunomagnetic cell sorting and CD133 expression was modulated by transfection of CD133 gene or CD133 small interfering ribonucleic acid. To assess the 5-FU cytotoxicity, Cell Counting Kit-8 was used. Expression of CD133, P-glycoprotein (P-gp), B-cell lymphoma 2 (Bcl‑2), Bcl-2-associated X protein (Bax), phospho-Akt (p-Akt) and phospho-p70S6 kinase (p-p70S6K) were analyzed by western blotting. CD133, P-gp, Bcl-2 and Bax messenger ribonucleic acids were evaluated using semi-quantitative reverse transcriptase-polymerase chain reaction. Cell apoptosis was assessed by Hoechst 33258 staining. CD133+ cells were more resistant to 5-FU than CD133- cells, and showed higher expression of P-gp and Bcl-2 with lower expression of Bax. Furthermore, CD133 silencing enhanced 5-FU cytotoxicity and apoptotic characteristics, whereas CD133 overexpression increased 5-FU resistance. CD133 silencing and activation directly decreased and increased the expression of P-gp, Bcl‑2, p-Akt and p-p70S6K, respectively. Notably, Akt inhibition by LY294002 restored the 5-FU cytotoxicity suppressed by CD133 overexpression, while Akt activation by epidermal growth factor reversed the 5-FU cytotoxicity enhanced by CD133 silencing. Therefore, CD133 may inhibit 5-FU-induced apoptosis by regulating the expression of P-gp and Bcl-2 family mediated by phosphoinositide 3-kinase/Akt/p70S6K pathway in GC cells.

Altered expression of alpha 2 beta 1 integrin in kidney fibroblasts: a potential mechanism for CsA-induced nephrotoxicity.

Nephrotoxicity is considered a significant cause of patient morbidity following chronic Cyclosporine A (CsA) treatment. The exact mechanism of CsA-induced nephrotoxicity remains to be fully clarified. Tubulointerstitial fibrosis is widely regarded as a major pathway of CsA toxicity; therefore, the role of integrins as regulators of collagen in the extra-cellular matrix can be deemed pivotal. The objective of the present study was to observe the expression levels of alpha2beta1 integrin following CsA treatment +/- antioxidants. Adhesion assay, immunofluorescent and flow cytometric analyses were performed on kidney fibroblasts obtained from rats after administration of CsA (25 mg/kg/day) +/- Vitamin E (vit. E) and Quercetin (Q) for 4 weeks. Total RNA was collected from the aforementioned fibroblasts for semi-quantitative reverse transcriptase-polymerase chain reaction analysis of α2 and β1 integrins. We found that α2 and β1 integrins were both markedly reduced following treatment with CsA, i.e., 25% and 13%, respectively, but were normal following subsequent consumption of the antioxidants vit. E and Q. Attachment and spreading of the CsA-treated fibroblasts declined from 82% to 50%; however, this effect was partially reversed to 70% following antioxidant treatment. Similar results were observed in the spreading assay in which the level of spreading decreased from 73% to 21% and was subsequently restored to 46%. We conclude that cell adhesion, mediated by binding of integrin to collagen, which is a prerequisite of normal cell viability and collagen regulation, may be a novel pathway further explaining the nephrotoxic effects of CsA.

Expression of Glycosyltransferases; ST3GAL1, FUT3, FUT5, and FUT6 Transcripts in Oral Cancer

Oral carcinogenesis process is frequently accompanied by alterations in glycosylation, regulated by sialyltransferase (ST) and fucosyltransferase (FUT) enzymes. The study aimed to assess ST3GAL1, FUT3, FUT5, and FUT6 mRNA expression by semi-quantitative reverse transcriptase PCR in 50 oral cancer and 50 adjacent normal tissues. The results indicated increased ST3GAL1 mRNA levels in malignant tissues as compared to adjacent normal tissues. A significant decrease in FUT3 and FUT5 transcripts was observed in malignant tissues as compared to adjacent normal tissues. Survival analysis of FUT3 transcript levels depicted significant lower survival with values above cutoff. The levels of ST3GAL1 and FUT6 were found to be higher in metastatic tissues as compared to the non-metastatic tissues and were also higher in advanced disease as compared to the early disease. The results indicated potential clinical utility of ST3GAL1, FUT3, FUT5, and FUT6 transcript levels in oral cancer pathogenesis.

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.Electronic supplementary materialThe online version of this article (doi:10.1007/s13353-014-0222-0) contains supplementary material, which is available to authorized users.

Effects and Safety of Xylitol on Middle Ear Epithelial Cells

OBJECTIVE: Xylitol is a natural sugar known to reduce dental decay. Recently, several epidemiology studies have also shown that xylitol can prevent acute otitis media in children. It is assumed that these effects are related to the inhibition of bacterial growth in the nasopharynx. However, the effects and safety of xylitol in middle ear epithelial cells, which play a key role in otitis media, have not been investigated. The present study was performed to investigate whether xylitol shows antiinflammatory or antioxidative effects on human middle ear epithelial cells and to assess its safety profile for clinical use. MATERIALS and METHODS: We induced inflammation in human middle ear epithelial cells (HMEEC) with lipopolysaccharide (LPS). After xylitol treatment, we measured expression levels of the inflammatory cytokines TNF-α and COX-2 and the mucin gene MUC5AC using semiquantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Cell viability and morphological changes following xylitol treatment were investigated by 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MMT) assay, flow cytometry, and Hoechst 33342 staining in human middle ear epithelial cells and mouse inner ear cells. RESULTS: Inflammatory cytokines and mucin gene expression were unaffected by xylitol. In addition, xylitol showed neither antioxidative effects nor cytotoxicity and did not stimulate apoptosis or necrosis of human middle ear epithelial cells or inner ear cells, even at high doses.