Real-time RT-PCR Technique Research Articles

Specific Viruses Detected in Nigerian Children in Association with Acute Respiratory Disease

Occurrence of different viruses in acute respiratory tract infections of Nigerian children was examined. Respiratory swabs were collected from 246 children referred to hospital clinics because of acute respiratory symptoms from February through May 2009. Validated real-time RT-PCR techniques revealed nucleic acids of at least one virus group in 189 specimens (77%). Human rhinoviruses and parainfluenza viruses were present each in one third of the children. Adenoviruses, enteroviruses, human metapneumovirus, human bocavirus, and influenza C virus were also relatively common. Possibly due to their seasonal occurrence, influenza A and B virus, and respiratory syncytial virus were detected rarely. We conclude that all major groups of respiratory tract viruses are causing illness in Nigerian children.

Polyunsaturated Fatty Acids and Modulation of Cholesterol Homeostasis in THP-1 Macrophage-Derived Foam Cells

Transformation of macrophages to foam cells is determined by the rates of cholesterol uptake and efflux. This study uses a real time RT-PCR technique to investigate the role of conjugated linoleic acid (CLA), α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) in the regulation of the ATP-binding cassette A1 (ABCA1) and liver X receptor α (LXR) genes, which are involved in cholesterol homeostasis. Accordingly, these fatty acids significantly reduced the total, free and esterified cholesterols within the foam cells. While the expression of the ABCA1 and LXRα genes was increased in the presence of the pharmacological LXRα ligand, T0901317, their mRNA expression was not significantly affected by CLA, ALA and EPA. These results suggest that although polyunsaturated fatty acids have an effect on cholesterol homeostasis, they cannot change the expression of the ABCA1 and LXRα genes. Alternatively, several other genes and proteins may be involved.

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1α partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B 1 and B 2 production levels. Among the isolated strains, 96 were identified as Fusarium verticillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1α sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verticillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r = 0.95 and r = 0.79 (correlation of FUM1 with FB 1 and FB 2, respectively, P < 0.0001); r = 0.93 and r = 0.78 (correlation of FUM19 with FB 1 and FB 2, respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity.

Coexpression of adrenomedullin and its receptor component proteins in the reproductive system of the rat during gestation

BackgroundAdrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries and uteri. Plasma ADM level increases in pregnant women and pregnant rats.MethodsThe gene expression levels of Adm and its receptor components - Crlr, Ramp1, Ramp2 and Ramp3, the ADM peptide concentration and localization in the rat female reproductive system during gestation were studied by real-time RT-PCR, EIA and immunohistochemical techniques.ResultsThe mRNAs of Adm and its receptor component and ADM were differentially distributed between implantation sites and inter-implantation sites of the pregnant uterus. The day on which vaginal sperm were found was taken to be pregnancy day 1. The Adm mRNA levels in the implantation sites of the uteri in mid- (day 12) and late pregnancy (day 17) were more than 10-fold higher than those in nonpregnancy, pre-implantation (day 3) or early (day 7) pregnancy. ADM was localized in the endometrial stroma with increased immunoreactivity from nonpregnancy to pregnancy. The ADM level and the mRNA levels of Adm, Crlr, Ramp2 and Ramp3 in the corpus luteum all increased in late pregnancy compared with early pregnancy. The gene expression of Adm and it receptor components and intense immunostaining of ADM were also found in the oviduct during pregnancy.ConclusionsThe gene expressions levels of Adm and its receptor components - Crlr, Ramp1, Ramp2 and Ramp3, and ADM peptide concentration exhibited a spatio-temporal pattern in the rat female reproductive system during gestation and this suggests that ADM may play important roles in gestation.

Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

BackgroundSeveral genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H2O2-induced oxidative stress and γ-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.ResultsHDFs with different experimental treatments exhibited diverse cell morphology with different expression of senescence-associated β-galactosidase (SA β-gal) activity. However the expression level of GAPDH was consistent in all treatment groups.ConclusionThe study demonstrated that GAPDH is reliable as reference gene for quantitative gene expression analysis in HDFs. Therefore it can be used as housekeeping gene for quantitative real time RT-PCR technique in human diploid fibroblasts particularly in studying cellular senescence.

Glioma Stem/Progenitor Cells Contribute to Neovascularization via Transdifferentiation

Previous studies suggest that tumor cells might be the progenitor for tumor vasculature. Whether vascular tube formation from transdifferentiation of human glioma stem/progenitor cells (hGSPCs) contribute to angiogenesis of gliomas remain largely uncertain. hGSPCs were isolated from thirteen surgical specimens of gliomas and cultured in medium favored for stem cell growth. In vitro transdifferentiation of hGSPCs was performed under hypoxia. Expression of vascular endothelial cells (VECs) markers CD31, CD34, kinase insert domain receptor (KDR), and von Willebrand factor (vWF) were analyzed with real-time quantitative RT-PCR and immunofluorescence techniques. Vasculogenic mimicry of hGSPCs was evaluated in a tumor stem cell xenograft model in vivo. Relationships between content of hGSPCs and expression levels of both VECs markers and proangiogenic factors in large number of clinical specimens were further investigated in glioma tissue microarray. In vitro, hGSPCs can transdifferentiate into VECs under hypoxia, they manifested typical "flagstone" pattern when cultivated in medium containing VEGF for a few days; when cultivated on Matrigel they were capable of forming capillary-like structures. Expression of VECs markers including CD31, CD34, KDR, and vWF were significantly up-regulated after transdifferentiation. Human leukocyte antigen (HLA) positively stained vessels were observed inside the xenograft tumors after intracerebral transplantation of hGSPCs in athymic nude mice, implied part of tumor cells with human origin were involved in formation of tumor vessels. In surgical specimens of human glioma, tumor vascular cells coexpressing the markers of early VECs (CD34) and markers of hGSPCs (ABCG2 and nestin) suggest that these vascular cells may stemmed from hGSPCs. Our observations suggest the functional role of hGSPCs as endothelial progenitors, which have properties that give rise to VECs, and have the ability to form vascular endothelial tubes. However, unspecific markers (ABCG2, nestin) that stain for both endothelial as well as glioma stem cells, were found to be expressed in tumor vasculature of human specimen, and limit further interpretation of this finding.

MODERN LABORATORY DIAGNOSTIC METHODS IN VETERINARY MEDICINE AND THE POSSIBILITY OF ITS APPLICATION

One of the greatest challenges of modern laboratory diagnosis is the selection of methods for fast, highly sensitive and specific detection of the infective agents. This challenge is present also in molecular diagnosing of causative agents in veterinary medicine. An example of molecular detection, the application of molecular diagnostic methods and procedures in the epizootiology of some viral infections is presented in the paper. The molecular methods play an important role in virus detection and surveillance. Out of a large number of molecular methods most frequently used are classical gel-based PCR (PCR, RT-PCR and nested PCR) and real-time PCR or RT-PCR techniques. Due to highly specificity and sensitivity these methods have been introduced as internationally recognized methods for virus detection in clinical materials. The advantage of the aforementioned molecular methods is that they are very fast and highly sensitive, and able to analyse a high number of samples. The obtained results may be used in molecular epizootiology, possibly for differentiation of filed isolates and vaccine virus strains, and for examining the samples not suitable for virus isolation. Alongside, these methods provide accurate quantification of viral particles in sample material. The detection with high sensitivity and specificity is of utmost importance in detection and typisation of the agents of highly contagious diseases and zoonoses. An example of rapid detection and sensitivity of RT-PCR and real-time RT-PCR in detection and characterization of avian influenza virus in clinical material as well as BVD virus in native bull semen is presented. Besides, molecular methods may be used for other purposes. Genome fragments amplified by PCR and RT-PCR, may be sequenced and used for classification, i.e. for virus isolate genotypisation. The results obtained in this way may be used for basic molecular research in epizootiology, that will point on the source of infections, their correlation and the prevalence of the causative agents what can help in finding the answer to the question why the diseases have occurred and what are the perspective for diseases outcome in the future. Also, these examination may help in determining pathogenicity, virulence and the spread of the pathogen agents. These information are of immeasurable importance for all the procedures in disease prevention and control. The possibilities of sequencing, molecular typisation and epizootiology of BVD virus and CSF virus isolated in Vojvodina in the last years are given as an example how this method can be used.

Expression of cathepsin G in photoaged fibroblasts

Objective To investigate the role of cathepsin G in photoaged fibroblasts. Methods Human fibroblasts were cultured and induced to premature senescence using UVA + MOP methods. Senescence-associated-β-galactosidase (SA-β-gal) stain was used to evaluate the positive rate of aged cells. The mRNA and protein expression of cathepsin G in photoaged fibroblasts were detected by real-time RT-PCR and Western blot techniques. Results Over 98 % induced cells presented a positive SA-β-gal straining. The expression of cathepsin G, detected by Western blot, was increased to (1. 70±0. 028) times of the control. And RT-PCR revealed that the synthesis of cathepsin G mRNA was also up-regulated to 1. 42±0. 09. Conclusion The results of our study demonstrates a significant correlation between photoaged fibroblasts and cathepsin G. The up-regulation of cathepsin G may play an important role in the damages of extracellular matrix and activation of MMPS in photoaged human skin. Key words: Photoaging; Cathepsin G; Skin; Fibroblast

Impact of diagnostic procedures on patient management and hospitalization cost during the 2000 and 2005 enterovirus epidemics in Marseilles, France

Enteroviruses are frequent aetiological agents of central nervous system infections in humans. In 2000 and 2005, two large outbreaks of Echovirus 30 (a member of species human enterovirus B) were observed in the University Hospitals of Marseilles (France). Between the two epidemics, the diagnostic protocols for enterovirus infection were modified, moving from viral cultures and classic RT-PCR in 2000 to real-time RT-PCR in 2005. We compared some viral and epidemiological characteristics of the 2000 and 2005 outbreaks with special attention to diagnostic procedures and to the subsequent clinical management of patients. Despite similar virological and epidemiological characteristics during both outbreaks, our results show that real-time RT-PCR techniques used in 2005 noticeably shortened the period of time necessary to deliver diagnostic results and suggest that this was associated with a decrease in the duration of hospitalization for -positive cases. In conclusion, this study suggests that the improvement of enterovirus diagnosis had a major financial impact on the management of the 2005 epidemic in Marseilles and may constitute an interesting example of how new diagnostic methods in microbiology can be self-financed through improvement in patient management.

Expression of Neuronal Nitric Oxide Synthase in Rabbit Carotid Body Glomus Cells Regulates Large-Conductance Ca2+-Activated Potassium Currents

Our previous studies show that a decrease in endogenous nitric oxide (NO) is involved in the blunted outward K(+) currents in carotid body (CB) glomus cells from chronic heart failure (CHF) rabbits. In the present study, we measured the effects of the neuronal nitric oxide synthase (nNOS) transgene on the K(+) currents in CB glomus cells from pacing-induced CHF rabbits. Using single-cell real-time RT-PCR and immunofluorescent techniques, we found that nNOS mRNA and protein are expressed in the rabbit CB glomus cells and CHF decreased the expression of nNOS mRNA and protein in CB glomus cells. After 3 days of an adenoviral nNOS (Ad.nNOS) gene transfection, the expression of nNOS protein was increased to the level found in sham CB glomus cells. In whole cell patch-clamp experiments, Ad.nNOS markedly reversed the attenuated K(+) currents in CB glomus cells from CHF rabbits. The specific nNOS inhibitor (S-methyl-l-thiocitrulline [SMTC]) and large-conductance Ca(2+)-activated K(+) (BK) channel blocker (iberiotoxin) fully abolished the effect of Ad.nNOS on the K(+) currents in the CB glomus cells from CHF rabbits. However, neither CHF nor Ad.nNOS altered the protein expression of BK channel alpha-subunit. These results suggest that a decrease of NO induced by an attenuated nNOS activity lowers the activation of the BK channels but not the protein expression of the BK channel alpha-subunit in the CB glomus cells during CHF.

Rosiglitazone Attenuates Endothelin-1–Induced Vasoconstriction by Upregulating Endothelial Expression of Endothelin B Receptor

Thiazolidinediones improve insulin resistance and endothelial dysfunction. However, the mechanisms underlying the vasoprotective effects of thiazolidinediones remain to be fully elucidated. The present study aimed to examine the molecular mechanism for the anti-vasoconstrictive effects of rosiglitazone in response to endothelin (ET) 1. Mouse aortas were treated with rosiglitazone for 24 hours, and ET-1-induced vasoconstriction was assessed by wire myography. The results showed that rosiglitazone attenuated ET-1-induced contraction in mouse aortas; this effect was abolished by ET-B receptor (ET(B)R) antagonist, NO synthase inhibitor, and by the removal of endothelium. By using Northern blotting, real-time RT-PCR, Western blotting, and immunohistochemical techniques, we found that rosiglitazone upregulated expression of ET(B)R at both mRNA and protein levels in mouse aortas and human vascular endothelial cells. The induction of ET(B)R was prevented by peroxisome proliferator-activated receptor-gamma antagonism. Luciferase reporter assay showed that rosiglitazone enhanced ET(B)R gene promoter activity. Furthermore, chromatin immunoprecipitation assays demonstrated that peroxisome proliferator-activated receptor-gamma can directly bind to ET(B)R gene promoter. Furthermore, in vivo treatment with rosiglitazone also attenuated the ET-1-induced vasoconstrictions and increased the ET(B)R expression in mouse aortas and mesenteric arteries. In conclusion, these results demonstrate that rosiglitazone attenuated ET-1-induced vasoconstriction through the upregulation of endothelial ET(B)R, which is a peroxisome proliferator-activated receptor-gamma direct target.

The effect of pigment epithelium-derived factor on expression of glutamine synthetase in diabetic rat retina

Objective To investigate the effect of pigment epithelium-derived factor (PEDF)on the expression of glutamine synthetase in retinal Mailer cells of diabetic rats. Methods Diabetic rats were induced with streptozotocin injection. Before and after injection of 10 μ1 (0. 1 μg/μl) PEDF (experimental group) or 10 μl PBS (control group) into the vitreous cavities of diabetic rats respectively for 48 hours, the expressions of GS and IL-1β in retina were analyzed by immunohistochemistry and real time RT-PCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions, the expressions of GS and IL-1β in cultured MOiler ceils were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V-fluorescein isothiocyanate/Propidium idoium (Annexin V-FITC/PI) staining. Results By immunohistochemistry (the protein level) and real time RT-PCR (the mRNA level), it was found that the expression of GS decreased and the expression of IL-1β increased obviously (real time RT-PCR:GS: t=4. 23, P<0. 01 ;IL-1β: t= 16. 73,P<0. 01 ;immunohistochemistry: GS: t=5.13,P<0. 01;IL-1β: t= 9. 32, P<0. 01) in diabetic rats. After injection of 10 μl (0. 1 μg/μl) PEDF into the vitreous cavities of diabetic rats for 48 hours, it was found that the expression of GS increased and the expression of IL-1β decreased significantly(RT-PCR GS: t= 3. 87, P<0.01 ; IL-1β: t= 3. 61,P<0. 05 ;immunohistochemistry:GS: t = 3. 32, P< 0.05; IL-1β: t = 2. 63, P<0.05). Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1β and increasing expression of GS significantly (RT-PCR:GS: t=2.89, P<0. 05;IL-1β: t=3. 37, P<0. 05;Western blot:GS: t=2.66, P< 0. 05;IL-1β: t=3.23, P<0. 05). Apoptosis of Muller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF (t=3.21, P<0. 05). Conclusions In diabetic rats, PEDF may decrease expression of IL-1β in rat retinal Mailer cells, which may result in increasing expression of GS. To some degree, it inhibits possibly the death of retinal ganglion cells. Key words: Diabetic retinopathy/Phisiopathology; Cytokines/physiology; Glutamate synthase/physiology; Diabetes mellitus, experimental

Short-term exposure to ethanol causes a differential response between nerve growth factor and brain-derived neurotrophic factor ligand/receptor systems in the mouse cerebellum

Alcohol ingestion affects both neuropsychological and motor functions. We hypothesized that one of the key factors involved in such functions are neurotrophins and their receptors. We have therefore examined the effects of short-term ethanol exposure on the mRNA expression and protein levels of neurotrophin ligands and receptors in the cerebellum using real-time RT-PCR and Western blotting techniques. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. The pair-fed control mice were fed an identical liquid diet except that sucrose was substituted isocalorically for ethanol. The cerebellum of mice exhibiting intoxication signs of stage 1 or 2 were used in the present study. We found that exposure to ethanol resulted in elevated levels of nerve growth factor (NGF) and TrkA mRNA expression but a decreased level of brain-derived neurotrophic factor (BDNF) mRNA expression. The expression of TrkB and p73 mRNA was unchanged. Changes in the level of these proteins were found to mirror these mRNA expression levels. We conclude that exposure to ethanol for a short period can cause a differential responsive in the various neurotrophin ligand/receptor systems. The functional consequences of these changes are unknown at present.

Quantitative estimation of plum pox virus targets acquired and transmitted by a single Myzus persicae

The viral charge acquired and inoculated by single aphids in a non-circulative transmission is estimated using plum pox virus (PPV). A combination of electrical penetration graph and TaqMan real-time RT-PCR techniques was used to establish the average number of PPV RNA targets inoculated by an aphid in a single probe (26,750), approximately half of the acquired ones. This number of PPV targets is responsible for a systemic infection of 20% on the inoculated receptor plants. No significant differences were found between the number of PPV RNA targets acquired after one and after five intracellular punctures (pd), but the frequency of infected receptor plants was higher after 5 pd. The percentage of PPV-positive leaf discs after just 1 pd of inoculation probe (28%/4,603 targets) was lower than after 5 pd (45.8%/135 x 10(6) targets). The methodology employed could be easily extended to other virus-vector-host combinations to improve the accuracy of models used in virus epidemiology.

Expression of insulin-like growth factor-1 and alkaline phosphatase in the reconstruction of cleft palate with distraction osteogenesis in rhesus

To investigate the osteogenesis mechanism by analysis of the expression of insulin-like growth factor-I (IGF-1) and alkaline phosphatase (ALP) in the reconstruction of cleft palate (CP) with distraction osteogenesis (DO) in rhesus. The CP animal models were established surgically. 21 rhesus in experimental group underwent DO to close the soft and bony defect, followed by consolidations. Every 3 animals were killed and the specimen were taken out after consolidation of 1, 2, 4, 6, 8, 12, 24 weeks. The mRNA of IGF-1 and ALP were detected with Real-time RT-PCR technique. The expression of IGF-1 and ALP was quantitatively analyzed by ELISA. The results were compared with those in control and sham groups (each of 2 animals), respectively. Since consolidation, the mRNA of IGF-1 and ALP increased significantly at one week and reached the peak at two weeks, but decrease to control level after 12 weeks of consolidation. The expression of IGF-1 also increased to peak level after two weeks of consolidation. The expression of ALT increased significantly since consolidation and reach the peak value after six weeks. They all decreased to nearly control level after 8-12 weeks. The palate cleft can be successfully closed with new formed bone after DO. The mechanism of bone consolidation is intramembranous bone formation.

Diminished expression of ICOS, GITR and CTLA-4 at the mRNA level in T regulatory cells of children with newly diagnosed type 1 diabetes.

Diabetes mellitus is one of the most common chronic diseases in children. T regulatory cells (Tregs) modulate response to autoantigens and probably play a role in pathogenesis of type 1 diabetes (T1DM). The aim of the present study was the assessment of T regulatory cells including their percentages and expression of critical genes in these cells in children with newly diagnosed type 1 diabetes. The examined group consisted of 50 children with T1DM. A flow cytometric analysis of T-cell subpopulations was performed using the following markers: anti-CD4, anti-CD25 and anti-CD127 (=IL-7R). Additionally, T regulatory cells were isolated for assessment of mRNA levels for chosen genes with the real-time RT-PCR technique. The percentages of CD4(+)CD25(high)CD127(dim/-) were very low and did not differ between T1DM and control children. We did not observe any statistically significant differences between healthy and diabetic children in mRNA expression for FoxP3, IL-7R (CD127), IL-8RA, IL-10RA, IL-12A, IL-2RA (CD25), IL-21, STAT1, STAT3, SOCS2, SOCS3, TGF-beta1-R1, TGF-beta-R2 and TBX-21 genes. Interestingly the mRNA level for CTLA-4, ICOS1, IL-23, IL-27, SMAD3 and GITR were lower in Treg cells of children with diabetes compared to the control patients. No disturbances in the percentages of T regulatory cells in patients with diabetes but diminished expression of some elements important in Treg function could be the result of an immunologic imbalance accompanying the onset of the diabetes. The results of our study should be used in future research in the field of immunotherapy in pediatric diabetes.

Emprego da RT-PCR em tempo real para a quantificaÇão da expressão de genes associados à resposta imune em bezerros bovinos experimentalmente infectados por Neospora caninum

Neospora caninum is one of the main causes of abortion and natimortality in cattle. Host immune defense is capable to inhibit tachyzoite activity during acute infection, but there is no action against bradyzoites in tissue cysts. Activation and modulation of this response is controlled by cell mediators. The real-time RT-PCR technique was employed to detect some of those mediators during N. caninum infection. Holstein and Nelore calves intramuscularly infected with tachyzoites and uninfected controls were slaughtered at the sixth day post-infection and popliteal lymph node, liver and brain cortex samples were analyzed. Real-time RT-PCR detected gene expression in all tissues. No significant variation of GAPDH gene expression was detected among groups, its amplification efficiency was similar to the other genes tested and it was used as the endogenous control for the analysis. Comparisons between infected and uninfected groups allowed the relative gene expression quantification. IFN-gamma and TNF-alpha genes showed increased expression in some samples. iNOS and TGF-beta1 genes had some non-significant variations and IL-4 and IL-10 stayed practically unaltered.

Plasma Glucose Lowering Effect of the Wild Satureja khuzestanica Jamzad Essential Oil in Diabetic Rats: Role of Decreased Gluconeogenesis

This study was to evaluate the effect of the wild SKEO on activities and genes expression of hepatic Glycogen Phosphorylase (GP) and phosphoenolpyruvate carboxykinase (PEPCK) in normal and diabetic rats. The wild SKEO was orally administered at different doses (50 and 100 mg/kg/day) to normal as well as diabetic rats for 21 days. The levels of mRNA were determined using the quantitative real-time RT-PCR technique. The plasma glucose concentrations of diabetic rats receiving SKEO (100 mg kg(-1)) compared with diabetic control were significantly decreased. Hepatic GP activity and its mRNA levels of diabetic rats treated with SKEO moderately increased. The activity of hepatic PEPCK and its mRNA levels were significantly decreased in normal rats treated with SKEO (100 mg kg(-1)). The enhancement of PEPCK activity and its mRNA levels of diabetic treated rats with SEKO (100 mg kg(-1)) was significantly decreased compared with diabetic control. In conclusion, an excessive inhibition of PEPCK in liver of diabetic rats treated with the wild SKEO may contribute to the plasma glucose lowering action of SKEO that seems to be in relation with antioxidant properties of SKEO.

Significance of BAALC, MN1, ERG, CEBPa Gene Expression as New Prognostic Factors, in the Context of Other Molecular Markers in Intermediate Risk AML Group According to Cytogenetics, Including Patients Younger Than 60 Years

Background: High expression of BAALC,MN1, ERG and low CEBPA have been shown to be an adverse risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). However intermediate (IM) risk group of AML includes not only patients with normal karyotype, but also with other aberrations, of unknown clinical significance. That's why it is important to evaluate prognostic impact of the above mentioned markers in the IM group of AML patients as total. So far, the prognostic significance of expression level of the studied markers has not been yet compared with each other, as well as evaluated in the context of other molecular biomarkers, such as FLT3 ITD (internal tandem duplication), MLL-PTD (partial tandem duplication) mutations, and EVI1 expression level.Aims: To evaluate the prognostic impact of BAALC,MN1, ERG, CEBPA expression in the context of FLT3-ITD, MLL-PTD and EVI1 expression on the complete response to induction therapy (CR), and the relapse rate in the patients group representing intermediate risk group of AML according to cytogenetics.Methods: 86 patients (16 to 60 years of age) with untreated primary IM-risk AML were included in this study: 55 with normal karyotype, and 31 with other aberrations. Patients were treated similarly according to the Polish Acute Leukemia Group (PALG) protocol (induction chemotherapy: daunorubicin, cytarabine plus fludarabine or cladribine, and two cycles of consolidation high-dose cytarabine with mitoxantrone and high- dose cytarabine). BAALC, MN1, ERG, CEBPA expressions were evaluated in the bone marrow cells from IM-AML patients by real-time RT-PCR technique, using TaqMan probes, as recently described.Results: High and low BAALC, as well asERGandMN1 expression was determined by dichotomizing delta Ct values at the median (high expression of ERG, MN1 referred further as negative or positive cases). High BAALC correlated significantly with the group positive for MN1 (p=0.01), ERG (p=0.01) as well as MLL-PTD (p=0.045), while no correlation was found between: high BAALC and FLT3-ITD, as well as CEBPA. There was a trend towards significance towards lower CR rates in the groups with high BAALC, positive MN1, positive EVI1 expression or MLL-PTD, while low CEBPA showed negative correlation with CR rate (p=0.02). Next we analysed cumulative presence of the subsequent markers: MLL-PTD, positive ERG, positive MN1, and positive EVI1. Cases negative for all of above markers presented 85 % of CR rate, patients with one or two markers present: 55% CR rate, and a group with more than three of them positive: only 20% CR rate. Further comparing DFS after 2nd and 3rd year of follow up, with each marker separately did not show significant correlation, although a trend towards significance was observed for most of them. However again, cumulative presence of high BAALC, positive MN1, positive ERG, positive EVI1, and MLL-PTD showed significant correlation with 2 and 3 years' DFS (respectively 70% for the cases with 0 –2 markers, while 22% for the group with more than 2 markers present; p=0.035).Conclusions: In our study, we analysed a series of patients with intermediate risk according to the cytogenetics (NC and other aberrations). Cumulative presence of high BAALC, and positiveERG, MN1, EVI1 expression levels as well as MLL-PTD, showed their prognostic significance.

Gene Expression Profiling Distinguishes Essential Thrombocythemia from Polycythemia Vera Patients and Identifies a Common Expressed Set of Genes in Relation to JAK2V617F Status

Introduction. The existence of the JAK2V617F mutation in a high proportion of Myeloproliferative Disorders (MPD) BCR-ABL-negative has provided important insight into the pathogenesis of these diseases. However, much of the molecular abnormalities associated to BCR-ABL-negative MPD remain unknown, specially in those which do not display JAK2V617F. In a previous study, we performed gene expression analysis by using the microarray technique in 20 essential thrombocythemia (ET) patients (44K whole human genome oligo microarrays, Agilent Technologies) and the results were confirmed in 40 ET patients by using TaqMan® Low Density Arrays Arrays (LDA, Applied Biosystems). In our previous experience the results showed different gene expression patterns in ET and a supervised clustering of the data identified genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients, and a characteristic gene expression profile for JAK2V617F-negative patients (Puigdecanet et al.,2008).Aim. Our aim was to confirm the ET gene expression profile in an extended group of patients and to explore the differences and similarities in polycythemia vera (PV) and reactive thrombocytosis (RT) patients by real-time quantitative RT-PCR (RQ-PCR) technique using the LDA platform. In addition, we wanted to analyze the relationship between gene expression data and JAK2V617F status.Patients and Methods. The following patients were included in the study: 58 ET (23 JAK2V617F-negative, 34 JAK2V617F in heterozygosity and one JAK2V617F in homozygosity) and 41 PV (7 JAK2V617F-negative, 25 JAK2V617F in heterozygosity and 9 JAK2V617F in homozygosity) patients, diagnosed according to the WHO criteria (2001) and who had never received cytoreductive treatment, and six patients with RT. Based on the previous results, we designed a new LDA platform containing 96 assays in duplicate, which included the most expressed genes in ET in relation to healthy controls and the most differentially expressed genes between JAK2V617F-negative and JAK2V617F-positive ET patients. The RQ-PCR analysis was performed in RNA from peripheral blood granulocytes and the relative gene expression quantification was achieved using GAPDH as the endogenous control and a pool of 10 healthy individuals as the calibrator.Results. ETvsPV: The majority of the genes studied presented significant higher expression in ET than in PV patients. Interestingly, FOSB was one of the most differentially expressed gene (FC= 8.3), and CISH and C13orf18 did not distinguish between the two groups.ET: We confirmed the differentially expression of the majority of the genes previously detected between JAK2V617F-negative and JAK2V617F-positive ET patients and we extended the set of genes. Among them, we highlight the differential expression of CISH, FOSB and C13orf18 genes (p<0.01).PV: Supervised analysis showed that CD44, BATF and CISH clearly distinguish JAK2V617F-negative PV patients from the JAK2V617F-positive.ET and PVvsRT: Some differentially expressed genes between ET and RT patients were detected, but the most significant gene was TNF (p<0.001), which presented a higher expression in RT (FC=5.9). The same difference was observed between PV and RT.Conclusions. We have detected a different gene expression pattern in ET and in PV patients. However, we also identified a set of genes which expression was related to JAK2V617F status, both in ET and PV patients. These findings would be interesting to identify other signal transduction pathways besides JAK-STAT involved in the pathogenesis of ET and PV.