scholarly journals Changes in the expression of galanin and galanin receptors in the wall of the colon in pigs experimentally infected with Brachyspira hyodysenteriae

2014 ◽  
Vol 58 (1) ◽  
pp. 23-28 ◽  
Author(s):  
Krzysztof Wąsowicz ◽  
Piotr Podlasz ◽  
Małgorzata Chmielewska ◽  
Katarzyna Łosiewicz ◽  
Jerzy Kaleczyc ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Experimental Colitis ◽  
Expression Level ◽  
Colonic Wall ◽  
Brachyspira Hyodysenteriae ◽  
Expression Levels ◽  
Gapdh Expression ◽  
Galanin Receptors ◽  
Different Levels

Abstract The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.

High FLT3 mRNA Expression in Acute Myeloid Leukemia May Be Functionally an Alternative to Mutational Activation of the Receptor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3374-3374 ◽  
Author(s):  
Florian C. Kuchenbauer ◽  
Wolfgang Kern ◽  
Claudia Schoch ◽  
Alexander Kohlmann ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
De Novo ◽  
Expression Level ◽  
Alternative Mechanism ◽  
Expression Levels ◽  
Flt3 Mutation ◽  
Flt3 Mutations ◽  
Flt3 Expression ◽  
Mutational Activation

Abstract Mutations of the FLT3 gene are detectable in approximately 30% of all adult AML. These mutations lead to an autoactivation of the receptor inducing increased proliferation of the leukemic clone. An alternative mechanism of FLT3 activation might be mRNA overexpression. In the presented study the FLT3 expression level in 208 adult AML patients and 8 healthy donors was assessed by real time PCR and correlated to several parameters. In all patients cytomorphology, cytogenetics, and FLT3 mutation status was assessed. Significant differences of FLT3-expression levels were found in certain AML subgroups. The highest expression levels were found in FAB subtypes M5 and the lowest in M3. In total, increasing levels were shown in the following order: M3 <M3v <M6 <M2 <M4eo <M4 <M0 <M1 <M5a <M5b. Independent analysis of FLT3 expression in different cytogenetic AML subgroups showed the lowest median in the t(15;17) group, followed by t(8;21), inv(16), normal, complex karyotype and the highest median in the t(11q23) group. No difference was observed between the group of secondary AML following MDS, therapy related AML and the de novo AML (p=0.868, p=0.562, and p=0.570, respectively). Compared to clinical parameters, FLT3 expression correlated with high percentage of bone marrow blasts (p=0.0005) and high leukocyte count (p<0.001). In contrast to previous studies no difference in FLT3 expression levels was detected between AML with (n=74) and without (n=130) any FLT3 mutation. Assessment of FLT3 RNA by microarray analysis and FLT3 receptor surface expression (CD135) detected by flow cytometry correlated significantly with FLT3-expression as assessed by real time PCR (p<0.001, each). To analyze whether high FLT3 expression is a prognostic parameter 118 AML cases with normal karyotype were devided into two groups. Group 1 (n=75) was defined to have less and group 2 (n=43) more than the median of the FLT3 expression level found in the total group. No impact on OS and EFS could be shown (608 vs. 311 days, p=0.1283 and 398 vs. 208 day, p=0.3056). In conclusion, these data support the hypothesis that FLT3 activation through mRNA overexpression is an alternative mechanism to FLT3 mutations. Especially as it was found extremely high in 11q23 AML, that rarely reveal FLT3 mutations.

scholarly journals The Effects of miR-136-5p-Mediated Regulation of A20 in Astrocytes from Cultured Spinal Cord Cultured Cells In Vitro

2017 ◽  
Vol 41 (4) ◽  
pp. 1596-1604 ◽  
Author(s):  
Xiaoming Peng ◽  
Xiongzhi Shi ◽  
Jinmin Zhao ◽  
Jichen He ◽  
Keke Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Inflammatory Cytokine ◽  
Cultured Cells ◽  
Expression Level ◽  
Control Group ◽  
Interleukin 17 ◽  
Expression Levels ◽  
Inhibition Group

Background/Aims: This study focused on investigating the regulatory mechanism of miR-136-5p in mouse astrocytes stimulated with interleukin-17(IL-17). Methods: C57BL/6 mouse astrocytes were stimulated with IL-17 (100ng/ml) for various periods of time (0-48 hours) and at various doses (0-200 ng), and the expression levels of inflammatory cytokine and chemokine genes (IL-6, TNF-α, MCP-1, MCP-5 and MIP-2) were then detected by real-time PCR. The expression of the A20 gene was measured with real-time PCR in cells that were stimulated with IL-17 (50 ng/ml) for various periods of time (0-48 hours). C57BL/6 mouse astrocytes were transfected with Ctrl-anti-miR-136-5p or LNA -anti-miR-136-5p for 48 h. Thereafter, the cells were stimulated with or without IL-17 (50ng/ml) for 6 h. The level of A20 protein (TNFα-induced protein 3, TNFAIP3) was detected by Western blot analysis. Results: (1) Compared with the DMEM control group, within six hours, IL-17 stimulation significantly increased the expression levels of inflammatory cytokine and chemokine genes and clearly decreased the expression level of the A20 protein. (2) Without IL-17 stimulation, the expression level of the miR-136-5p gene was significantly decreased, whereas in the miR-136-5p-inhibition group, the A20 protein expression was elevated. IL-17 stimulation slightly decreased the expression of the A20 protein in the miR-136-5p-inhibition group, but it was still slightly higher than in the control group. Conclusion: This study demonstrated that miR-136-5p affected the expression of A20 in IL-17-stimulated astrocytes.

scholarly journals A survey on the occurrence of Brachyspira pilosicoli and Brachyspira hyodysenteriae in growing-finishing pigs

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1702
Author(s):  
Arkadiusz Dors ◽  
Ewelina Czyżewska-Dors ◽  
Grzegorz Woźniakowski
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Finishing Pigs ◽  
Lawsonia Intracellularis ◽  
Fecal Samples ◽  
Brachyspira Pilosicoli ◽  
Brachyspira Hyodysenteriae ◽  
Veterinary Research ◽  
Two Samples ◽  
Intestinal Pathogens

Background: The major pathogenic intestinal spirochetes affecting pigs during the growing- finishing stage of production include Brachyspira hyodysenteriae and Brachyspira pilosicoli. Infections by these pathogens, which affect the economics of pig production, can result in mortality, growth rate losses and substantial antibiotic costs. The aim of this study was to assess the current occurrence of B. hyodysenteriae and B. pilosicoli in Polish pig herds. Moreover, associations between the presence of diarrhea or other intestinal pathogens and occurrence of B. hyodysenteriae and B. pilosicoli in pigs were investigated. Methods: Between January 2017 and August 2019, a total of 401 samples of pig feces from 95 different herds were submitted to the National Veterinary Research Institute of Poland. These samples were obtained from pigs older than 7 weeks. All the received fecal samples were examined for the presence of B. hyodysenteriae, B. pilosicoli and Lawsonia intracellularis by real-time PCR. Results: For B. pilosicoli, 4.5% (95% CI, 2.5–7.0%) of samples and 13.7% (95% CI, 7.5–22.3%) of herds were positive. Out of 12 samples, B. pilosicoli was detected simultaneously with L. intracellularis, B. hyodysenteriae and B. pilosicoli were detected alone in two samples each. In terms of B. hyodysenteriae, 7.0% of samples (95% CI, 4.7–9.9%) from 18.9% of herds (95% CI, 11.6–28.3%) were positive in real time PCR. The presence of B. hyodysenteriae in fecal samples was associated with the presence of diarrhea in pigs. Conclusions: This study confirmed that B. pilosicoli infections occur in Polish pig herds, but the prevalence is at a low level and the presence of B. pilosicoli is not associated with the development of diarrhea in pigs. B. hyodysenteriae is still a common cause of diarrhea among pigs from Polish herds.

46 EXPRESSION OF TRANSCRIPTION FACTORS SPECIFIC TO THE TROPHOBLAST LINEAGE IN MOUSE SOMATIC NUCLEAR TRANSFER EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 103
Author(s):  
T. Mitani ◽  
M. Nishiwaki ◽  
M. Anzai ◽  
H. Kato ◽  
Y. Hosoi ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Ectopic Expression ◽  
Expression Level ◽  
Pcr Analysis ◽  
Cell Stage ◽  
Cdx2 Expression ◽  
Morula Stage

Somatic cell nuclear transfer (SCNT) embryos can develop at relatively high rates during the preimplantation period; however, most of these fail after implantation. Development of extraembryonic tissue is indispensable for normal embryonic development. Hence, an abnormality of trophoblast development might be a significant factor in post-implantation lethality of SCNT embryos. A transcription factor, caudal-related homeobox 2 (Cdx2), appears to be involved in the segregation of ICM and trophectoderm (TE) in preimplantation embryos (Niwa et al. 2005 Cell 123, 917–929). Both Cdx2 and Oct3/4 are expressed in all cells at the morula stage, and then Cdx2 expression becomes restricted to the TE and Oct3/4 to the ICM as the blastocyst develops. Mouse embryos deficient in Cdx2 are able to develop to normal blastocysts but die soon after implantation, probably because of defects in the TE lineage. Moreover, dysplasia of the spongiotrophoblast layer might attribute to an abnormality of Tpbpa expression in mouse SCNT embryos (Wakisaka-Saito et al. 2006 Biochem. Biophys. Res. Commun. 349, 106–114). In this study, we examined the expression profiles of transcription factors implicated in trophoblast development in mouse SCNT embryos and intracytoplasmic sperm injection (ICSI) embryos by immunohistochemistry and real-time PCR analysis. SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998 Nature 394, 369–374). In brief, B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. After nuclear transfer, the oocytes were activated and cultured in KSOM to the morula and blastocyst stages. Immunohistochemical analysis demonstrated that in ICSI embryos Cdx2 was only partially expressed at the 8-cell stage but completely in early morulae. In contrast, in SCNT embryos, it was absent at the 8-cell stage and appeared partially at the early morula stage. Thereafter, Cdx2 expression became restricted to the TE cells in both the ICSI and the SCNT blastocysts. However, ectopic expression of Oct3/4 was observed in the TE cells of SCNT, but not in ICSI blastocysts. Real-time PCR analysis showed that at the 8-cell stage, Cdx2 was expressed in ICSI but not in SCNT embryos. In addition, the expression level of Cdx2 in SCNT embryos at the blastocyst stage was only half that in ICSI embryos (P < 0.05). However, there was no significant difference in expression level of Oct3/4 between ICSI and SCNT embryos. Eomesodermin (Eomes) is also implicated in trophoblast development and its expression depends on Cdx2, BMP4, and FGF4. In SCNT embryos, the expression level of Eomes was also only half that in ICSI embryos. These results indicate that the delayed expression of Cdx2 in SCNT embryos may lead to the ectopic expression of Oct3/4 in blastocysts and, along with the limited expression of Cdx2 and Eomes, may contribute to disorders in the function of the trophoblast lineage for normal placental development. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

scholarly journals Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load

2019 ◽  
Vol 200 ◽  
pp. 13-15 ◽  
Author(s):  
Júlio César Rente Ferreira Filho ◽  
Lucia Maria Almeida Braz ◽  
Marcos Luiz Alves Andrino ◽  
Lidia Yamamoto ◽  
Kelly Aparecida Kanunfre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Parasite Load ◽  
Different Levels

Real-Time PCR Gene Expression Profiling of Microarray Gene Signatures in Acute Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4401-4401
Author(s):  
Ebrahim Sakhinia ◽  
Mahboubeh Farahangpour ◽  
John A. Liu Yin ◽  
Gerard Brady ◽  
Judith A. Hoyland ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Cyclin D3 ◽  
Expression Level ◽  
Tumor Type ◽  
Gene Signatures ◽  
Significant Difference ◽  
The Mean ◽  
Microarray Gene

Abstract Cancer subtype discovery and classification using microarray gene signatures has the potential to transform pathological diagnosis but measurement of indicator genes in routine practice remains difficult. We tested use of real-time PCR measurement of indicator genes for AML and ALL (Golub et al, Science, 1999) as a method for validation and application of microarray gene signatures. Mononuclear cells (MC) were isolated from whole bone marrow (BM) aspirates by density gradient centrifugation and sorted into unselected (total), CD34+ve and CD34-ve fractions. The mRNA in each fraction was globally amplified using a PolyA PCR method. We measured the expression profile of the 17 top ranked genes (cystatin C, leptin receptor, fumarylacetoacetate, CD33, HoxA9, adipsin, proteoglycan 1, LTC4 synthase, LYN, C-myb, MB-1, cyclin D3, SNF2, RbAp48, proteasome iota, HkrT-1 and E2A) from Golub et al (1999) by real-time PCR. All values were calibrated against control standards and normalized to the mean of three housekeeping genes (IF2-beta, GAPDH and human ribosomal protein S9). Data for all 17 genes were obtained for 4 (ALL), 26 (AML), 12 (AML remission) and 9 (morphologically normal) BM samples, each fractionated into three fractions (total MC, CD34+ve MC & CD34−ve MC). There was no significant difference in the mean of three housekeeping gene expression levels between the diagnostic groups. Comparison of the expression level of the other genes confirmed ability to separate AML and ALL, whilst the direction of expression change (increased or decreased) for each gene between AML and ALL was the same as found by Golub et al. In particular, c-myb showed largest significant increase in ALL vs AML in the total BM fraction, whilst cystain c was increased in AML in the CD34−ve fraction. hSNF2b was significantly increased in the ALL total B.M fraction and Hox-A9 was significantly increased in the AML CD34+ve B.M fraction. Furthermore expression level of LYN and CD33 was significantly increased in AML compared to remission AML, indicating ability of the method to determine activity status of disease. In addition, several of the genes provided better separation between AML and ALL when measured in the CD34+ve and −ve fractions indicating more prominent expression in cells of different maturity and that prior fractionation is diagnostically more informative. The results demonstrate ability of the method to validate gene expression signatures by an independent method, which is simple, sensitive and robust, allowing translation to routine clinical use. Whilst the present study used AML and ALL, in principle the method could be extended to any other tumor type for which gene signatures exist.

Identification of Candidate Tumor Suppressor Genes Silenced Epigenetically in Mantle Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3001-3001
Author(s):  
Norihiko Kawamata ◽  
Takayuki Saitoh ◽  
Sakura Sakajiri ◽  
Phillip H. Koeffler
Keyword(s):  
Tumor Suppressor ◽  
Mantle Cell Lymphoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Tumor Suppressor Genes ◽  
Cell Lymphoma ◽  
Suppressor Genes ◽  
Expression Levels ◽  
Candidate Tumor Suppressor ◽  
Mantle Cell

Abstract Many tumor suppressor genes are silenced by epigenetic mechanisms in human cancers, including mantle cell lymphoma (MCL). In this study, we have used a variety of research tools to screen for genes that are epigenetically silenced in MCL. Changes in the global gene expression profile of the MCL cell line, Jeko1, were analyzed after treatment with the combination of the demethylating agent, 5-aza-2′-deoxycytidine, and the histone deacetylase inhibitor, suberoyl anilide bishydroxamide, by DNA microarray technique. By screening over 22,000 genes, we identified 26 candidate tumor suppressor genes, expression of which were enhanced by the treatment, in the MCL line. Basal expression of these 26 genes were low in Jeko1 cells. The treatment enhanced the expression more than 2 folds and the enhancement was also confirmed by real-time PCR. Methylation status of these 26 genes were examined by bisulfite sequencing and/or combined bisulfite and restriction enzyme digestion assay in Jeko1 cells. We found hypermethylation of a CpG island in the middle of the INPP5F gene. We also found the hypermethylation of that region of INPP5F in normal peripheral blood. We also examined expression levels of these 26 genes in normal mantle cells by real-time PCR and found only 11 genes showed high levels of transcription in laser-dissected normal mantle cells. We examined expression of these 11 genes in eight MCL clinical samples by real-time PCR and found that only three genes, INPP5F, DUSP10 and FGD2 showed very low expression levels. We conclude that expression of INPP5F, DUSP10 and FGD2 genes were suppressed in MCL cells although the expression of these genes are high in normal mantle cells. INPP5F is a inositol phosphatase and could be involved in PI3K pathway. DUSP10 is a dual specific phosphatase and could be involved in JNK pathway. FGD2 is a RAS-GAP gene and could be involved in RAS pathway. These three genes may be candidate tumor suppressor genes in MCL and further functional analysis is ongoing.

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