Expression and effect of resistin on bovine and rat granulosa cell steroidogenesis and proliferation

Reproduction ◽  
2011 ◽  
Vol 141 (4) ◽  
pp. 467-479 ◽  
Author(s):  
Virginie Maillard ◽  
Pascal Froment ◽  
Christelle Ramé ◽  
Svetlana Uzbekova ◽  
Sébastien Elis ◽  
...  
Keyword(s):  
Opposite Effect ◽  
Thymidine Incorporation ◽  
Rt Pcr ◽  
Mapk Phosphorylation ◽  
Ovarian Cells ◽  
Basal Proliferation ◽  
D2 Protein ◽  
Resistin Mrna

Resistin, initially identified in adipose tissue and macrophages, was implicated in insulin resistance. Recently, its mRNA was found in hypothalamo–pituitary axis and rat testis, leading us to hypothesize that resistin may be expressed in ovary. In this study, we determined in rats and cows 1) the characterization of resistin in ovary by RT-PCR, immunoblotting, and immunohistochemistry and 2) the effects of recombinant resistin (10, 100, 333, and 667 ng/ml)±IGF1 (76 ng/ml) on steroidogenesis, proliferation, and signaling pathways of granulosa cells (GC) measured by enzyme immunoassay, [3H]thymidine incorporation, and immunoblotting respectively. We observed that resistin mRNA and protein were present in several bovine and rat ovarian cells. Nevertheless, only bovine GC abundantly expressed resistin mRNA and protein. Resistin treatment decreased basal but not IGF1-induced progesterone (P<0.05; whatever the dose) and estradiol (P<0.005; for 10 and 333 ng/ml) production by bovine GC. In rats, resistin (10 ng/ml) increased basal and IGF1-induced progesterone secretion (P<0.0001), without effect on estradiol release. We found no effect of resistin on rat GC proliferation. Conversely, in cows, resistin increased basal proliferation (P<0.0001; for 100–667 ng/ml) and decreased IGF1-induced proliferation of GC (P<0.0001; for 10–333 ng/ml) associated with a decrease in cyclin D2 protein level (P<0.0001). Finally, resistin stimulated AKT and p38-MAPK phosphorylation in both species, ERK1/2-MAPK phosphorylation in rats and had the opposite effect on the AMPK pathway (P<0.05). In conclusion, our results show that resistin is expressed in rat and bovine ovaries. Furthermore, it can modulate GC functions in basal state or in response to IGF1in vitro.

Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽  
2011 ◽  
Vol 138 (14) ◽  
pp. 1832-1842 ◽  
Author(s):  
V. RISCO-CASTILLO ◽  
V. MARUGÁN-HERNÁNDEZ ◽  
A. FERNÁNDEZ-GARCÍA ◽  
A. AGUADO-MARTÍNEZ ◽  
E. JIMÉNEZ-RUIZ ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Cluster ◽  
Neospora Caninum ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Initial Characterization ◽  
Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

300 ESTABLISHMENT AND CHARACTERIZATION OF RAT MESENCHYMAL STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 247
Author(s):  
T. H. Kim ◽  
B. G. Jeon ◽  
S. L. Lee ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transcriptional Factors ◽  
Skin Tissue ◽  
Rt Pcr ◽  
Alternative Source ◽  
Differentiated Cells ◽  
Fetal Bovine

Mesenchymal stem cells (MSC) are regarded as an attractive source for tissue engineering and regeneration, and bone marrow extract has been commonly used as a source of pluripotent MSC. However, skin tissue has recently been identified as a convenient alternative source of MSC. The present study was focused on the effect of characterised MSC derived from rat on expression of early transcriptional factors, alkaline phosphate (AP) activity, and in vitro differentiation into selected cell lineages. The MSC were isolated from 8-week-old s.d. rat’s ear skin and cultured in advanced DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air. To evaluate AP activity, cells were fixed with 3.7% formaldehyde solution and stained with Western Blue® (Promega, Madison, WI, USA). Expressions of early transcriptional factors (Oct-4, Sox2, and Nanog) were evaluated by RT-PCR. Differentiation into distinct mesenchymal lineages such as adipogenic, osteogenic, and neuron was done by following previously described protocols and assessed by lineage-specific stains. The specific genes in the osteocytes (osteocalcin, osteonectin, osteopontin, and Runx2), adipocytes (pparγ2, adiponectin, and aP2) or neuron (nestin, neurogenin 1, β-tublin, and nerve growth factor) were characterised by RT-PCR. The MSC were positive for AP activity and expressed Oct-4, Sox2, and Nanog. Following induction, MSC were successfully differentiated into adipocytes, osteocytes, and neurons. As adipocytes markers, aP2, pparγ2, and adiponectin were strongly detected in the adipocyte induced cells. Osteonectin, osteocalcin, Runx2, and osteopontin were expressed in the adipocyte induced cells. Futhermore, neuron-specific markers were clearly expressed in the neuronal differentiated cells. In conclusion, MSC have the capability of differentiation into multilineages including adipocytes, osteocytes, and neurons under the specific induction conditions. Skin tissue in rat can serve as an easily accessible and expandable alternative source for MSC harvesting and preclinical applications using an animal model. This work was supported by Grant No. 2007031034040 from Bio-organ and 200908FHT010204005 from Biogreen21, Republic of Korea.

Characterization of Gene Expression Associated with Cyclophosphamide Resistance in Chronic Myeloid Leukemia (CML).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1532-1532
Author(s):  
Fei Bao ◽  
Mary L. Nordberg ◽  
Paula Polk ◽  
Amanda Sun ◽  
David Murray ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Resistant Cell ◽  
Parental Line ◽  
Resistant Line ◽  
Rt Pcr

Abstract Cyclophosphamide (CP) is one of the alkylating agents collectively referred to as oxazaphosphorines that are used to treat many types of cancers including myeloid leukemia. Tumor cell drug resistance is an important factor for clinical treatment failure. The mechanisms of drug resistance are multifactorial and incompletely understood. KBM-7 human CML cell line was established from blast cells from a patient in the terminal phase of CML. In the CP resistance model, the B5-180 sub-line was isolated following exposure to the in vitro active CP analog 4HC. B5-180 cells were cross-resistant to busulfan and γ-radiation. Total RNA was extracted and hybridized to Affymetrix Genechip HG-U95Av2 arrays. Each array contains 12,386 probes corresponding to approximately 9000 known human genes. Each cell line was arrayed in triplicate. Quantitative RT-PCR, Fluorescence In-Situ Hybridization (FISH) and cytogenetic analysis were performed in both cell lines. Both the KBM-7/B5 parental line and B5-180 resistant sub-line expressed high-levels of BCR-ABL transcripts by real-time RT-PCR. FISH and cytogenetic analysis revealed multiple copies of t(9;22) translocation and other additional chromosomal abnormalities such as trisomy 8, and abnormalities of chromosome 18 in both cell lines. Gene array identified 794 gene transcripts that were more than twofold (range from 2-fold to 2675-fold) over-expressed or under-expressed in the resistant line relative to the parental line. ALDH1A1 (aldehyde dehydrogenase 1 family) showed the most differential expression between sensitive and resistant cell lines, ALDH1A1 was upregulated more than 2000-fold in the resistant sub-line. ALDH-2 (aldehyde dehydrogenase 2 family mitochondrial) was also expressed substantially higher in the resistant line. This finding is consistent with the established fact that elevated ALDH activity is an important factor in the resistance of B5-180 cells to 4HC. The remaining differentially expressed genes encode proteins with a wide variety of biochemical functions, which include 44 apoptosis and 7 anti-apoptosis-related genes, 56 genes related to cell cycle and cell growth, 6 DNA repair genes, 13 genes involved in hemopoiesis and B-cell activation. We also tested the expression of the hematopietic transcription factor PU-1 and PUB, a novel PU-1 binding factor. Interestingly, the expression of PU-1 was decreased and PUB increased in the resistant clone. In conclusion, we have identified a large number of differentially expressed genes in a CP resistant cell line derived from CML blast crisis by microarray technology. Our results suggest that CP resistance is a complex phenotype that involves multiple genes and a variety of mechanisms. Real-time RT-PCR analysis and further characterization of selected genes associated with CP resistance as well as the response in vitro to tyrosine kinase inhibitors are currently under investigation.

Establishment and Characterization of a tel/abl Rearrangement Responsible for Imatinib Sensitivity in bcr/abl Negative Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4280-4280
Author(s):  
Jörg Bäumler ◽  
J.H. Frederik Falkenburg ◽  
Marianke L.J. van Schie ◽  
Karoly Szuhai ◽  
Martine Pape ◽  
...  
Keyword(s):  
Growth Factors ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Array Cgh ◽  
Lymphoblastic Leukemia ◽  
Chromosome 12 ◽  
Rt Pcr

Abstract Proliferation of Philadelphia (Ph) positive acute lymphoblastic leukemia (ALL) cells is driven by the bcr/abl oncoprotein which renders transformed cells independent of exogeneous growth factors. Bcr/abl can be targeted by fusion tyrosine kinase inhibitors such as imatinib. ALL is a heterogenous disease. Some cases of Ph negative ALL may be driven by oncogenic fusion kinases other than bcr/abl, with or without involvement of the abl-gene. Such kinases may also be susceptible to targeted inhibitors. At present however, few of such fusion kinases are known. This may be due to the lack of reliable in vitro culturing systems for primary ALL cells. Because of this, only a limited number of ALL cell lines are available to screen for alternative oncogenic fusion kinases. We have recently established an in vitro culturing system in which continuous proliferation (>1 year) was thus far induced in 11 out of 22 primary ALL samples, in the absence of exogenously added growth factors. Five of these 11 cultures were Ph negative by cytogenetics and bcr/abl negative by RT-PCR. Therefore, we first screened these lines for sensitivity to imatinib. One of the 6 Ph negative cell lines, LeidenALL-VG, derived from a 30-year old patient with Ph chromosome negative common ALL, displayed sensitivity to imatinib with an IC50 of 0.1 μM, comparable to the sensitivity of the bcr/abl positive cell lines. RT-PCR with primers specific for tel and abl revealed tel/abl fusion transcripts. Sequencing of the amplicons showed two splice variants of tel-abl: tel4/abl2 and tel5/abl2. Multicolor FISH based karyotyping using COBRA-FISH did not provide evidence for the suggested t(9;12) translocation, indicating cryptic tel/abl rearrangement. To map cryptic genomic aberrations, an array-comparative genomic hybridization array (array-CGH) was performed. A region at 9q34 spanning two array clones was gained (clones RP11-83J21 and RP11-143H20), corresponding with gain of a 300-1300 kb region containing the distal part of the abl gene. A region at 12p13 spanning one array clone was lost (clone RP11-59H1) corresponding with loss of a 1150 to 1750 kb region directly centromeric to the tel gene. To localize cryptic tel/abl rearrangement, fluorescent in situ hybridization (FISH) was performed using the distal tel and abl probes identified by array-CGH, as well as the clones proximal to these regions. FISH revealed an additional distal abl signal, co-localized with the proximal tel signal, on the short arm of an otherwise normal looking chromosome 12. With this, tel/abl rearrangement in LeidenALL-VG cells was characterized as a micro-insertion of one additional distal abl region into the tel region of a normal chromosome 12. Tel/abl rearrangement and imatinib sensitivity were confirmed in the primary cells. These results suggest that our serum-free approach to in vitro culture of primary ALL cells, in combination with molecular karyotyping tools such as array-CGH and COBRA-FISH, may allow identification and characterization of otherwise un-identified chromosomal aberrations, such as the cryptic tel/abl translocation illustrated here, that may be targeted by kinase inhibitors such as imatinib.

scholarly journals Identification and Functional Characterization of a new Sunflower Germacrene A Synthase (HaGAS3)

2010 ◽  
Vol 5 (5) ◽  
pp. 1934578X1000500 ◽  
Author(s):  
Jens Göpfert ◽  
Anna-Katharina Bülow ◽  
Otmar Spring
Keyword(s):  
Sesquiterpene Lactones ◽  
Glandular Trichomes ◽  
Functional Characterization ◽  
Plant Tissues ◽  
Rt Pcr ◽  
Plant Organs ◽  
Reverse Transcription Pcr ◽  
Engineered Yeast

Sesquiterpenes and sesquiterpene lactones are major natural compounds found in linear and capitate glandular trichomes of sunflower, Helianthus annuus L. In addition to two recently identified germacrene A synthases HaGAS1 and HaGAS2, found in capitate trichome gland cells, reverse transcription-PCR experiments have now allowed identification of a third enzyme of this type, HaGAS3. Its cDNA sequence was established and its functional characterization as a germacrene A synthase was achieved through in vitro expression in engineered yeast, and by GC-MS experiments. PCR and RT-PCR experiments with cDNA from different plant organs revealed that the new enzyme is expressed independently from the other two. While these latter two were expressed in plant organs bearing capitate glandular trichomes and in roots, the new enzyme occurred in plant tissues not linked to the presence of specific trichomes (for example, cotyledons), and was absent in roots. The experiments show that independently regulated pathways for the first cyclic sesquiterpene, germacrene A, are present in sunflower.

scholarly journals Characterization of the anthranilate degradation pathway in Geobacillus thermodenitrificans NG80-2

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Xueqian Liu ◽  
Yangpeng Dong ◽  
Xiaomin Li ◽  
Yi Ren ◽  
Yanxia Li ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Degradation Pathway ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Geobacillus Thermodenitrificans ◽  
Reservoir Conditions ◽  
Important Intermediate ◽  
First Time

Anthranilate is an important intermediate of tryptophan metabolism. In this study, a hydroxylase system consisting of an FADH2-utilizing monooxygenase (GTNG_3160) and an FAD reductase (GTNG_3158), as well as a bifunctional riboflavin kinase/FMN adenylyltransferase (GTNG_3159), encoded in the anthranilate degradation gene cluster in Geobacillus thermodenitrificans NG80-2 were functionally characterized in vitro. GTNG_3159 produces FAD to be reduced by GTNG_3158 and the reduced FAD (FADH2) is utilized by GTNG_3160 to convert anthranilate to 3-hydroxyanthranilate (3-HAA), which is further degraded to acetyl-CoA through a meta-cleavage pathway also encoded in the gene cluster. Utilization of this pathway for the degradation of anthranilate and tryptophan by NG80-2 under physiological conditions was confirmed by real-time RT-PCR analysis of representative genes. This is believed to be the first time that the degradation pathway of anthranilate via 3-HAA has been characterized in a bacterium. This pathway is likely to play an important role in the survival of G. thermodenitrificans in the oil reservoir conditions from which strain NG80-2 was isolated.

Human Pancreatic Mesenchymal Stem Cells Produce Blood in the Non-Injury Fetal Sheep Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2558-2558 ◽  
Author(s):  
Adel Ersek ◽  
Angelina Daisy Goodrich ◽  
Christopher D. Porada ◽  
John S. Pixley ◽  
Maria Graca Almeida-Porada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Culture System ◽  
Cultured Cells ◽  
Rt Pcr ◽  
Sheep Model ◽  
In Vitro Culture System ◽  
Human Specific

Abstract Mesenchymal stem cells (MSC) are known for their relatively easy isolation, expansion by culture, and low immunogenicity. Here we report the isolation and characterization of a human fetal pancreas-derived MSC population (pMSC), and its long-term engraftment and differentiation into hematopoietic elements. We started a three step in-vitro culture system with freshly digested human fetal pancreatic cells cultured on uncoated plastic in RPMI media supplemented with 2% FBS for 48 hours. Next, adherent cells were further cultured in restrictive media (0.5% FBS) for about 4 weeks where the ensued starvation depleted contaminating cells. In the third phase, human pMSC were expanded in the presence of 10% FBS, bFGF, and EGF. The pMSC maintained their proliferative capacity even after repeated freeze-thaw cycles. Characterization of the pMSC was done by FACS analyses, RT-PCR, and differentiation studies. Cultured cells became CD44+, CD29+, CD90+, CD105+, CD34−, and CD45−, a phenotype similar to the bone marrow-derived MSC. RT-PCR analyses revealed that the pMSC from our culture system did not express pancreatic tissue-specific markers including insulin, PDX1 and PAX6, but expressed the general stem cell marker Nestin and also the c-Met marker attributed to primitive endodermal and pancreatic stem cells. Using specific differentiation inductive media, the pMSC were able to differentiate in-vitro into adipocytes and osteocytes. Thus, the cultured cells could be labeled as pMSC as confirmed by their multi-lineage differentiation when induced, as well as their ability to remain undifferentiated in restrictive media with the expression of characteristic markers listed above. To investigate the engraftment and in-vivo differentiation potential of human pMSC we transplanted these cells into pre-immune sheep (1–2x10e6 cells/fetus) (n=8) at 55–60 days of gestation by intra-peritoneal injections. Repeated evaluation of the recipients up to 1 year of age confirmed the engraftment of human cells in the bone marrow of animals by PCR for the human-specific GAPDH gene and by flow cytometry using human-specific antibodies. Human hematopoietic markers including CD47, CD45, CD133, and CD15 were found in both the bone marrow and peripheral blood of chimeric animals. Sargramostim (human specific GM-CSF) treatment of one animal (5 micrograms/ kg /day for three days) induced up-regulation of the human CD47, CD45, CD36, and CD56 expressing human cells. Methylcellulose culture of a bone marrow sample from this animal generated colonies that tested positive for human GAPDH by PCR thereby confirming the presence of human hematopoietic progenitors. In summary, we established a three step in vitro culture system for the isolation and expansion of human fetal pMSCs. Using the non-injury sheep model we were able to demonstrate the plasticity of human pMSC which gave rise to hematopoietic elements for up to one year following engraftment. These animals will be further evaluated for the pMSC contribution in other tissues including the pancreas.

Cloning and Characterization of Adinbitor, a Novel Disintegrin from the Snake Venom of Agkistrodon halys brevicaudus stejneger

2004 ◽  
Vol 36 (6) ◽  
pp. 425-429 ◽  
Author(s):  
Ji-Hong Wang ◽  
Yu Wu ◽  
Feng Ren ◽  
Li Lü ◽  
Bao-Chang Zhao
Keyword(s):  
Amino Acids ◽  
Platelet Aggregation ◽  
Human Platelet ◽  
Venom Gland ◽  
Rt Pcr ◽  
E Coli ◽  
Human Platelet Aggregation

Abstract Adinbitor was cloned from Agkistrodon halys brevicaudus stejneger and characterized as a novel disintegrin. In this study, total RNA was extracted from venom gland and used in RT-PCR to generate a cDNA which is 219 bp long. The sequence encoded a polypeptide composed of 73 amino acids, including 12 cysteines, an RGD motif, and the signature motif of disintergrin. Recombinant Adinbitor (rAdinbitor) was expressed in E. coli and purified by using the His·Bind affinity chromatography. The IC50 for inhibiting human platelet aggregation and bFGF-induced proliferation of ECV304 cells was 6 μM and 0.89 μM respectively. Furthermore, Adinbitor significantly inhibited angiogenesis both in vivo and in vitro. Taken together, these results suggested that Adinbitor had typical functions of disintegrins.

scholarly journals Differential Expression of DUB Genes in Ovarian Cells Treated with Di-2-Ethylhexyl Phthalate

2020 ◽  
Vol 21 (5) ◽  
pp. 1755
Author(s):  
Da-Hye Lee ◽  
Jun-Hyeok Park ◽  
Jihye Choi ◽  
Kyung-Ju Lee ◽  
Bo-Seong Yun ◽  
...  
Keyword(s):  
Differential Expression ◽  
Ovarian Function ◽  
Expression Patterns ◽  
Ubiquitin Proteasome System ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Protein Levels ◽  
Ovarian Cells ◽  
Ethylhexyl Phthalate

Premature ovarian failure (POF) is defined as loss of ovarian function in women less than 40 years of age. The causes of POF are diverse and include environmental factors. Di-2-ethylhexyl phthalate (DEHP) is one factor that may cause POF. The ubiquitin-proteasome system maintains intracellular balance by promoting or inhibiting protein degradation. To investigate the differential expressions of deubiquitinating enzyme (DUB) genes in patients with POF, we developed two in vitro POF models by treating A2780 or OVCAR5 with DEHP. Using these models, a multiplex RT-PCR system for DUB genes was applied to identify biomarkers by comparing expression patterns and DUB mRNA levels; multiplex RT-PCR results were validated by qRT-PCR and Western blotting analyses. Observed differential expression levels of several DUB genes including USP12, COPS5, ATXN3L, USP49, and USP34 in A2780 and OVCAR5 cells at the mRNA and protein levels suggest that they should be investigated as potential biomarkers of POF.

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