Biomedical subjects

M W Pfaffl

Publications and source records attributed to M W Pfaffl.

At least 19 recordsLinked to original sources

PubMed2006

Effects of mycophenolic acid on inosine monophosphate dehydrogenase I and II mRNA expression in white blood cells and various tissues in sheep.

Mycophenolic acid (MPA) is a mycotoxin commonly found as Penicillium genus secondary metabolite in feedstuffs and silages. Feeding with MPA contaminated silages may modulate the immune system in the farm animals and can cause appetite lost, ketosis, paralysis and abortion. The aim of the present study was to characterize the long-term MPA effect on both the inosine monophosphate dehydrogenase (IMPDH) isoforms I and II mRNA expression in white blood cells (WBC) and various tissue of healthy sheep. In treated animals 300 mg MPA/day/sheep was applied. In all investigated tissues the IMPDH I and II mRNA was abundant: WBC, spleen, thymus, ileum, jejunum, kidney, liver, pharyngeal and mesenterial lymph node. An efficiency-corrected relative quantification of the IMPDH types I and II isoforms mRNA were performed by normalizing with the constant reference gene expression of beta-actin. High IMPDH I mRNA expression levels were seen in kidney > mesenterial lymph node > jejunum > spleen > pharyngeal lymph node. Medium and low abundance was found in ileum > WBC > liver > thymus. Type II mRNA was highly expressed in liver > thymus > jejunum. In pharyngeal lymph node > spleen > ileum > mesenterial lymph node > kidney > WBC medium to low IMPDH II mRNA concentrations were detected. Under MPA treatment the IMPDH I mRNA expression was not significantly regulated in WBC, only trends of down- and upregulation were observed. Surprisingly in jejunum an upregulation could be observed (P < 0.05). In pharyngeal lymph node a tendency to downregulation was shown. This may be due to frequent ruminant activities and frequent exposition of MPA to the pharyngeal lymph nodes. In contrast to type I mRNA expression, IMPDH II mRNA was significantly downregulated in ileum (3.4-fold, P < 0.01) and tendencies in downregulation could be seen in jejunum (5.1-fold, P = 0.14). In addition, significant downregulation of IMPDH II gene expression over the entire feeding experiment could be shown in WBC of MPA-treated animals compared with untreated animals (P < 0.05). In conclusion, the recent study demonstrates that feeding sheep with MPA-contaminated silage did not induce IMPDH I mRNA expression in various tissues and blood, except in jejunum, but has suppressive effects on IMPDH II mRNA expression in WBC and ileum.

Animals↗

PubMed2005

Short-term effects on pro-inflammatory cytokine, lactoferrin and CD14 mRNA expression levels in bovine immunoseparated milk and blood cells treated by LPS.

Leucocytes (WBC) are recruited from peripheral blood into milk as part of the inflammatory response, mediated through cytokines or interleukins (IL) synthesized by mammary tissue and the milk somatic cells (SC). The inflammatory response is related to the concentration of SC and the cytokines produced. To investigate and to compare the kinetics of cytokine production in SC and WBC during inflammation, cell culture models were established, where SC and WBC were cultured in parallel (n = 3). In addition, macrophages or monocytes were isolated from milk and blood with antibody-coated magnetic beads and cultivated separately. Isolated cells were pure, unaltered and viable. Cultures were activated with 10 microg/ml lipopolysaccharide (LPS). After 0, 1, 2, 3, 4 and 8 h cells were harvested for RNA isolation. Cytokine [tumour necrosis factor alpha (TNFalpha), IL-1beta, IL-6] mRNA expression responses and transcriptional activity of CD14 and lactoferrin (LF) were quantified via a one-step real-time RT-PCR. Significant cytokine mRNA increases were found in all four cell culture types and genes, with peaks after 1 and 2 h (TNFalpha > IL-6 > IL-1beta). In WBC or monocytes higher LPS responses and longer persistence could be found than in corresponding milk cells (IL-1beta > IL-6 > TNFalpha). SC and macrophages are less responsive to LPS stimulation than WBC or monocytes. The strength of the immune response in the blood system is much more prominent than in the mammary gland. This may be ascribed to the role of CD14 on the cytokine production of the investigated cells, or may be caused by the blood-to-milk diapedesis. The constitutive transcription of CD14 mRNA in WBC and monocytes was found to be 6 to 15 times higher than in adequate milk cells.

Animals↗

PubMed2005

Quantitative real-time RT-PCR--a perspective.

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.

Reverse Transcriptase Polymerase Chain Reaction↗

PubMed2004

Short-term changes of mRNA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis.

During mammary gland infection, non-specific responses are the predominant ones. The goal of this study was to investigate the mRNA expression of various soluble immune components and of the major milk proteins during the acute phase of mammary inflammation. Five healthy lactating cows were intramammary infused in one quarter with 100 microg Escherichia coli-endotoxin (lipopolysaccharide, LPS) and the contralateral quarter with saline (9 g/l) serving as control. Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of various factors was quantified via real-time RT-PCR. Blood samples for determination of leukocyte number were taken simultaneously with the biopsy samples and rectal temperature was measured at 1-h intervals. Rectal temperature increased until 5h (P < 0.05) after LPS administration and remained elevated until 9 h after LPS inoculation. Blood leukocyte number decreased (P < 0.05) from 0 to 3 h from 7.7 +/- 1.1 x 10(9)l(-1) to 5.7 +/- 1.0 x 10(9)l(-1) and thereafter recovered to pre-treatment levels until 12 h after LPS challenge. In LPS-treated quarters, tumor necrosis factor-alpha and cyclooxygenase-2-mRNA expression increased (P < 0.05) to highest values at 3h after LPS challenge. Lactoferrin, lysozyme, inducible nitric oxide synthase increased (P < 0.05) and peaked at 6 h after challenge, and platelet-activating factor acetylhydrolase-mRNA expression tended to increase (P = 0.07). mRNA expression of insulin-like growth factor-I and of alphaS1-casein (CN), alphaS2-CN, beta-CN and beta-lactoglobulin did not change significantly, whereas mRNA expression of 5-lipoxygenase and alpha-lactalbumin decreased (P < 0.05) in both quarters and that of kappa-CN only in the LPS quarter. mRNA expression of some investigated factors (tumor necrosis factor-alpha, lysozyme, 5-lipoxygenase, alpha-lactalbumin) changed in control quarters, however in all respective factors less than in the LPS quarters (P < 0.05). In conclusion, mRNA expression of most inflammatory factors increased within hours, whereas that of most milk proteins remained unchanged.

Animals↗

PubMed2003

Involvement of pro-inflammatory cytokines, mediators of inflammation, and basic fibroblast growth factor in prostaglandin F2alpha-induced luteolysis in bovine corpus luteum.

The process of luteolysis requires very subtly modulated coordination of different factors and regulation systems. Immune cells and cytokines were shown to be relevant for bovine luteolysis. The aim of this study was to investigate the detailed pattern of mRNA expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha), TNF receptor type 1 (TNF-R1), interleukin 1beta (IL-1beta), and interferon gamma (IFNgamma), and of the inducible nitric oxide synthase (iNOS) and the basic fibroblast growth factor (FGF-2) during prostaglandin (PG) F(2alpha)-induced luteolysis in the bovine corpus luteum (CL). In addition, the mRNA expression for the LH-receptor (LH-R) and the steroidogenic enzyme p450scc was determined. Cows in the midluteal phase (Days 8-12) were injected with the PGF(2alpha) analogue cloprostenol, and CL were collected by transvaginal ovariectomy before and 2, 4, 12, 48, and 64 h after PGF(2alpha) injection. Conventional and real-time reverse transcription polymerase chain reaction RT-PCR (LightCycler) using SYBR Green I detection were employed to determine the mRNA expression for the investigated factors. All cytokines were significantly up-regulated during induced luteolysis. LH-R and p450scc mRNA were down-regulated (P < 0.05) during structural luteolysis (after 12 h), and p450scc in addition at 2 h after PGF(2alpha) (P < 0.05). FGF-2 expression increased (P < 0.001) during functional luteolysis (until 12 h after PGF(2alpha)) and diminished thereafter. The mRNA expression for iNOS decreased (P < 0.05) after induction of luteolysis. In conclusion, cytokines may be involved not only in structural but also in functional luteolysis and the deprivation of luteal survival factors, leading to a situation where apoptosis can occur. FGF-2 may participate in the suppression of cytokine-induced iNOS mRNA expression and in the prevention of an inflammatory reaction in the surrounding tissues.

Animals↗

PubMed2003

[Effect of transforming growth factor beta 2 on phagocytosis in cultured bovine trabecular meshwork cells].

AIM: This study was performed to investigate the effect of transforming growth factor beta 2 (TGF-beta 2) on phagocytosis in bovine trabecular meshwork cells in vitro. METHODS: After cultured bovine trabecular meshwork cells were treated for 24 h with 0 ng/ml (control), 0.32 ng/ml, 1 ng/ml, and 3.2 ng/ml TGF-beta 2, latex beads were added to the incubation medium, and the numbers of latex beads in 20 adjacent cells were then counted under a microscope after treatment with Wright's stain. RESULTS: The average numbers of latex beads in the trabecular meshwork cells treated with TGF-beta 2 of different concentrations were 53.1+/-1.7 beads/cell, 56.4+/-2.9 beads/cell, and 77.9+/-6.5 beads/cell, respectively, compared to 45.5+/-3.3 beads/cell in the nontreated control group. Thus, TGF-beta 2 significantly increased the numbers of latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose-dependent manner. CONCLUSION: TGF-beta 2 can promote the phagocytosis of bovine trabecular meshwork cells in vitro. It may be involved in the reduced cellularity of the trabecular meshwork in patients with primary open angle glaucoma by promoting the phagocytosis of these cells.

Animals↗

PubMed2003

The mRNA expression of the members of the IGF-system in bovine corpus luteum during induced luteolysis.

The components of the IGF-system were shown to be differentially regulated in bovine antral follicles and corpora lutea (CL) during different stages of the estrous cycle, and to have important functions for specific stages. The aim of this study was to investigate the detailed pattern of mRNA expression of most constituents of the IGF-system and their possible involvement in prostaglandin (PG)F2alpha-induced luteolysis in the bovine CL. Therefore, cows in the mid-luteal phase (days 8-12) were injected with the PGF2alpha-analogue Cloprostenol, and CL were collected by transvaginal ovariectomy at 2, 4, 12, 48 and 64 h after PGF2alpha-injection. Real-time RT-PCR using SYBR Green I detection was employed to determine mRNA expressions of the following factors: ubiquitin (UBQ), insulin-like growth factor I (IGF I), IGF II, IGF-receptor type 1 (IGFR-1), growth hormone receptor (GH-R) and IGF-binding proteins-1-6 (IGFBP-1-6). Total extractable RNA decreased with ongoing luteolysis. IGFBP-1 mRNA was significantly up-regulated at 2h after PGF2alpha and maximal at 4h with a 34-fold increase. IGFBP-5 mRNA was significantly up-regulated after 12h with a maximum of an 11-fold increase at 64 h. For GH-R, IGFR-1, IGF II, IGFBP-3 and -4 mRNA expression, we found a significant down-regulation in certain stages. There was a significant up-regulation for IGFBP-2 and -6 mRNA at 64 h after induced luteolysis. There were no significant changes in IGF I mRNA expression. In conclusion, the IGF-system with all its components seems to play an important role in the very complex process of PGF2alpha-induced luteolysis in bovine CL.

Animals↗

PubMed2003

Detection and quantification of mRNA expression of alpha- and beta-adrenergic receptor subtypes in the mammary gland of dairy cows.

Adrenergic receptors are pharmacologically classified into the receptor types alpha(1), alpha(2), beta(1), beta(2), and beta(3). Structural differences and varying affinities in radioligand binding studies lead to a further classification of alpha(1)- and alpha(2)-receptors into subtypes which are termed alpha(1A) (formerly alpha(1C)), alpha(1B), and alpha(1D) (formerly alpha(1AD)), and alpha(2AD), alpha(2B), and alpha(2C), respectively. mRNA expression of all but one alpha-adrenergic receptor subtypes and of all beta-adrenergic receptor types was measured quantitatively in total RNA extracted from mammary tissue of 10 lactating dairy cows by real-time reverse transcription (RT) polymerase chain reaction (PCR). mRNA expression of alpha(1)-adrenergic receptors was highest for the alpha(1A)-subtype followed by alpha(1B), whereas the alpha(1D)-subtype could not be detected. The highest mRNA expression of alpha(2)-adrenergic receptors was found for the alpha(2AD)-subtype, followed by alpha(2B) and alpha(2C). Within the beta-adrenergic receptors, the beta(2)-receptor type was most highly expressed, followed by beta(1) and beta(3). In conclusion, eight of nine adrenergic receptors classified to date were detected and relatively quantified in the mammary gland of dairy cows.

Animals↗

PubMed2003

Milking characteristics and their relation to adrenergic receptor mRNA expression and ligand binding in the mammary gland of dairy cows.

Stimulation of alpha- and beta-adrenergic receptors in the bovine mammary gland affects milking characteristics such as milk yield and peak flow rate. The aim of this study was to detect possible correlations between milkability, adrenergic receptor binding capacity and receptor expression at the mRNA level. In addition, dose-response relationships of alpha- and beta-adrenergic receptor stimulation were evaluated after application of an alpha- and beta-adrenergic receptor agonist, respectively in different dosages. Density and distribution of adrenergic receptor binding sites in the region around the large mammary ducts were investigated as well as adrenergic receptor mRNA expression. Milk flow of one-quarter was recorded in 10 cows without or with additional alpha- and beta-adrenergic receptor stimulation in three dosages each. After slaughter, mammary tissue was taken from the region around the large mammary ducts in the previously investigated quarters. Protein and RNA were extracted for measuring alpha(1)-, alpha(2)-, and beta(2)-adrenergic receptor binding sites and mRNA expression levels by real-time reverse-transcription polymerase chain reaction (RT-PCR). Peak flow rate without additional adrenergic receptor stimulation was negatively correlated with alpha(2)-adrenergic receptor binding (maximal binding capacity, B(max)) and positively correlated with alpha(2)-adrenergic receptor expression at the mRNA level (crossing point (CP) of the real-time PCR). During alpha-adrenergic receptor stimulation, there was a positive correlation between milkability and alpha(2)-adrenergic receptor mRNA expression, whereas during beta-adrenergic receptor stimulation no correlations were detected. Dose-response relationships were existing during alpha-adrenergic receptor stimulations, but not during beta-adrenergic receptor stimulations at four dosages each including control milking. Significant changes in milk yield and peak flow rate mainly occurred after application of an alpha-adrenergic receptor agonist. In conclusion, high mRNA expression levels or binding capacities of adrenergic receptors do not necessarily lead to according reactions in vivo, concerning milk yield and peak flow rate. To influence milking characteristics, individual reactions of the cow on adrenergic stimulation have to be considered.

Adrenergic alpha-Agonists↗

PubMed2003

The gastrointestinal tract as target of steroid hormone action: quantification of steroid receptor mRNA expression (AR, ERalpha, ERbeta and PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR.

We have examined the tissue-specific mRNA expression pattern of androgen receptor (AR), both estrogen receptor (ER) subtypes ERalpha and ERbeta and progestin receptor (PR) in 10 bovine gastrointestinal compartments. Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation Ralgro on the compartment-specific expression regulation. Ralgro contains Zeranol which shows strong estrogenic and anabolic effects. Eight heifers were treated for 8 weeks with Ralgro at different dosages (0, 1, 3, and 10 times). To quantify the very low abundant steroid receptor mRNA transcripts sensitive and reliable real-time (kinetic) reverse transcription (RT)-PCR quantification methods were validated on the LightCycler. Expression results indicate the existence of AR and both ER subtypes in all 10 gastrointestinal compartments. PR receptor was expressed at very low abundancy. Gastrointestinal tissues exhibit a specific ERalpha and ERbeta expression pattern with high expression levels for both subtypes in rectum, colon and ileum. With increasing Zeranol concentrations a significant down-regulation for ERalpha and ERbeta was observed in jejunum (P<0.001 and <0.05, respectively). Significant up-regulations under estrogen treatment could be shown in abomasum for ERalpha (P<0.05) and in rectum for ERbeta (P<0.001). The authors conclude, that especially estrogens and the expression of their corresponding receptor subtypes may play an important role in the modulation and regulation in gastric as well as gut functions, cell proliferation and possibly in the pathophysiology of cell cancer. The different expression patterns of ERalpha and ERbeta can be regarded as support of the hypothesis that the subtype proteins may have different biological functions in the gastrointestinal tract. AR and PR seem to be not estrogen dependent.

Animals↗

PubMed2003

Characterisation of gene expression patterns in 22RV1 cells for determination of environmental androgenic/antiandrogenic compounds.

Alteration of androgen receptor function due to hormonally active compounds in the environment, may be responsible for impaired reproductive function in aquatic wildlife. Based on human prostate carcinoma 22RV1 cells, a cell culture expression system was established to test effects of putative androgenic/antiandrogenic compounds on endogenous gene expression. 22RV1 cells were shown to express human androgen receptor, but not human progestin (hPR) or human oestrogen receptor (hER) alpha and beta. Six androgen-regulated genes (ARGs) were chosen to determine androgenic/antiandrogenic action using highly sensitive real-time RT-PCR. Results showed that gene expression is altered in a time-dependent manner. After stimulation of cells by DHT (10nM), synthetic androgen R1881 (1 nM), or organic pesticides (difenoconazole, fentinacetate, tetramethrin) TMPRSS2 mRNA expression was down-regulated by the factor 0.6 after 24h of DHT treatment. Similar results were obtained when cells were assayed for mRNA expression of PSA after fentinacetate and R1881 stimulation. In contrast, TMPRSS2 expression was up-regulated by the factor 0.9 when cells were stimulated by tetramethrin. Final goal of the work is a sensitive determination of differential gene expression by different compounds under study, achievement of substance-specific expression patterns and function related analysis of potential androgens/antiandrogens.

Androgen Antagonists↗

PubMed2003

Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution.

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.

Animals↗

PubMed2003

Abundance of message for insulin-like growth factors-I and -II and for receptors for growth hormone, insulin-like growth factors-I and -II, and insulin in the intestine and liver of pre- and full-term calves.

The somatotropic axis and insulin are involved in pre- and postnatal development. In pre- and full-term calves (GrP0 and GrN0; born after 277 and 290 d of pregnancy, respectively) and in preterm calves on d 8 of life after being fed for 7 d (GrP8), we studied whether there are differences in the abundance of messenger RNA (mRNA) of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II, and insulin among different intestinal sites (duodenum, jejunum, ileum, and colon) and whether there are ontogenetic differences during the perinatal period in intestine and liver. Intestinal site differences (P < 0.05) existed in mRNA levels of IGF-I and IGF-II and receptors for GH, IGF-I, IGF-II, and insulin. Abundance of mRNA of IGF-I and -II and of receptors for IGF-I and GH was highest (P < 0.05) in the colon, abundance of the receptor for IGF-II was comparably high in the colon and ileum, and that of the receptor for insulin was similarly high in colon, ileum, and jejunum. Among GrP0, GrN0, and GrP8 groups, there were differences (P < 0.05) in mRNA levels of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II and insulin. Abundance of mRNA of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II and insulin was highest (P < 0.05) in GrP0 calves immediately after birth and was primarily seen in the ileum. In liver, the mRNA levels differed (P < 0.05) among groups for IGF-II and receptors for IGF-I, IGF-II, and insulin, and were highest (P < 0.05) for IGF-II in GrP0, for receptors of IGF-I in GrN0, and were higher (P < 0.05) in GrP0 than GrP8 for receptors of IGF-II. In conclusion, mRNA levels of IGF-I and IGF-II and of receptors for GH, IGF-I, IGF-II, and insulin were different at different intestinal sites and in intestine and liver and changed during the perinatal period.

Animals↗

PubMed2003

Gene expression of immunologically important factors in blood cells, milk cells, and mammary tissue of cows.

Cytokines, eicosanoids, and lactoferrin are involved in the mammary gland's immune response to invading microorganisms. The goal of this work was to investigate the synthesis of these immunologically important factors in somatic milk cells, blood cells, and mammary tissue of cows with different somatic cell count levels, i.e., different immunological activity. On the level of mRNA expression, the cytokine tumor necrosis factor alpha (TNFalpha), lactoferrin (Lf), and specific key enzymes of leukotriene and prostaglandin biosynthesis, 5-lipoxygenase (5-LO), and cyclooxygenase-1 (COX-1) and -2 (COX-2), respectively, were determined. All 15 experimental cows were clinically healthy with no visible mammary disease. Eight cows were defined as control group with all quarters <150,000 cells/ml (C), whereas seven cows had partially elevated quarter somatic cell counts, with at least one quarter >150,000 cells/ml (H) and one quarter <150,000 cells/ml (L). Total quarter milk from one quarter of control group and from two quarters of cows with partially elevated cell counts (one of H and one of L) was collected at one milking and a blood sample was taken simultaneously. In addition, mammary tissue samples were taken from the respective quarters on the following day during slaughter. Total RNA from milk, blood, and tissue cells was isolated and reverse transcription and quantitative polymerase chain reaction was carried out. All factors investigated were not significantly different between groups in blood cells and between C and L quarters in milk cells and mammary tissue. TNFalpha and COX-2 mRNA expression was higher in milk cells and mammary tissue of H than in L quarters, except for COX-2 in mammary tissue. Generally, TNFalpha and COX-2 showed their highest expression in milk cells, 5-LO in blood cells, whereas lactoferrin was mainly expressed by the mammary tissue. COX-1 was similarly expressed in all tested samples.

Animals↗

PubMed2002

[Insulin-like growth factor 1 (IGF-1) mRNA and IGF-1 protein. Expression in cells of the trabecular meshwork of the bovine eye].

BACKGROUND: The purpose of the study was to determine whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin-like growth factor-1 (IGF-1) mRNA and protein. METHODS: The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of IGF-1 mRNA. To detect the protein on the cells an IGF-1-specific immunohistochemical stain was used on trabecular meshwork cells. RESULTS: A single 240 bp RT-PCR product was obtained, the RT-PCR product was verified by sequencing and the derived sequence was homologous to the known bovine sequence. IGF-1 immunostaining was positive in the cytoplasm of trabecular meshwork cells. CONCLUSIONS: We conclude that trabecular meshwork cells produce IGF-1 mRNA and contribute to the presence of IGF-1 protein in the trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by IGF-1 not only through paracrine but also through autocrine action. Whether regulations in IGF-1 production may contribute to the pathogenesis of primary open-angle glaucoma and the possibility of promoting the autocrine action of IGF-1 by trabecular meshwork cells to treat the disease is worth further investigation.

Animals↗

PubMed2002

Real-time RT-PCR quantification of insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species.

Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analysing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain highly accurate and reliable results in a real-time RT-PCR a highly defined calibration curve is needed. We designed and developed nine different calibration curves, based on recombinant DNA plasmid standards and established them on a constant real-time PCR platform for the following factors: growth hormone receptor (GHR), insulin-like growth factor (IGF)-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor (INSR), and IGF-binding proteins (IGF-BP) 1, 2 and 3. Developed assays were applied in the LightCycler system on bovine ileum and liver total RNA and showed high specificity and sensitivity of quantification. All assays had a detection limit of under 35 recombinant DNA molecules present in the capillary. The SYBR Green I determination resulted in a reliable and accurate quantification with high test linearity (Pearson correlation coefficient r > 0.99) over seven orders of magnitude from <10(2) to >10(8) recombinant DNA start molecules and an assay variation of maximal 5.3%. Applicability of the method was shown by analysing mRNA levels in newborn calves: mRNA concentrations per gram tissue of mRNAs of IGF-1, IGF-1R, IGF-2, IGF-2R, GHR, INSR, and IGF-BP1, 2 and 3 were all different between in liver and ileum and the traits all exhibited individual differences.

Animals↗

PubMed2002

Effects of synthetic progestagens on the mRNA expression of androgen receptor, progesterone receptor, oestrogen receptor alpha and beta, insulin-like growth factor-1 (IGF-1) and IGF-1 receptor in heifer tissues.

Synthetic progestagens like melengestrol acetate (MGA) are widely used for oestrus synchronization and for growth promotion in cattle production. The metabolic effects exceed its primary potency as a progestagen. It is speculated that MGA stimulates follicle development and thereby endogenous oestrogen production, but inhibits ovulation. To investigate the dose-dependent effects on mRNA expression levels, six heifers were fed for 8 weeks with different levels of MGA (0.5, 1.5, 5 mg) daily and two heifers served as controls. The expression of steroid receptor mRNA [androgen receptor (AR), progesterone receptor (PR), oestrogen receptor (ER) ERalpha and ERbeta], insulin-like growth factor-1 (IGF-1) and its receptor were quantified in liver, neck (m. splenius) and shoulder muscularity (m. deltoideus). Plasma concentrations of IGF-1 were quantified by radioimmunoassay. In treated animals the MGA plasma levels were elevated over the complete treatment period, corresponding to the MGA treatment concentrations. IGF-1 concentrations of control animals were at constant levels. Plasma levels for oestradiol (E2) and IGF-1 were increased in the low MGA treatment group. Overdosed MGA decreased progesterone (P4) and E2 levels. To quantify the IGF-1 and all receptor mRNA transcripts, sensitive and reliable real-time reverse transcription-polymerase chain reaction (RT-PCR) quantification methods were developed and validated in the LightCycler. A dose-dependent relationship between increasing MGA concentration and mRNA expression was observed in liver for AR and IGF-1 receptor, and in neck muscularity for IGF-1. ERalpha in liver and neck muscle showed a trend of increasing expression.

Animals↗

PubMed2001

A new mathematical model for relative quantification in real-time RT-PCR.

Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.

Animals↗