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Biomedical subjects

G Brem

Publications and source records attributed to G Brem.

At least 127 records · Page 7Linked to original sources
PubMed1996

Three year results of in vitro production of bovine embryos in serum-poor bovine oviduct conditioned medium. An overview.

This paper presents a synthesis of 3 year results of in vitro production of bovine embryos in medium previously conditioned by bovine oviduct epithelial cells. In Louvain-la-Neuve, Belgium, a total of 18356 oocytes were matured and inseminated in vitro: 13967 (76%) had cleaved at 3 days post-insemination and 3593 (26%) became blastocysts using this culture system. Our data show that conditioned medium can be stored frozen for up to 3 years without significant loss of activity and is resistant to lyophilization. One single batch of conditioned medium was tested within the same period in four different laboratories and yielded variable results: 27 and 37% blastocysts/cleaved embryos in two of them and only 7 and 0% in the two others whereas in each case more than 30% blastocysts were obtained with the local reference co-culture system. In one laboratory, the batch of oil used to overlay the culture drops had a detrimental effect on the blastocyst rate in conditioned medium but not in co-culture.

Animals↗
PubMed1996

Evaluation of seven microsatellite loci in Simmental cattle.

Seven microsatellite loci were evaluated for their suitability for parentage control. The polymerase chain reaction (PCR) was used to amplify the short tandem repeat (STR) loci in separate reactions. The microsatellite polymorphisms were visualized by radioisotopic autoradiographic detection. The microsatellite loci showed extensive polymorphism with allele numbers ranging from 4-23 and polymorphism information content (PIC) values in the range of 0.57-0.87. The analysis of these loci also revealed that they have a 99.9% combined probability of exclusion (PE) of erroneous parentage. The results of this study revealed that a very high probability of exclusion could be reached with only four microsatellite loci.

Alleles↗
PubMed1996

Epigenetic modification of transgenes under the control of the mouse mammary tumor virus LTR: tissue-dependent influence on transcription of the transgenes.

Transgenic mice expressing human urokinase, as well as animals expressing human urokinase receptor under the control of the murine mammary tumor virus (MMTV) long terminal repeat, were established. In the vast majority of the founder animals and their descendants, the transgene was completely methylated, corresponding to down-regulation of transgene expression in the mammary gland. Two lineages with human urokinase receptor as the transgene with mixed methylation of the transgenes were analyzed in more detail. We show here for the first time that the methylation status of the transgene is identical in different organs of an animal, but may differ from animal to animal among the descendents. In the mammary gland, complete methylation of the transgene was incompatible with expression; unmethylated and mixed methylation transgenes gave rise to expression at the RNA as well as at the protein level. The methylation observed was not the consequence of an imprinting process. Surprisingly, in organs other than the mammary gland, such as liver, kidney and spleen, weak expression of the transgene was noted independent of the methylation status of the MMTV promoter. With respect to the molecular mechanism it is unresolved whether the human growth hormone sequence of the transgene harbors a methylation inducing element responsible for the observed methylation pattern.

3T3 Cells↗
PubMed1996

Granulosa-cumulus-corona expansion and aromatase localization in preovulatory follicles in superovulated heifers.

Granulosa-cumulus and cumulus-corona expansion as well as aromatase localization within ovarian follicles were monitored during the preovulatory period in superovulated cattle that were blood sampled every 2'nd h for LH analyses. Granulosa-cumulus as well as cumulus-corona expansion were studied by means of transmission electron microscopy and computerized image analysis. Localization of aromatase, an enzyme involved in estrogen synthesis, was determined immunocytochemically using anti-human placental aromatase cytochrome P-450 antisera. Nuclear oocyte maturation was determined by aceto:orcein staining. Significant cell dissociation within the granulosa-cumulus stalk occurred before the breakdown of the germinal vesicle, i.e. the oocyte nucleus, during the period up to 5-7 h after the LH peak, i.e. the highest LH concentration during the surge. Significant increase in intercellular spacing between the cumulus-corona cells occurred at 13-15 and 19-21 h after the LH peak. Before the LH peak all layers of granulosa cells were immunocytochemically stained for aromatase. At 5-7 h after the LH peak, however, only the granulosa cell layers located near the basal lamina were stained, and at all later intervals staining was absent. The granulosa cells of primary and secondary follicles, the interstitial gland cells, the theca interna cells and the oocytes in all follicles were immunocytochemically unstained.

Animals↗
PubMed1996

[The use of pluripotent mouse embryo stem cells for the production of chimeric animals].

Embryonic stem (ES) cells derived from pluripotent cells of the early mouse embryos provide a powerful tool for genome manipulation in mammals. Conditions for maintaining and preservation of pluripotent properties of embryonic stem (ES) cells in culture using different tests are described. A simple aggregation of pluripotent ES cells with morulae-stage embryos for derivation of chimaeras is considered.

Alkaline Phosphatase↗
PubMed1995

Characterization of a protein that binds a negative regulatory element in the mammary-specific whey acidic protein promoter.

Whey Acidic Protein (WAP) gene expression is restricted to the pregnant and lactating mammary gland. We have recently defined a negative regulatory element (NRE) in the WAP promoter which interacts with a factor (NBF) present in all nonWAP expressing cells (Kolb et al., 1994; J. Cell. Biochem. 56:245-261). Here we characterise this factor and show that although it is not related to a number of known transcription factors, including AP-1, NF-1 and SP-1, it may also be involved in controlling the expression from the mouse mammary tumour virus promoter. Three proteins that bind to the WAP-NRE have been identified, one of which is a 53kDa nuclear protein. This protein is present in nonWAP expressing cells, suggesting that it is responsible for limiting WAP expression to the pregnant and lactating mammary gland. This protein has been partially purified and its binding to the WAP-NRE is not appreciably affected by high salt concentrations.

Animals↗
PubMed1995

Detection of membrane-bound HLA-G translated products with a specific monoclonal antibody.

A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.

Animals↗
PubMed1995

Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF) gene family, has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes such as long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF gene. We then used hippocampal slices from these mice to investigate whether LTP was affected by this mutation. Homo- and heterozygous mutant mice showed significantly reduced LTP in the CA1 region of the hippocampus. The magnitude of the potentiation, as well as the percentage of cases in which LTP could be induced successfully, was clearly affected. According to the criteria tested, important pharmacological, anatomical, and morphological parameters in the hippocampus of these animals appear to be normal. These results suggest that BDNF might have a functional role in the expression of LTP in the hippocampus.

Animals↗
PubMed1995

Does the overexpression of pro-insulin-like growth factor-II in transfected human embryonic kidney fibroblasts increase the secretion of lysosomal enzymes?

Insulin-like growth factor-II (IGF-II) and lysosomal enzymes bearing the mannose 6-phosphate (Man6P) recognition marker, bind to two distinct binding sites of the IGF-II/M6P receptor. The two classes of ligands reciprocally modulate the binding of the other class of ligand to the receptor [Kiess, W., Thomas, C. L., Greenstein, L., Lee, L., Sklar, M. M., Rechler, M. M., Sahagian, G. G. & Nissley, S. P. (1989) J. Biol. Chem. 264, 4710-4714]. We asked whether or not overexpression of pro-IGF-II by cells in culture leads to missorting of lysosomal enzymes. Human embryonal kidney fibroblasts were transfected with the full-length human IGF-II cDNA or a control cDNA. Solution hybridization/RNase protection experiments using a human IGF-II riboprobe showed that two transfectants expressed large quantities of IGF-II mRNA, whereas the non-transfected cells did not. The analysis of conditioned media revealed that these cells secrete approximately 0.15 micrograms and 1.0 micrograms immunoreactive IGF-II/ml and 22 x 10(6) cells and 24 x 10(6) cells within 24 hours. Immunoreactive IGF-II was shown by Western blotting to represent 17-kDa pro-IGF-II. The amount of the lysosomal enzyme, beta-hexosaminidase, was approximately twofold increased in the conditioned media from pro-IGF-II overexpressing cells compared with control media, as shown by Western-blot analysis and immunoprecipitation of media extracts of metabolically labeled cells. The synthesis rate of beta-hexosaminidase was not affected by pro-IGF-II overexpression. In addition, the basal amount of another newly synthesized lysosomal enzyme, the cathepsin D precursor, was also twofold higher in pro-IGF-II overexpressing cells than in control cells. In contrast, the surface binding and cellular uptake rate of a Man6P-containing neoglycoprotein did not differ between the cell lines. The results indicate that the overexpression of pro-IGF-II doubles the secretion and/or reduces the re-uptake of beta-hexosaminidase and cathepsin D to approximately 20% of the total synthesized enzymes in human embryonal kidney fibroblasts compared to control cells. We hypothesize that, in cells synthesizing high amounts of pro-IGF-II, the growth factor may modulate the targeting of a portion of lysosomal enzymes, mainly by partially enhancing the secretion of newly synthesized enzymes and, in addition, possibly by affecting the re-uptake mechanism.

Cathepsin D↗
PubMed1995

Akv murine leukemia virus enhances lymphomagenesis in myc-kappa transgenic and in wild-type mice.

The contribution of endogenous retroviruses to the multistep process of lymphomagenesis was investigated in wild-type mice and in two different myc-kappa transgenic mouse lines by infection with Akv. This retrovirus is derived from the endogenous ecotropic provirus of the AKR mouse and was previously considered to be nonlymphomagenic. The mice of the two myc-k transgenic lines are predisposed to B-cell lymphomagenesis and were therefore considered to be more susceptible to Akv. For comparison, the same mouse strains were also infected with the exogenous Moloney murine leukemia virus (MoMuLV). Both MoMuLV and Akv increased the tumor incidence and shortened the tumor latency period in wild-type mice and in the transgenic mouse lines. The differences in pathogenicity, number of provirus integrations, and level of virus expression between MoMuLV and Akv indicate different mechanisms of lymphomagenesis: while MoMuLV induced tumors apparently by insertional mutagenesis involving common integration sites similar to previous reports, the enhancement of lymphomagenesis by Akv seems to be directed by other mechanisms.

AKR murine leukemia virus↗
PubMed1995

Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning.

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25-34), intermediate (40-55), and high (60-81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50-83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40-55) or high (60-81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40-55 cells) and 41% versus 25% (60-81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning.

Animals↗
PubMed1995

Construction of a porcine YAC library and mapping of the cardiac muscle ryanodine receptor gene to chromosome 14q22-q23.

Large-scale physical mapping of the porcine genome has been limited because up to now no suitable genomic libraries for this purpose have been available. Therefore, we have constructed a yeast artificial chromosome (YAC) library from porcine lymphocytes. The library was cloned in the amplifiable vector pCGS966. A total of 10080 YAC clones was obtained and has been ordered into 105 96-well microtiter plates. An average insert size of 300 kb was calculated from the analysis of 78 randomly selected clones, giving a one-fold coverage of the porcine genome. To analyze the complexity, we have screened the library for five different genes and isolated four different clones containing parts of three of these genes. One YAC clone harboring parts of the porcine cardiac muscle ryanodine receptor (RYR2) gene allowed us to assign this locus to Chromosome (Chr) 14q22-q23. The data were confirmed by PCR analysis of a rodent-porcine hybrid cell panel.

Animals↗
PubMed1995

Secretion of biologically active interferon tau by in vitro-derived bovine trophoblastic tissue.

Secretion of interferon tau (IFN tau) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Ménézo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-microliters drops of BRL cell-conditioned medium, mean IFN tau secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFN tau secretion by trophoblastic tissue increased to mean levels of > 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.

Animals↗
PubMed1995

Effects of growth hormone overproduction on grip strength of transgenic mice.

Growth hormone (GH) is used by athletes like bodybuilders to increase muscle strength and weight gain. On the other hand, chronic hypersecretion of GH in active acromegaly may result in outwardly hypertrophied but functionally weaker muscles. As a model for studying long-term effects of GH on muscle strength, we analysed transgenic mice (TM) carrying rat phosphoenolpyruvate carboxykinase-bovine GH (PEPCKbGH) fusion genes, which are expressed in liver and kidney but not in skeletal muscle. Circulating GH levels in TM ranged between 0.5 and 3 micrograms/ml, resulting in increased (p < 0.001) body weight (wt) as well as increased (p < 0.01) weights of forelimb and hindlimb muscles. However, muscle weight/body wt ratios of TM were 16-20% smaller than in controls (p < 0.05). Forelimb grip strength of hemizygous TM (16 males, 132 +/- 45 days old, body wt = 56.8 +/- 8.3 g; 32 females, 146 +/- 38 days old, body wt = 54.9 +/- 6.1 g) and non-transgenic controls (28 males, 127 +/- 47 days old, body wt = 40.5 +/- 2.9 g; 33 females, 126 +/- 47 days old, body wt = 32.1 +/- 3.6 g) was determined using an automated grip strength meter. Data were computed by analysis of variance, taking into account effects of group, sex and age. Least-squares means estimated for the grip strength (N) of male TM (1.91) and controls (1.92) were significantly (p < 0.05) greater than those of female TM (1.78) and controls (1.61). A significant difference between groups was only seen in females (p < 0.01). Least-squares means estimated for grip strength/body wt ratios (N/10 g) of male (0.34) and female TM (0.33) were 29% and 35% lower than those of male (0.48) and female controls (0.51), respectively (p < 0.001). In summary, long-term elevated GH levels in TM increased muscle weight less efficiently than body weight, and muscle strength did not increase proportionally with muscle weight.

Analysis of Variance↗
PubMed1995

Skeletal growth of transgenic mice with elevated levels of circulating insulin-like growth factor-II.

Insulin-like growth factor-II (IGF-II) is a major factor produced by skeletal tissues. To evaluate endocrine effects of IGF-II on bone growth, we measured skeletal dimensions of 12-week-old transgenic mice harbouring fusion genes where a human IGF-II cDNA is transcriptionally controlled by rat phospheonolpyruvate carboxykinase (PEPCK) promoter sequences. Transgene expression in liver, kidney and intestine resulted in circulating IGF-II levels in transgenic mice which were 2-3-fold higher than in controls. Serum IGF-I concentrations of transgenic mice were lower than in controls. Body weight was not influenced by the expression of the IGF-II transgene. Only 1 out of 5 measurements taken from the radius was significantly affected by the presence of the transgene, while in 60 measurements taken from eight other bones there was no difference between transgenic mice and controls. Furthermore, serum levels of calcium and phosphate as well as alkaline phosphatase activity were not significantly altered in PEPCK-IGF-II transgenic mice. Our findings demonstrate that moderately increased levels of circulating IGF-II do not cause major changes in skeletal growth and turnover in mice. This may be due to a lack of activity of circulating IGF-II on bone growth or to physiological consequences of elevated IGF-II, like a reduction of circulating IGF-I or an increase in IGF binding proteins.

Animals↗
PubMed1994

Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits.

We have developed an expression system where foreign proteins are synthesized specifically in the mammary gland of transgenic rabbits and secreted into the milk. Regulatory elements were isolated from the bovine alpha S1-casein-encoding gene and combined with a synthetic DNA coding for human IGF-1 and for [Gln58]IGF-1, an IGF-1 analogue. The resulting hybrid DNA constructs were used to generate transgenic rabbits. Females of seven transgenic lines tested were positive for synthesis of IGF-1. Transmission of the transgene to progeny and IGF-1 production in female offspring was observed in all transgenic lines analysed. As expected, expression of transgene mRNA could only be detected in the mammary gland. Production levels of transgenic protein were as high as 1 g IGF-1 per liter rabbit milk. IGF-1, as well as [Gln58]IGF-1, when secreted into rabbit milk, was correctly processed and biologically active. IGF-1 was purified from the milk of transgenic rabbits to a nearly homogenous active form.

Animals↗