Biomedical subjects

Gottfried Brem

Publications and source records attributed to Gottfried Brem.

28 records · Page 2Linked to original sources

PubMed2002

Plasminogen activator activity in cortical granules of bovine oocytes during in vitro maturation.

In this study, we provide evidence that plasminogen activator of tissue-type (t-PA), at least, is present in extracts of bovine oocyte cortical granules, and that its activity varies significantly with the duration of oocyte in vitro maturation. Cortical granules were collected from bovine oocytes by means of micromanipulation, after 0, 12, or 24 h of IVM. Our results show that plasminogen activator activity of cortical granule extracts was significantly higher after 24 h of IVM than after 12 h of IVM or before IVM. This activity was apparently due, at least partly, to tissue-type plasminogen activator as shown immunologically. No evidence was found for the presence of urokinase-type plasminogen activator, plasminogen activator inhibitors or plasmin inhibitors in bovine oocyte cortical granule extracts. Our findings further support the hypothesis that t-PA activity of oocyte origin may have a role in oocyte maturation or fertilization, as well as in post-fertilization events, such as cortical reaction and formation of the zona block to polyspermy.

Amiloride↗

PubMed2002

Identification of HLA-B27-restricted cytotoxic T lymphocyte epitope from carcinoembryonic antigen.

Characterization of epitopes recognized by cytotoxic T lymphocytes (CTLs) in the sequence of tumor antigens is an important step in the development of tumor therapies. Because carcinoembryonic antigen (CEA) is a protein expressed in a high number of epithelial tumors, it is an interesting target to study. We screened for the presence of HLA-B27-restricted CTL epitopes from CEA by studying the binding to HLA-B27 of 31 synthetic peptides predicted to bind to this molecule. This afforded 16 peptides with moderate or high binding affinity. Immunization of HLA-B27 transgenic mice with the best binder peptides yielded 4 immunogenic peptides: CEA(9-17), CEA(9-18), CEA(138-146) and CEA(360-369). However, splenocytes from mice immunized with a vaccinia virus-expressing CEA recognized only CEA(9-18). These CTLs were of the CD8(+) phenotype, which upon stimulation with peptide specifically produced IFN-gamma. Moreover, they did not cross-react against peptides of region 9-18 from proteins of the CEA family. Our results show that CEA(9-18) may induce specific CTL responses against CEA.

Animals↗

PubMed2002

Reduced body growth and excessive incisor length in insertional mutants mapping to mouse Chromosome 13.

Phenotypic and molecular genetic examinations of a transgenic mouse line showing developmental defects caused by a recessive insertional mutation were carried out. The mutant phenotype is characterized by general retardation of postnatal body growth and by the appearance of increased incisor length in the upper and lower jaw. The mutation causing the aberrant phenotype was mapped to Chromosome 13, 40 cM. Examination of the expression of the candidate genes did not show any alterations. This mutant mouse line provides a reproducible model for the identification and examination of gene(s) involved in growth and in the craniofacial development, including that of the jaws and teeth.

Animals↗

PubMed2002

Aging of animals produced by somatic cell nuclear transfer.

Cloning by somatic cell nuclear transfer (NT) has been used to successfully generate viable mammalian embryos in many species. Despite the successful production of cloned animals, NT remains an inefficient technique at best. High rates of developmental failure and abnormalities are associated with NT-derived animals. Issues such as cell cycle regulation, epigenetic modification and oocyte activation have been examined in order to ultimately find ways to improve the efficiency of somatic cell NT. In reviewing the available literature regarding the generation of various mammalian species by using somatic cell NT, it becomes apparent that there are not only differences in the protocols used to generate such animals, but significant differences in the phenotypes of resultant animal clones as well. In this review we discuss the current findings about how aging and senescence are affected by somatic cell NT in mammals.

Aging↗

PubMed2002

The recent history of somatic cloning in mammals.

The history of somatic cell nuclear transfer (NT) in mammals is full of exciting experiments and findings regarding the technique and outcome of NT, despite only covering a period of 6 years. The production of Dolly, for the first time demonstrating cloning from an adult somatic cell, had a great impact on subsequent studies. However, the more progress we make, the more obvious it becomes how little we know about the processes during NT, specifically how reprogramming events occur. Therefore, it is certainly challenging to continue investigating every step of somatic cell NT more intensively, starting from the donor cell, (type, cell cycle, synchronization, population doublings) and continuing until the cloned offspring are born and even further, to see how and if NT has an influence on health, viability, quantitative traits, and reproduction of cloned individuals.

Animals↗

PubMed2002

Coexistence of Bos taurus and B. indicus mitochondrial DNAs in nuclear transfer-derived somatic cattle clones.

We investigated the mitochondrial DNA (mtDNA) composition in one of the largest adult somatic mammalian clones (n = 20) reported so far. The healthy cloned cattle were derived from nuclear transfer of an identical nuclear genetic background (mural granulosa donor cells including surrounding cytoplasm) into enucleated oocytes with either Bos indicus or B. taurus mtDNA. Here we report the first cases of coexisting mtDNAs of two closely related subspecies following nuclear transfer. Heteroplasmy (0.6-2.8%) was found in 4 out of 11 cross-subspecies cloned cattle. Quantitation was performed using "amplification refractory mutation system (ARMS) allele-specific real-time PCR." We determined that the ratio of donor cell to recipient cytoplast mtDNA copy number was 0.9% before nuclear transfer. Therefore, we concluded that the percentage of donor cell mtDNA in the heteroplasmic intersubspecific cloned animals is in accordance with neutral transmission of donor mtDNA. We determined an amino acid sequence divergence of up to 1.3% for the two subspecies-specific mtDNA haplotypes. In addition, intrasubspecific B. indicus heteroplasmy of approximately 1% (but up to 7.3 and 12.7% in muscle and follicular cells of one animal) was detected in 7 out of the 9 B. indicus intrasubspecific cloned cattle.

Animals↗

PubMed2002

Bovine somatic cell nuclear transfer using recipient oocytes recovered by ovum pick-up: effect of maternal lineage of oocyte donors.

The efficiency of bovine nuclear transfer using recipient oocytes recovered by ultrasound-guided follicle aspiration (ovum pick-up [OPU]) was investigated. Oocyte donors were selected from 2 distinct maternal lineages (A and B) differing in 11 nucleotide positions of the mitochondrial DNA control region. A total of 1342 cumulus-oocyte complexes (COCs) were recovered. The numbers of total COCs and class I/II COCs recovered from donors of lineage A were higher (P < 0.001) than those obtained from lineage B. Follicle aspiration once per week yielded a higher (P < 0.001) total number of COCs per session than aspiration twice per week, whereas the reproduction status of donors (heifer vs. cow) had no effect on OPU results. Of the 1342 oocytes recovered, 733 (55%) were successfully matured in vitro and used for nuclear transfer. Fusion was achieved in 550 (75%) karyoplast-cytoplast complexes (KCCs), resulting in 277 (50%) cleaved embryos on Day 3. On Day 7 of culture, 84 transferable embryos (15% based on fused KCCs) were obtained. After 38 transfers (10 single, 22 double, and 6 triple transfers), 9 recipients (8 double and 1 triple transfer) were diagnosed as pregnant on Day 28, corresponding to a pregnancy rate of 24%. The proportion of transferable embryos on Day 7 was significantly (P < 0.05) influenced by maternal lineage of oocyte donors and by the frequency of follicle aspiration. Our study demonstrates the feasibility of generating nuclear transfer embryos with defined cytoplasmic background. These will be valuable tools to experimentally dissect the effects of nuclear and cytoplasmic components on embryonic, fetal, and postnatal development.

Animals↗

PubMed2002

Characterization of porcine endogenous retrovirus gamma pro-pol nucleotide sequences.

Endogenous retroviral sequences in the pig genome (PERV) represent a potential infectious risk in xenotransplantation. All known infectious PERV have been asssigned to the PERV gamma1 family, consisting of the subfamilies A, B, and C. The aim of the study was the concise examination of PERV gamma by the analysis of the retroviral pro-pol sequences. The analysis of 52 pro-pol clones amplified in this study revealed eight PERV gamma families. In addition to four already-described families (gamma1, gamma4, gamma5, gamma6), four novel families (gamma7, gamma8, gamma9, gamma10) were identified. Quantitative analysis of the novel PERV gamma sequences in selected breeds revealed variations in the endogenous retroviral load. Open reading frames (ORF) in the amplified proviral fragment were only found for PERV gamma1. In addition, novel ORF-containing PERV gamma1 clones consisting of hybrid sequences were revealed. Sequence comparison from published full-length PERV gamma1 clones of the PERV subfamilies A, B, and C resulted in a lack of strict correlation of the classification of pro-pol and env. The results indicated the occurrence of causative recombination events between retroviral genomes. Thus, our study on PERV gamma provides new data for the evaluation and selection of pigs intended to be used in xenotransplantation.

Animals↗

PubMedDate not supplied

Somatic gene transfer into the lactating ovine mammary gland.

BACKGROUND: Somatic gene therapy requires safe and efficient techniques for the gene transfer procedure. The ovine mammary gland is described as a model system for the evaluation of somatic gene transfer methods. METHODS: Different gene delivery formulations were retrogradely injected into the mammary gland of lactating sheep. The efficiency of the gene transfer was subsequently measured by the detection of the secreted transgene products in the milk. To counteract the milk flow in the lactating gland caused by the permanent milk production, a newly developed pretreatment of the mammary gland with hyperosmotic solutions was applied. In addition, in vivo electroporation of DNA into the mammary gland is described. RESULTS: Gene transfer using naked DNA or simple complexes of DNA with polycations did not result in traceable amounts of reporter gene products. However, utilizing the complex cationic lipid DOSPER, a peak expression of about 400 ng/ml was observed 6 days after transfection. Maximum expression rates of more than 1 microg/ml were obtained by combining hyperosmotic pretreatment and receptor-mediated gene transfer. For the in vivo electroporation, the proof of principle for this technique in the mammary gland is reported. CONCLUSIONS: The ovine mammary gland turned out to be a very well suited as a model system for evaluation and optimization of various gene transfer protocols.

Animals↗

PubMedDate not supplied

History of Lipizzan horse maternal lines as revealed by mtDNA analysis.

Sequencing of the mtDNA control region (385 or 695 bp) of 212 Lipizzans from eight studs revealed 37 haplotypes. Distribution of haplotypes among studs was biased, including many private haplotypes but only one haplotype was present in all the studs. According to historical data, numerous Lipizzan maternal lines originating from founder mares of different breeds have been established during the breed's history, so the broad genetic base of the Lipizzan maternal lines was expected. A comparison of Lipizzan sequences with 136 sequences of domestic- and wild-horses from GenBank showed a clustering of Lipizzan haplotypes in the majority of haplotype subgroups present in other domestic horses. We assume that haplotypes identical to haplotypes of early domesticated horses can be found in several Lipizzan maternal lines as well as in other breeds. Therefore, domestic horses could arise either from a single large population or from several populations provided there were strong migrations during the early phase after domestication. A comparison of Lipizzan haplotypes with 56 maternal lines (according to the pedigrees) showed a disagreement of biological parentage with pedigree data for at least 11% of the Lipizzans. A distribution of haplotype-frequencies was unequal (0.2%-26%), mainly due to pedigree errors and haplotype sharing among founder mares.

Animals↗