Biomedical subjects

Bernd Fischer

Publications and source records attributed to Bernd Fischer.

13 recordsLinked to original sources

PubMed2006

Semi-supervised LC/MS alignment for differential proteomics.

MOTIVATION: Mass spectrometry (MS) combined with high-performance liquid chromatography (LC) has received considerable attention for high-throughput analysis of proteomes. Isotopic labeling techniques such as ICAT [5,6] have been successfully applied to derive differential quantitative information for two protein samples, however at the price of significantly increased complexity of the experimental setup. To overcome these limitations, we consider a label-free setting where correspondences between elements of two samples have to be established prior to the comparative analysis. The alignment between samples is achieved by nonlinear robust ridge regression. The correspondence estimates are guided in a semi-supervised fashion by prior information which is derived from sequenced tandem mass spectra. RESULTS: The semi-supervised method for finding correspondences was successfully applied to aligning highly complex protein samples, even if they exhibit large variations due to different biological conditions. A large-scale experiment clearly demonstrates that the proposed method bridges the gap between statistical data analysis and label-free quantitative differential proteomics. AVAILABILITY: The software will be available on the website http://people.inf.ethz.ch/befische/proteomics.

Algorithms↗

PubMed2006

Differential expression of glucose transporter isoforms during embryonic stem cell differentiation.

In mouse blastocysts six facilitative glucose transporter isoforms (GLUT)1-4, 8 and 9 are expressed. We have used the mouse embryonic stem (ES) cell line D3 and spontaneously differentiating embryoid bodies (EB) to investigate GLUT expression and the influence of glucose during differentiation of early embryonic cells. Both ES cells and EBs (2d-20d) expressed GLUT1, 3, and 8, whereas the isoforms 2 and 4 were detectable exclusively in EBs. Differentiation-associated expression of GLUT was analyzed by double staining with stage-specific embryonic antigen (SSEA-1), cytokeratins (CK18, 19), nestin, and desmin. Similar to trophoblast cells in mouse blastocysts the outer cell layer of endoderm-like cells showed a high GLUT3 expression in early EBs. In 20-day-old EBs no GLUT3 protein and only minor GLUT3 mRNA amounts could be detected. A minimal glucose concentration of 5 mM applied during 2 and 8 days of EB culture resulted in up-regulated GLUT4, Oct-4 and SSEA-1 levels and a delay in EB differentiation. We conclude that GLUT expression depends on cellular differentiation and that the expression is modulated by glucose concentration. The developmental and glucose-dependent regulation of GLUT strongly suggests a functional role of glucose and glucose transporters in ES cell differentiation and embryonic development.

Animals↗

PubMed2005

NovoHMM: a hidden Markov model for de novo peptide sequencing.

De novo sequencing of peptides poses one of the most challenging tasks in data analysis for proteome research. In this paper, a generative hidden Markov model (HMM) of mass spectra for de novo peptide sequencing which constitutes a novel view on how to solve this problem in a Bayesian framework is proposed. Further extensions of the model structure to a graphical model and a factorial HMM to substantially improve the peptide identification results are demonstrated. Inference with the graphical model for de novo peptide sequencing estimates posterior probabilities for amino acids rather than scores for single symbols in the sequence. Our model outperforms state-of-the-art methods for de novo peptide sequencing on a large test set of spectra.

Amino Acid Sequence↗

PubMed2005

Apoptosis in bovine cumulus-oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation.

Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocyte in vitro maturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs during in vitro maturation has been investigated. Results indicate that exposure of cumulus-oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested.

Animals↗

PubMed2004

Constitutive expression of CYP1A1 in bovine cumulus oocyte-complexes in vitro: mechanisms and biological implications.

The arylhydrocarbon receptor (AhR) is known to mediate toxic responses to dioxin (2,3,7,8-tetrachlorodibenzo-p- dioxin) and related compounds and has been extensively characterized from a toxicological viewpoint. However, it has recently been reported that the AhR may have a central role in ovarian physiology. To investigate the role of AhR during oocyte maturation, we analyzed the expression of AhR, its nuclear partner AhR nuclear translocator, and the major target gene CYP1A1, in bovine cumulus-oocyte complexes (COCs) by semiquantitative RT-PCR and Western blot. Coexpression of AhR and AhR nuclear translocator was observed in both oocytes and surrounding cumulus cells before and after in vitro maturation (IVM). Furthermore, after IVM, both cell types showed a clear up-regulation of AhR mRNA compared with the expression at 0 h. Constitutive expression of CYP1A1 mRNA was observed in immature oocytes at the background level, whereas no expression was observed in the surrounding cumulus cells. Interestingly, a significant increase in CYP1A1 expression level was observed in both oocytes and cumulus cells after IVM. To further investigate the role of AhR in CYP1A1 up-regulation and oocyte maturation, COCs were treated throughout IVM with the AhR antagonists, alpha-naphthoflavone and resveratrol. Both antagonists decreased the level of CYP1A1 in COCs compared with controls. Furthermore, CYP1A1 down-regulation was accompanied by a reduced ability of oocytes to complete in vitro maturation until metaphase II stage. These results suggest that CYP1A1 induction in COCs is necessary for correct proceeding of in vitro oocyte maturation in bovine and suggest a physiological role of AhR during resumption of meiosis.

Animals↗

PubMed2004

Two insulin-responsive glucose transporter isoforms and the insulin receptor are developmentally expressed in rabbit preimplantation embryos.

Glucose is the most important energy substrate for mammalian blastocysts. Its uptake is mediated by glucose transporters (GLUT). In muscle and adipocyte cells insulin stimulates glucose uptake by activation of the insulin receptor (IR) pathway and translocation of GLUT4. GLUT4 is expressed in bovine preimplantation embryos. A new insulin-responsive isoform, GLUT8, was recently described in mouse blastocysts. Thus, potentially, two insulin-responsive isoforms are expressed in early embryos. The mechanism of insulin action on embryonic cells, however, is still not clear. In the present study expression of IR, GLUT1, 2, 3, 4, 5 and 8 was studied in rabbit preimplantation embryos using RT-PCR, Western blotting and immunohistochemistry. The rabbit mRNA sequences for the complete coding region of IR, GLUT4 and a partial GLUT8 sequence were determined by RACE-PCR and sequencing. GLUT4 was expressed in 3-day-old morulae and in 4- and 6-day-old blastocysts. IR and GLUT8 transcripts were detectable only in blastocysts. Blastocysts also expressed GLUT1 and 3, but not GLUT2 and 5. Transcript numbers of GLUT4 and 8 were higher in trophoblast than in embryoblast cells. Translation of IR, GLUT4 and 8 proteins in blastocysts was confirmed by Western blotting. GLUT4 was localized mainly in the membrane and in the perinuclear region in trophoblast cells while in embryoblast cells its localization was predominantly in the perinuclear cytoplasm. The possible function(s) of two insulin-responsive isoforms, GLUT4 and GLUT8, in rabbit preimplantation embryos needs further investigation. It may not necessarily be linked to insulin-stimulated glucose transport.

Amino Acid Sequence↗

PubMed2004

Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts.

The addition of insulin during in vitro culture has beneficial effects on rabbit preimplantation embryos leading to increased cell proliferation and reduced apoptosis. We have previously described the expression of the insulin receptor (IR) and the insulin-responsive glucose transporters (GLUT) 4 and 8 in rabbit preimplantation embryos. However, the effects of insulin on IR signaling and glucose metabolism have not been investigated in rabbit embryos. In the present study, the effects of 170 nM insulin on IR, GLUT4 and GLUT8 mRNA levels, Akt and Erk phosphorylation, GLUT4 translocation and methyl glucose transport were studied in cultured day 3 to day 6 rabbit embryos. Insulin stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) Erk1/2 and levels of IR and GLUT4 mRNA, but not phosphorylation of the phosphatidylinositol 3-kinase-dependent protein kinase, Akt, GLUT8 mRNA levels, glucose uptake or GLUT4 translocation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control.

Animals↗

PubMed2003

Visual field representations and locations of visual areas V1/2/3 in human visual cortex.

The position, surface area and visual field representation of human visual areas V1, V2 and V3 were measured using fMRI in 7 subjects (14 hemispheres). Cortical visual field maps of the central 12 deg were measured using rotating wedge and expanding ring stimuli. The boundaries between areas were identified using an automated procedure to fit an atlas of the expected visual field map to the data. All position and surface area measurements were made along the boundary between white matter and gray matter. The representation of the central 2 deg of visual field in areas V1, V2, V3 and hV4 spans about 2100 mm2 and is centered on the lateral-ventral aspect of the occipital lobes at Talairach coordinates -29, -78, -11 and 25, -80, -9. The mean area between the 2-deg and 12-deg eccentricities for the primary visual areas was: V1: 1470 mm2; V2: 1115 mm2; and V3: 819 mm2. The sizes of areas V1, V2 and V3 varied by about a factor of 2.5 across individuals; the sizes of V1 and V2 are significantly correlated within individuals, but there is a very low correlation between V1 and V3. These in vivo measurements of normal human retinotopic visual areas can be used as a reference for comparison to unusual cases involving developmental plasticity, recovery from injury, identifying homology with animal models, or analyzing the computational resources available within the visual pathways.

Brain Mapping↗

PubMed2003

Epithelial cells in the oviduct and vagina and steroid-synthesizing cells in the rabbit ovary express AhR and ARNT.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Beside exogenous ligands, an increasing list of endogenous ligands has recently been described. The AhR and its dimerization partner AhR nuclear translocator (ARNT) were found in embryos, fetuses and in genital tract tissue. Its role in reproduction and fertility is not known. In the current study, AhR and ARNT expression and co-localization were studied immunohistochemicaly during the pre-implantation period in various genital tract organs (ovary, oviduct, vagina) of the rabbit. In the ovary, the AhR was expressed in the steroid-secreting interstitial cells, in follicular and granulosa cells, and in lutein cells. The receptor was localized in the cytoplasm. A cytoplasmic localization was also found in the oviduct epithelium with a diffuse cytoplasmic staining in the ampulla and a localized cytoplasmic localization in the isthmus. In the vagina, AhR localization changed from cytoplasmic in the non-pregnant animal to nuclear staining in the basal layer of the vaginal epithelium on day 6 of pregnancy. The ARNT protein was found in all AhR expressing cells except for oocytes within primordial follicles. Its localization was in the cytoplasm and/or nuclei. Therefore, the full complement for AhR/ARNT transcriptional activity was found in the studied organs. AhR expression showed stage-specific changes in both the uterus (Hasan and Fischer 2001) and the vagina during the pre-implantation period, while the ARNT protein exhibited no change in expression during this period. In summary, these findings indicate a functional AhR/ARNT complex in the rabbit genital tract epithelia. Its precise role and activation mechanism in reproductive tissue is not clear. It is supposed, however, that this complex may be involved in both hormone production and hormone-induced cellular changes during early pregnancy of the rabbit.

Animals↗

PubMed2003

Toxic effects of in vitro exposure to p-tert-octylphenol on bovine oocyte maturation and developmental competence.

Alkylphenolic compounds are a widespread family of xenoestrogens. High concentrations of these substances are present in sewage sludge that is spread on arable land and pasture as fertilizer. Because of their known endocrine system-disrupting activity, alkylphenols represent a potential risk for the reproductive health of farm animals. In this study, the impact of p-tert-octylphenol (OP) on the developmental competence of bovine oocytes was evaluated. Endocrine activity of OP was investigated for its effect on estrogen receptors alpha and beta (ERalpha and ERbeta) and progesterone receptor (PR) mRNA levels. Cumulus-oocyte complexes (COCs) were exposed during in vitro maturation to serial concentrations of OP (1-0.0001 microg/ml) and were compared with vehicle-treated controls and a group of COCs treated with 17 beta-estradiol (E2). A dose-related decrease in the percentage of oocytes that completed maturation after 24 h and in oocyte fertilization competence was observed at doses of OP as low as 0.01 microg/ml. Groups treated with > or =0.001 microg/ml OP showed impaired embryo development. No adverse effects of E2 were observed. In the E2-treated COCs, ERalpha mRNA was decreased but PR mRNA was upregulated compared with controls. Treatment with 0.001 and 0.0001 microg/ml OP induced a decrease in ERalpha mRNA, but ERbeta and PR mRNA were not affected. Treatment with 0.01 microg/ml OP did not produce changes in the expression of any of the mRNAs studied. OP impairs meiotic progression and developmental competence of bovine oocytes without demonstrating clear estrogen-mimic activity.

Animals↗

PubMed2003

Polychlorinated biphenyls affect gene expression in the rabbit preimplantation embryo.

Polychlorinated biphenyls (PCBs) have been shown to be embryotoxic. The mechanism(s) of action is not clearly understood. The toxic effects could be either direct or indirect. Furthermore, PCB congeners vary in their toxic potential. They can be classified in coplanar PCBs binding to the transcription factor aryl hydrocarbon receptor (AhR), which induce subsequent changes in gene expression, and noncoplanar PCBs exhibiting AhR-independent effects. In order to investigate possible mechanisms, 5 and 6 days old preimplantation rabbit embryos were exposed in vitro to low levels of coplanar (PCB 77, 126, and 169) or noncoplanar PCBs (PCB 28, 52, 101, 118, 138, 153, and 180). The PCB effects were studied by semiquantitative RT-PCR analysis of AhR target genes (cytochrome P450 (CYP) 1A1, 1A2, UDP-glucuronosyl transferase 1, glutathione S-transferase pi1 and aldehyde dehydrogenase) and dioxin-responsive genes (IL 1beta, PAI 2, Cox 2, TGFalpha, EGF, erbB 1-4, c-fos, c-jun, HSP 90, cyclophilin 40), and by differential display (DD) RT-PCR. CYP 1B1 mRNA and AhR protein were localized by in situ hybridization and immunohistochemistry, respectively. From the AhR target genes studied only CYP 1B1, and cyclooxygenase 2 showed an increase in mRNA levels after coplanar and noncoplanar PCB. Interleukin 1beta and plasminogen activator inhibitor 2 were downregulated. CYP 1B1 mRNA showed a stage specific inducibility at day 6, but not at day 5. By DD RT-PCR we identified six new genes previously not reported to be regulated by PCBs. The mRNAs encoding the subunits 1, 2, 4, and 5 of the NADH ubiquinone oxidoreductase and beta-globin showed a decrease, whereas trichohyalin mRNA was increased after PCB exposure. Coplanar and noncoplanar PCB congeners elicited similar responses on the mRNA levels of the studied genes. Exposure to coplanar PCBs did not result in the AhR being translocated to the nucleus. Our results show that (i). PCBs induce changes in gene expression in rabbit day 5 and 6 preimplantation embryos and imply (ii). that the transcriptional changes observed were not mediated by the nuclear AhR.

Animals↗

PubMed2003

INSL-3 is expressed in human hyperplastic and neoplastic thyrocytes.

The insulin-like hormone INSL-3, also named relaxin-like factor (RLF) or Leydig-derived insulin-like peptide (LEY-IL), is expressed in various reproductive tissues and is regarded a marker of differentiation in human testicular Leydig cells. Recently, we have identified differential expression of human INSL-3 in neoplastic Leydig cells and mammary epithelial cells suggesting an involvement of INSL-3 in tumor biology. Here we have investigated the expression of INSL-3 in human thyroid carcinoma cell lines and in the human thyroid gland which has been shown to express transcripts for the G protein coupled INSL-3 receptor LGR8. When we determined the expression of INSL-3 in eight human thyroid carcinoma cell lines, a novel INSL-3 splice variant containing a 95 bp out-of-frame insertion at the beginning of exon II of the INSL-3 gene was discovered. Treatment of the human anaplastic thyroid carcinoma cell line 8505C with diethylstilbestrol (DES) caused a significant dose-dependent transcriptional down-regulation of INSL-3 and a marked up-regulation of LGR8. Employing in situ hybridization to detect INSL-3 transcripts and specific rabbit antisera against the INSL-3 proteins, both INSL-3 isoforms were detected in patients with Graves' disease (n=10), follicular carcinomas (FTC; n=12), papillary carcinomas (PTC; n=9) and undifferentiated anaplastic carcinomas (UTC; n=15). By contrast, thyrocytes of all 15 benign goiter tissues studied were devoid of both INSL-3 isoforms, mRNA and protein. Our data indicate that INSL-3 hormone is up-regulated in hyperplastic and neoplastic human thyrocytes suggesting that the INSL-3 isoforms may serve as additional markers for hyperplastic and neoplastic human thyrocytes. In the anaplastic thyroid carcinoma cell line 8505C, the regulation of both INSL-3 and LGR8 by estrogen may be the first indication of a novel hormonally responsive, auto-/paracrine INSL-3 LGR8 ligand receptor system active in human thyroid carcinoma cells.

Alternative Splicing↗

PubMedDate not supplied

Lineage-specific effects of polychlorinated biphenyls (PCB) on gene expression in the rabbit blastocyst.

The ubiquitous toxic environmental contaminants polychlorinated biphenyls (PCBs) change gene expression in preimplantation embryos. Cell lineage-specific effects of PCB are not known. Rabbit day 6 blastocysts were exposed in vitro to low (0.1 ng/congener/mL medium) and high (1 microg) PCB levels of coplanar (PCB 77, 126, and 169) or non-coplanar PCBs (PCB 28, 52, 101, 118, 138, 153, and 180). Embryoblast (ICM) and trophoblast cells (TE) were separated and analyzed for transcriptional changes of PCB-and implantation-associated genes by semiquantitative RT-PCR. PCBs increased CYP 1A1 mRNA only in the ICM. CYP 1B1, VEGFR2, and COX-2 transcript numbers were elevated in both ICM and TE. Transcripts for HIF-1alpha were decreased in the ICM. No obvious differences in gene expression following exposure to coplanar and non-coplanar PCBs were detected. Our results show that transcriptional responses to PCBs differ between the cell lineages of the rabbit blastocyst, indicating that PCBs can influence the highly sensitive process of early mammalian development.

Animals↗