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Biomedical subjects

A Zimmer

Publications and source records attributed to A Zimmer.

At least 73 records · Page 4Linked to original sources
PubMed1998

POU transcription factors control expression of CNS stem cell-specific genes.

Multipotential stem cells throughout the developing central nervous system have common properties. Among these is expression of the intermediate filament protein nestin and the brain fatty acid binding protein (B-FABP). To determine if common mechanisms control transcription in CNS stem cells, the regulatory elements of these two genes were mapped in transgenic mice. A 257 basepair enhancer of the rat nestin gene is sufficient for expression throughout the embryonic neuroepithelium. This enhancer contains two sites bound by the class III POU proteins Brn-1, Brn-2, Brn-4, and Tst-1. Only one of the two POU sites is required for CNS expression. An adjacent hormone response element is necessary for expression in the dorsal midbrain and forebrain. The regulatory sites of the B-FABP gene are strikingly similar to those of the nestin gene. A hybrid POU/Pbx binding site is recognized in vitro by Pbx-1, Brn-1 and Brn-2. This site is essential for expression in most of the CNS. In addition, a hormone response element is necessary for forebrain expression. Both the nestin and B-FABP genes therefore depend on POU binding sites for general CNS expression, with hormone response elements additionally required for activity in the anterior CNS. These data indicate that regulation by POU proteins and hormone receptors is a general mechanism for CNS stem cell-specific transcription.

Animals↗
PubMed1997

Discrimination and association processes for faces and non-faces: the effect of rotation.

Most current theories of face perception claim that inversion leaves the coding of non-face stimuli largely unaffected, while causing a qualitative change in the coding of faces. Empirical support for this hypothesis mainly stems from recognition studies which typically show a larger inversion decrement for faces than for other stimuli. Several recent studies using experimental paradigms that do not contain a substantial memory component have however yielded contradicting results. This observation suggests that the disproportionate effect of inversion for faces might be related to the presence, or absence, of a memory component in the experimental task. In order to explore this hypothesis we investigated the effect of inversion within a discrimination learning paradigm, which contains a memory component comparable to that included in a recognition paradigm. We compared the effect of rotation on discrimination and association processes for faces and cars, Subjects learned to discriminate pairs of similar faces and similar cars and to associated them with neutral responses. The stimulus pairs were presented upright, inverted, and additionally in two intermediate orientations. We found that discrimination performance was generally better for faces than for cars and that associations were learned faster for faces than for cars. However, we did not find any evidence that rotation affected discrimination and association processes for faces differently than for cars. In this sense, our results provide no evidence for the hypothesis that memory processes are responsible for disproportionate effect of inversion which is found in recognition experiments.

Adult↗
PubMed1997

Endothelial apoptosis in Braf-deficient mice.

Tyrosine kinase growth factor receptors and Ras/Raf/MEK/MAPK signalling have been implicated in the suppression as well as augmentation of programmed cell death. In addition, a Ras-independent role for Raf as a suppressor of programmed cell death has been suggested by the recent finding that Craf1 interacts with members of the Bcl-2 family at mitochondrial membranes. However, genetic studies of C. elegans and Drosophila, as well as the targeted mutagenesis of the murine Araf gene, have failed to support such a role. Here we show that mice with a targeted disruption in the Braf gene die of vascular defects during mid-gestation. Braf -/- embryos, unlike Araf -/- or Craf1 -/- embryos (L.W. et al., unpublished), show an increased number of endothelial precursor cells, dramatically enlarged blood vessels and apoptotic death of differentiated endothelial cells. These results establish Braf as a critical signalling factor in the formation of the vascular system and provide the first genetic evidence for an essential role of Raf gene in the regulation of programmed cell death.

Animals↗
PubMed1997

Cationic polyhexylcyanoacrylate nanoparticles as carriers for antisense oligonucleotides.

After antisense oligodeoxynucleotides (ODNs) were suggested for therapeutic use in 1978, major advances were made in developing modified oligonucleotides with increased nuclease resistance and improved cellular uptake. In the present report, positively charged nanoparticles prepared from diethylaminoethyl (DEAE)-dextran and polyhexylcyanoacrylate (PHCA) were evaluated as carriers for ODNs. The oligonucleotides were analyzed by anion-exchange HPLC. The nanoparticles exhibited a high loading capacity, with approximately 35 mumol ODNs adsorbed per gram of polymeric material. The adsorption efficacy was found to be dependent on the pH, on the ionic strength of the medium, and on the amount of DEAE-dextran. Highest loading for ODNs was achieved at pH 5.5, using a 10 mM phosphate buffer. Oligonucleotides adsorbed to the surface of the nanoparticles were nearly completely protected against degradation by the endonuclease DNase I and under in vitro cell culture conditions, whereas unprotected ODNs were totally digested under these conditions. Nanoparticles led to a 20-fold increase in cellular uptake of FITC-oligonucleotides. The internalized oligonucleotides were frequently localized as vesicular structures in the cytoplasmatic compartment. Because of their temperature-dependent uptake, we propose an active uptake mechanism, such as endocytosis, for the internalization of the ODN-nanoparticle formulations.

Animals↗
PubMed1997

Retinoid-induced chromatin structure alterations in the retinoic acid receptor beta2 promoter.

Transcription of the retinoic acid receptor beta2 (RARbeta2) gene is induced by retinoic acid (RA) in mouse P19 embryonal carcinoma (EC) cells. Here we studied RA-induced chromatin structure alterations in the endogenous RARbeta2 promoter and in an integrated, multicopy RARbeta2 promoter in EC cells. RA markedly increased restriction site accessibility within the promoter, including a site near the RA responsive element (RARE) to which the nuclear receptor retinoid X receptor (RXR)-RAR heterodimer binds. These changes coincided with RA-induced alterations in the DNase I hypersensitivity pattern in and around the promoter. These changes became undetectable upon removal of RA, which coincided with the extinction of transcription. Analyses with receptor-selective ligands and an antagonist showed that increase in restriction site accessibility correlates with transcriptional activation, which parallels the RA-induced in vivo footprint of the promoter. Despite these changes, the micrococcal nuclease digestion profile of this promoter was not altered by RA. These results indicate that concurrent with the binding of the RXR-RAR heterodimer to the RARE, the local chromatin structure undergoes dynamic, reversible changes in and around the promoter without globally affecting the nucleosomal organization.

Animals↗
PubMed1996

Pain responses, anxiety and aggression in mice deficient in pre-proenkephalin.

Enkephalins are endogenous opioid peptides that are derived from a pre-proenkephalin precursor protein. They are thought to be vital in regulating many physiological functions, including pain perception and analgesia, responses to stress, aggression and dominance. Here we have used a genetic approach to study the role of the mammalian opioid system. We disrupted the pre-proenkephalin gene using homologous recombination in embryonic stem cells to generate enkephalin-deficient mice. Mutant enk-/- animals are healthy, fertile, and care for their offspring, but display significant behavioural abnormalities. Mice with the enk-/- genotype are more anxious and males display increased offensive aggressiveness. Mutant animals show marked differences from controls in supraspinal, but not in spinal, responses to painful stimuli. Unexpectedly, enk-/- mice exhibit normal stress-induced analgesia. Our results show that enkephalins modulate responses to painful stimuli. Thus, genetic factors may contribute significantly to the experience of pain.

Aggression↗
PubMed1996

Understanding mammary gland development through the imbalanced expression of growth regulators.

Functional differentiation of mammary tissue progresses in distinct phases spanning puberty and pregnancy. Here we have analyzed and compared the effects of transforming growth factor beta 1 (TGF beta 1), TGF alpha, and whey acidic protein (WAP), the Notch-related cell fate protein Int3, and p53 and pRb on mammary development. We chose transgene expression from the WAP gene promoter which is only active in mammary alveolar cells. The imbalanced expression of these molecules specifically altered development and differentiation of the gland. While TGF alpha did not disturb alveolar outgrowth, little or no alveolar structures developed in the presence of Int3. TGF beta 1, WAP, and the expression of SV40 T-antigen-which inactivates p53 and PRb-reduced overall alveolar development. The expression of individual milk protein genes was affected differentially by the transgenes. A WAP-lacZ transgene served as an additional indicator of terminal differentiation of alveolar cells, Homogeneous expression of lacZ was seen in mice transgenic for lacZ, or for TGF alpha and lacZ. In contrast, only a few differentiated cells were observed in the presence of TGF beta 1 and Tag. Thus, the expression of growth regulators in the same defined subset of mammary cells results in distinct developmental changes and a specific pattern of alveolar differentiation.

Animals↗
PubMed1996

Embryonic expression of glial cell-line derived neurotrophic factor (GDNF) suggests multiple developmental roles in neural differentiation and epithelial-mesenchymal interactions.

We describe the cloning of the mouse glial cell line-derived neurotrophic factor (GDNF) gene and its expression during embryogenesis. GDNF is a distant member of the superfamily of TGF-beta related genes that was originally identified on the basis of its striking neurotrophic activity. GDNF is expressed in a highly dynamic pattern in the anterior neuroectoderm during early stages of neurogenesis between E7.5 and E10.5. Beginning at E10.5 GDNF is also expressed in several organs that develop through inductive epithelial-mesenchymal interactions. In those organs, GDNF expression is strictly confined to mesenchymal tissues and is not found in epithelia. Our results suggest multiple roles for GDNF during early stages of neuronal development and in epithelial-mesenchymal interactions.

Amino Acid Sequence↗
PubMed1996

Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region.

Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR beta 2 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111-1119). To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions. uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major ORF.

Amino Acid Sequence↗
PubMed1994

Gene targeting constructs: effects of vector topology on co-expression efficiency of positive and negative selectable marker genes.

Targeting of DNA into specific chromosomal loci often involves the use of a negative selectable marker gene to enrich for cell clones that have undergone homologous recombination. In this study we asked if the arrangement of the positive and negative markers in 'knockout' constructs can influence the expression of a lacZ gene that was used as a negative marker. We show that constructs which differ only in their topology have vastly different co-expression efficiencies. The site of DNA linearization was critical. While linearizing at the 5'-end of the lacZ gene was compatible with efficient lacZ expression, linearizing at the 3'-end was always detrimental. We also demonstrate that the topology of the template was more important than the promoter used to drive lacZ expression.

Animals↗
PubMed1994

Pharmacokinetic and pharmacodynamic aspects of an ophthalmic pilocarpine nanoparticle-delivery-system.

The regional pharmacokinetics as well as the pharmacodynamics of pilocarpine-loaded nanoparticles for the treatment of glaucoma were investigated and compared to a solution of this drug. Polybutylcyanoacrylate nanoparticles were prepared by an emulsion polymerization process. Formulations with different drug concentrations (2-6%) as well as different particle concentrations were investigated and analyzed for size and drug loading. Drug binding to the particles was achieved at a level of 10-18% of the total drug content. The colloidal nanoparticles were sufficiently small (diameter: 100-300 nm) for a non-irritating application to the eye. All preparations were applied to the eyes of New Zealand white rabbits which were treated with betamethasone before to create an elevated intraocular pressure (IOP). Pilocarpine concentrations, assayed from aqueous humor using gaschromatography, increased by 23% (AUC) for nanoparticle suspensions compared to aqueous reference solutions. Additionally, t1/2 was prolonged and the elimination coefficient was significantly decreased. Pharmacodynamic effects such as miosis and IOP reduction were investigated. tmax values of aqueous humor concentration were observed to be in a similar time range as miosis tmax readings. It was found that at lower drug contents a more pronounced prolongation of miosis was achieved with nanoparticles versus a standard solution. The IOP-reduction was significantly prolonged with nanoparticles preparations; whereas maximum reduction was obtained with a reference solution after 1-2 hours, it was reached with nanoparticles at about 2-3 hours. Differences between nanoparticles and aqueous solutions were most pronounced at lower drug concentrations.

Animals↗
PubMed1994

Tissue specific expression of the retinoic acid receptor-beta 2: regulation by short open reading frames in the 5'-noncoding region.

The 40-S subunit of eukaryotic ribosomes binds to the capped 5'-end of mRNA and scans for the first AUG in a favorable sequence context to initiate translation. Most eukaryotic mRNAs therefore have a short 5'-untranslated region (5'-UTR) and no AUGs upstream of the translational start site; features that seem to assure efficient translation. However, approximately 5-10% of all eukaryotic mRNAs, particularly those encoding for regulatory proteins, have complex leader sequences that seem to compromise translational initiation. The retinoic-acid-receptor-beta 2 (RAR beta 2) mRNA is such a transcript with a long (461 nucleotides) 5'-UTR that contains five, partially overlapping, upstream open reading frames (uORFs) that precede the major ORF. We have begun to investigate the function of this complex 5'-UTR in transgenic mice, by introducing mutations in the start/stop codons of the uORFs in RAR beta 2-lacZ reporter constructs. When we compared the expression patterns of mutant and wild-type constructs we found that these mutations affected expression of the downstream RAR beta 2-ORF, resulting in an altered regulation of RAR beta 2-lacZ expression in heart and brain. Other tissues were unaffected. RNA analysis of adult tissues demonstrated that the uORFs act at the level of translation; adult brains and hearts of transgenic mice carrying a construct with either the wild-type or a mutant UTR, had the same levels of mRNA, but only the mutant produced protein. Our study outlines an unexpected role for uORFs: control of tissue-specific and developmentally regulated gene expression.

Amino Acid Sequence↗
PubMed1994

The expression pattern of a novel gene encoding brain-fatty acid binding protein correlates with neuronal and glial cell development.

Fatty acid binding proteins (FABPs) are a multigene family of small intracellular proteins that bind hydrophobic ligands. In this report we describe the cloning and expression pattern of a novel member of this gene family that is specifically expressed in the developing and adult nervous system and thus was designated brain (B)-FABP. B-FABP is closely related to heart (H)-FABP with 67% amino acid identity. B-FABP expression was first detected at mouse embryonic day 10 in neuroepithelial cells and its pattern correlates with early neuronal differentiation. Upon further development, B-FABP was confined to radial glial cells and immature astrocytes. B-FABP mRNA and protein were found in glial cells of the peripheral nervous system such as satellite cells of spinal and cranial ganglia and ensheathing cells of the olfactory nerve layer from as early as embryonic day 11 until adulthood. In the adult mouse brain, B-FABP was found in the glia limitans, in radial glial cells of the hippocampal dentate gyrus and Bergman glial cells. These findings suggest a function of B-FABP during neurogenesis or neuronal migration in the developing nervous system. The partially overlapping expression pattern with that of cellular retinoid binding proteins suggests that B-FABP is involved in the metabolism of a so far unknown hydrophobic ligand with potential morphogenic activity during CNS development.

Amino Acid Sequence↗