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  • 1
    Publication Date: 2008-11-16
    Description: The pathogenesis of osteonecrosis of the jaws (ONJ) in patients treated with intravenous bisphosphonates is still not completely understood, and likely involves both reduction in blood supply and the activity of oral bacteria. We present an animal model of ONJ, which will significant contribute to the understanding of the aetiopathogenesis of this condition. Five skeletally-mature female Wistar rats (weight 450 g) were given intravenous zoledronic acid 0.04 mg in saline solution (0.2 mg/ml) once a week for 5 weeks. After 2 weeks, the animals underwent the extraction of the upper right first molar, followed by the creation of a 4 mm-diameter bone defect on the same site under general anaesthesia. After 7 weeks from the extraction, the animals were clinically examined and a static bone-scintigraphy using 99mTc-MDP methylene diphosphonate was performed with a gamma camera equipped with pinhole collimator (Siemens-Ecam). After an additional week, the rats were sacrificed and a Computed Tomography (CT) was carried out. Samples obtained from the bone defect were decalcified and prepared for histological assessment. Five rats, not treated with zoledronic acid and exposed to the same surgical treatment, were used as controls. Histological and imaging features were assessed blindly. At 7 weeks after the extraction, all the rats treated with zoledronic acid showed impair healing, expansion of the defect and bone exposure. These features were confirmed by the scintigraphy, which showed abnormal localized activity in comparison with the surrounding tissues. On clinical examination, the rats of the control group demonstrated epithelialization of the bone defect and a normal uptake of the contrast medium during the scan. The CT scan disclosed irregularity of the cortical margin and destruction of the cortical bone, which were not evident in the control group. The histology showed sheets of necrotic bone, with loss of osteocytes from their lacunae and peripheral resorption. No inflammatory infiltrate was observed. The control group instead demonstrated normal bone healing. All animals were treated according with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Based on this study, the rat treated with zoledronic acid can be considered a novel, reliable and reproducible model to better understand the pathophysiology of ONJ and to develop a therapeutic approach.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
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  • 2
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated “CpG island” at the 5′-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 220 (2003), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The human immunodeficiency virus (HIV-1) (transactivator of transcription (Tat)) protein is a pleiotropic factor that induces a broad range of biological effects in numerous cell types. At the HIV promoter, Tat is a powerful transactivator of gene expression, which acts by both inducing chromatin remodeling and by recruiting elongation-competent transcriptional complexes onto the viral LTR. Besides these transcriptional activities, Tat is released outside the cells and interacts with different cell membrane-associated receptors. Finally, extracellular Tat can be internalized by cells through an active endocytosis process. Here we discuss some of the molecular mechanisms involved in intracellular and extracellular Tat function.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 79 (2001), S. 3353-3355 
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: A single-biomolecule optical toggle switch is demonstrated based on a mutated green fluorescent protein (GFP). We have exploited molecular biology techniques to tailor the GFP molecular structure and photophysical properties and to give it optically controlled bistability between two distinct states. We present optical control of the fluorescence dynamics with two laser beams at 476 and 350 nm down to the ultimate limit of single molecules. These results indicate that GFP-class fluorophores are promising candidates for the realization of biomolecular devices such as volumetric optical memories and optical switches. © 2001 American Institute of Physics.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Genetica 94 (1994), S. 255-266 
    ISSN: 1573-6857
    Keywords: DNA replication ; origin ; competitive polymerase chain reaction ; transcription ; lamin B2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Attempts at identifying DNA replication origins in human cells have been performed with a variety of molecular genetic and biochemical approaches, with often controversial results. The combination of bromodeoxyuridine labelling, immunopurification of newly synthesized labelled DNA, measurement of the relative abundance of markers in this DNA by quantitative competitive PCR, has allowed the identification within 450 bp of the start-site of DNA replication located at the human lamin B2 gene. The origin is located near the non-transcribed spacer between two highly transcribed genes and shows evidence of a number of specific protein-DNA interactions, the most prominent of which disappears when the cells are differentiated into a non-proliferating state.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Genetica 106 (1999), S. 125-130 
    ISSN: 1573-6857
    Keywords: chromatin ; transcription ; HIV-1 ; Tat ; histone acetyltransferase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Upon infection of susceptible cells, the RNA genome of the human immunodeficiency virus type 1 (HIV‐1) is reverse transcribed into double-stranded DNA, which can be subsequently integrated into the cellular genome. After integration, the viral long terminal repeat (LTR) promoter is present in a nucleosome-bound conformation and is transcriptionally silent in the absence of stimulation. Activation of HIV-1 gene expression is concomitant with an acetylation-dependent rearrangement of the nucleosome positioned at the viral transcription start site. Thus, similar to most cellular genes, the transcriptional state of the integrated HIV-1 provirus is closely linked to histone acetylation. This enzymatic activity results from the function of histone-specific nuclear acetyltransferase (HAT) enzymes. Efficient viral transcription is strongly dependent on the virally-encoded Tat protein. The mechanism by which Tat increases the rate of transcriptional initiation has been recently demonstrated and involves the interaction of Tat with the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase activity.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 0730-2312
    Keywords: nuclear matrix ; replication origin ; topoisomerase II-mediated DNA loop excision ; DNA loop anchorage sites ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions. J. Cell. Biochem. 69:13-18, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Publication Date: 2015-08-24
    Description: Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors’ broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 9
  • 10
    Publication Date: 2015-12-30
    Description: Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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