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  • 1
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Journal of chemical & engineering data 10 (1965), S. 386-386 
    ISSN: 1520-5134
    Source: ACS Legacy Archives
    Topics: Chemistry and Pharmacology , Process Engineering, Biotechnology, Nutrition Technology
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  • 2
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We screened the putative rap-phr (response regulator aspartyl-phosphate phosphatase-phosphatase regulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE (alkaline protease gene) expression by using a multicopy plasmid. We found that rapG was involved in the regulation of aprE, which belongs to the regulon of DegU, the response regulator of the DegS-DegU two-component system. Disruption of rapG and phrG resulted in enhancement and reduction of aprE-lacZ expression, respectively, suggesting that PhrG inhibits RapG activity. Addition of 1–30 nM of a synthetic pentapeptide (PhrG; NH2-EKMIG-COOH) to the phrG disruptant completely rescued aprE-lacZ expression, indicating that the PhrG peptide is indeed involved in aprE-lacZ expression. Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100–300 nM) to the phrG cells decreased aprE-lacZ expression. These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA-lacZ expression directed by ComA, the regulator of the ComP-ComA two-component system. Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of DegU-P (phosphorylated DegU) in vitro. RapG, however, did not affect dephosphorylation of DegU-P under the adopted experimental conditions. Therefore, we hypothesized that RapG might inhibit the binding activity of DegU to the target promoters. We analysed the interaction of DegU and RapG using the aprE promoter and another target, a comK promoter. Gel shift analysis revealed that RapG served as the inhibitor of DegU binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 115 (1994), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract A Bacillus subtilis response regulator, DegU9, carrying an amino acid alteration caused by the degU9(Hy) mutation was partially purified, and phosphorylation and dephosphorylation of the protein was studied. The extent of phosphorylation was not as high as the level attained with wild-type DegU, but the DegU9-phosphate once formed was more stable than the wild-type DegU-phosphate. An in vivo study with a degU9 mutant showed that degS was necessary for the overproduction of exoproteases. These results suggest that phosphorylation is necessary for the mutant DegU9 to exert its effect and that the higher stability of phosphorylated DegU9 is responsible for the overproulation phenotype.
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  • 5
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 175 (1979), S. 235-237 
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r-m-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec + strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r+ or r- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r-m- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.
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  • 7
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Adrenergic stimulation induces contraction of hypertrophied prostatic tissue via the α1 adrenoceptor, and the results of pharmacological studies suggested the existence of adrenoceptor subtypes. Recently three subtypes (α1a, α1b, and α1d) were cloned. Using probes for these subtypes, we demonstrated their expression in the tissues of ten cases of benign prostatic hypertrophy, usingin situ hybridization. To determine the ratio between these subtypes, an RNase protection assay was also performed in three cases. Expression of the α1a and α1d adrenoceptors was diffuse in the smooth muscles of the interstitium, but was absent in glandular epithelial cells. On the contrary, the α1b adrenoceptor was hardly detectable. The RNase protection assay confirmed the absence of the α1b adrenoceptor, the ratio of α1a and α1d being 4∶1. These results supported the idea that the differences in prostatic contractile response to several adrenergic drugs are based on the affinities of these drugs for the different subtypes.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 223 (1990), S. 268-272 
    ISSN: 1617-4623
    Keywords: Neomycin ; Chloramphenicol ; Recombination ; Cotransformation ; Negative selection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB −, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB + pro+)→(leuB: : cat pro +)→ (leuB − pro: : neo)→(leuB − pro +) resulted in a leuB − single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD − and pro −, as well as the double mutation leuB − pro − can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.
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  • 9
    Electronic Resource
    Electronic Resource
    Hoboken, NJ : Wiley-Blackwell
    Journal of Biomedical Materials Research 30 (1996), S. 523-533 
    ISSN: 0021-9304
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: Bone-titanium contact was examined in young and mature rats on various days after insertion of pure titanium into the tibia. Under light microscopy, on the 14th day, lamellar mature bone was initially formed, and was seen to make direct contact with the titanium in both groups. In young rats on the 28th day, bone-titanium contact was greater than that in mature animals. On 1-μm sections, an amorphous zone 0.5-1.0 μm thick was found around the titanium, and a slender cell layer lay parallel to the implant, forming the superficial layer of the amorphous zone. Ultrastructurally, these slender cells were identified as osteoblastlike cells and made direct contact with the implant via a 20-50-nm thin amorphous zone. Below this cell layer, a collagen-containing, poorly mineralized zone was present and bordered by lamellar bone with a lamina limitans-like structure. However, this cell layer was absent in places, and therefore the thick amorphous zone without slender cell layer consisted ultrastructurally of a 20-50-nm thin amorphous zone and a poorly mineralized zone bordered by the lamellar bone. Sometimes this poorly mineralized zone was absent, and in such cases, the lamellar bone contacted the titanium by the thin amorphous zone formed on the lamina limitans-like structure. Thus, although bone was seen to make contact with the titanium implant, ultrastructurally a 20-50-nm thin amorphous zone, a slender cell layer, and/or a poorly mineralized zone were interposed between the bone and titanium. © 1996 John Wiley & Sons, Inc.
    Additional Material: 17 Ill.
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  • 10
    ISSN: 0021-9304
    Keywords: titanium ; osteocalcin ; osteopontin ; bone-titanium interface ; amorphous zone ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine , Technology
    Notes: This study was designed to investigate by postembedding immunogold method the localization and distribution of osteocalcin (Ocl) and osteopontin (Opn) at the bone-titanium interface in rat tibiae 14 and 28 days postimplantation to determine which bone proteins are present at this interface. Both proteins were widely distributed on the newly formed bone and accumulated predominantly in the region of bone close to the titanium, in electron-dense patches in the bone, and at the osteocytic lacunae. Collagenous osteoid showed little or no labeling for either Ocl or Opn. An amorphous zone (20-50 nm) was interposed between the titanium and interfacial slender cells, osteoid, or bone, and was labeled strongly for Ocl but only weakly for Opn. Furthermore, a second electron-dense layer, the lamina limitans, which faces the titanium, was labeled strongly for Opn but weakly for Ocl. Ocl as a marker protein of osteoblasts was sometimes found in the granules and vesicles of the interfacial cells and extracellularly in their intercellular spaces, close to the titanium. However, Opn was not detected in any granules. This is the first report to show that the amorphous zone contains large amounts of Ocl and small amounts of Opn, and that bone contacts titanium through this Ocl-rich amorphous zone. Furthermore, it is suggested that the interfacial cells seem to be osteoblasts, and that Ocl in the amorphous zone is produced and secreted by these cells and functions with Opn as a regulator of the mineralization front close to the titanium, and as a mediator of cell-matrix and matrix-matrix/mineral adhesion along the titanium. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 41, 111-119, 1998.
    Additional Material: 10 Ill.
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