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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Numerische Mathematik 12 (1968), S. 416-428 
    ISSN: 0945-3245
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mathematics
    Notes: Abstract Calculation in unnormalized arithmetic provides, as has been described elsewhere, an estimate of the error of each quantity, initial, intermediate, and final, in a calculation. In an acceptable computational algorithm, this error estimate should be close to the true probable error. (The probable error is defined as follows: each computed quantity is defined, in the statement of the problem, explicitly or implicitly as some function of the input data, which are however known only up to errors having some distribution. As the input data vary in their joint distribution, the value of this function varies in a distribution whose variance determines the probable error of the result.) Many computational algorithms fail to achieve this goal, because whenever two numbers are combined arithmetically (+, -, ×, or ÷) in a computer, anycorrelation of the errors of the two numbers is perforce ignored; in consequence, the error estimate of a computed result may be greater than, comparable with, or smaller than the probable error, in which case the algorithm is called “conservative”, “faithful”, or “liberal”, respectively. In this article, a matrix-inversion algorithm based on an assignment strategy is described, which is very close to faithful, as evidenced by extensive numerical tests, which are also described. A test matrix has elements with varying numbers of leading zeros, the remaining bits in a 43-bit pattern are regarded as meaningful; its inverse is computed. A series of perturbed matrices are formed by adding or subtracting a one-bit in the 31st-stage of each input matrix element; this corresponds to amaximum change in the last twelve bits; the statistical aspect is then in the choice of addition-subtraction. By comparing the results of the successive inversions, the magnitude of the probable error (as defined above) resulting from these perturbations can be rather closely determined. (These tests cover the case in which the errors of the input matrix elements are uncorrelated.) If the calculation is regarded as one in which the 30th bit is the last significant one and the remaining 13 bits are guard digits, then, for a faithful algorithm, the observed probable error should appear starting in the 31st place. This is found to be the case quite accurately for the algorithm here proposed, but not for other commonly used algorithms. It should be pointed out that the algorithm here described is optimal also for normalized arithmetic (in which errors are ignored), because the precision on the one hand cannot be increased by retaining insignificant digits and on the other hand is in any case limited by the true probable error, no matter what algorithm is used (unless the input data are infinitely precise). However, the results are likely to be less satisfactory in normalized arithmetic, because explicit use is made of the relative precisions of certain quantities at each pivoting in the matrix inversion, and the machine indication of this relative precision is falsified when numbers are unduly normalized.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Biochemical genetics 27 (1989), S. 153-166 
    ISSN: 1573-4927
    Keywords: Aspergillus nidulans ; DNase A ; deoxyribonuclease secretion ; phosphate repression ; regulator genepalcA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract High levels of nuclease activities were identified in filtrates ofAspergillus cultures after growth in low- but not in high-phosphate media. Deoxyribonuclease activities, characterized extensively by column chromatography, showed a coincident single peak for ss- and ds-DNase which was distinct from the peak for RNase. Both ss-DNase and ds-DNase are endonucleolytic and showed the highest activity in the presence of Ca2+ and Mn2+ (atpH 8.0). They also showed identical heat sensitivities suggesting that a single, phosphate-repressible DNase was secreted. This enzyme, therefore, corresponds to the well-characterized extracellular DNase A ofNeurospora. However, theAspergillus DNase A did not cross-react with antisera to secretedNeurospora nucleases and showed different chromatographic properties, and active peptides of different sizes were visualized on DNA activity gels. The increasing derepression ofAspergillus DNase A by decreasing phosphate levels was similar to that of secreted alkaline phosphatase and these increases were both abolished by the regulatory mutantpalcA.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Biochemical genetics 27 (1989), S. 153-166 
    ISSN: 1573-4927
    Keywords: Aspergillus nidulans ; DNase A ; deoxyribonuclease secretion ; phosphate repression ; regulator genepalcA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract High levels of nuclease activities were identified in filtrates ofAspergillus cultures after growth in low- but not in high-phosphate media. Deoxyribonuclease activities, characterized extensively by column chromatography, showed a coincident single peak for ss- and ds-DNase which was distinct from the peak for RNase. Both ss-DNase and ds-DNase are endonucleolytic and showed the highest activity in the presence of Ca2+ and Mn2+ (atpH 8.0). They also showed identical heat sensitivities suggesting that a single, phosphate-repressible DNase was secreted. This enzyme, therefore, corresponds to the well-characterized extracellular DNase A ofNeurospora. However, theAspergillus DNase A did not cross-react with antisera to secretedNeurospora nucleases and showed different chromatographic properties, and active peptides of different sizes were visualized on DNA activity gels. The increasing derepression ofAspergillus DNase A by decreasing phosphate levels was similar to that of secreted alkaline phosphatase and these increases were both abolished by the regulatory mutantpalcA.
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  • 4
    ISSN: 1573-675X
    Keywords: Apoptosis ; caspase ; etoposide ; hydroxychloroquine ; nuclease.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Apoptosis induced by etoposide (VP-16) in HL-60 cells was confirmed to be caspase-dependent. It was fully inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk. However, the caspase-3-specific inhibitor Z-DEVD-fmk only partially inhibited apoptosis. This indicated that a second caspase is required in vivo for full activation of the apoptotic nucease CAD. Aurin tricarboxylic acid (ATA) did not inhibit VP-16-induced apoptosis. In contrast, apoptosis induced by hydroxychloroquine (HCQ) in HL-60 cells was caspase-3 independent and was fully inhibited by ATA. Thus, CAD does not appear to be involved in chromatin DNA degradation in this case. A second apoptotic nuclease is postulated to degrade the DNA, likely endo-exonuclease, an abundant nuclear enzyme that acts on both DNA and RNA and is present in latent form. HCQ, but not VP-16, stimulated DNA degradation (“laddering”) in isolated nuclei. This indicates that the drug can act directly in the nuclei to trigger activation of the second latent apoptotic nuclease.
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  • 5
    ISSN: 1573-675X
    Keywords: apoptosis ; caspase-3 ; nuclease ; endo-exonuclease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Single-strand DNase and poly rAase, activities characteristic of endo-exonuclease, were co-activated in nuclear fractions of HL-60 cells by caspase-3. Activation was accompanied by cleavages of large soluble polypeptides (130–185 kDa) and a 65 kDa inactive chromatin-associated polypeptide related to the endo-exonuclease of Neurospora crassa as detected on immunoblots. The major products seen in vitro were a 77 kDa soluble polypeptide and an active chromatin-associated 34 kDa polypeptide. When HL-60 cells were induced to undergo apoptosis by treating with 50 μM etoposide (VP-16) for 4 hours, 77 kDa and 40 kDa polypeptides accumulated in nuclear fractions. Chromatin DNA fragmentation activity was also activated in cytosol and nuclear extract either by pre-treating the cells in vivo with VP-16 or by treating the cytosol in vitro with caspase-3 or dATP and cytochrome c. Endo-exonuclease activated by caspase-3 in cytosol-derived fractions augmented chromatin DNA fragmentation activity in vitro. Endo-exonuclease is proposed to act in vivo in conjunction with the caspase-activated DNase (CAD) to degrade chromatin DNA during apoptosis of HL-60 cells.
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  • 6
    ISSN: 1573-675X
    Keywords: Apoptosis ; etoposide ; hydroxychloroquine ; mitochondria ; nuclease ; transmembrane potential.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Since mitochondrial factors have been implicated in apoptosis, experiments were designed to assess whether or not the potent mitochondrial nuclease could be one of these factors. Nuclei isolated by two different methods were found to contain mitochondrial nuclease in masked form. This nuclease was released by treatment with the non-ionic detergent NP-40 and rendered trypsin-sensitive. It was not removed appreciably from the nuclei by washing and sedimentation of the nuclei through a sucrose cushion. Levels of the mitochondrial nuclease were followed during drug-induced apoptosis. Time courses of apoptosis in cultures of HL-60 cells were monitored by flow cytometry of propidium iodide-stained cells and by agarose gel electrophoresis of extracted DNA. Changes in the inner mitochondrial transmembrane potential were monitored by flow cytometry of chloromethyl-X-Rosamine-stained cells. Apoptosis was induced by treatment with either the chemotherapeutic agent etoposide (VP-16 at 10 μM) over an 8 h period or with the anti-rheumatic agent hydroxychloroquine (HCQ at 0.28 mM) over a 24 h period. These two drugs likely act in different pathways of apoptosis. VP-16 caused loss of the mitochondrial transmembrane potential 1.0–1.5 h before apoptosis was detected. On the other hand, treatment with HCQ caused these processes to occur in parallel possibly indicating that the mitochondrial changes are secondary events. No losses of masked mitochondrial nuclease were detected with either drug treatment during the course of apoptosis. HL-60 mitochondrial DNA was also not degraded during apoptosis induced by either agent. These observations likely explain why the mitochondrial DNA is not degraded and make it unlikely that mitochondrial nuclease plays any role in vivo in chromatin DNA fragmentation.
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  • 7
    ISSN: 1573-6857
    Keywords: piggyBac ; Lepidopteran ; transposable element
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The piggyBac Lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured TN-368 cells. We have constructed genetically tagged piggyBac elements that permit analysis of excision when transiently introduced on plasmids into the piggyBac-deficient Spodoptera frugiperda IPLB-SF21AE cell line. Precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumably supplies the piggyBac transposase. The results suggest that the piggyBac transposon encodes a protein that functions to facilitate not only insertion, but precise excision as well. This is the first demonstration of piggyBac mobility from plasmid sources in uninfected Lepidopteran cells.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Methods in cell science 12 (1989), S. 7-11 
    ISSN: 1573-0603
    Keywords: transfection ; baculovirus ; nuclear polyhedrosis virus ; insect cell cultures
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Several protocols are presented for preparation and transfection of Baculovirus and plasmid DNAs into Lepidopteran insect cells using the calcium-phosphate co-precipitation technique. Important parameters for optimum efficiency include the inherent susceptibility of the recipient cell line for transfection, and the method of preparation of viral and plasmid DNAs. The protocols presented provide reproducible high efficiencies for transfection of several Lepidopteran cell lines.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Methods in cell science 7 (1982), S. 43-46 
    ISSN: 1573-0603
    Keywords: plaque assay ; insect cells ; nuclear polyhedrosis virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 0.75% agarose overlay procedure is described which has the advantages of being easy to prepare and perform, and is applicable to several commonly used insect cell lines and viruses. In addition, plaque variants which do not produce polyhedra can be easily detected.
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  • 10
    ISSN: 1435-0653
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Type of Medium: Electronic Resource
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