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  • 1
    ISSN: 0173-0835
    Keywords: Trypanosoma brucei ; Free-flow electrophoresis ; Endosomes ; Lysosomes ; Transferrin ; Percoll ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conlusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0173-0835
    Keywords: Capillary electrophoresis ; DNA ; Mitochondrial DNA ; Forensic DNA typing ; Polymerase chain reaction analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The polymorphic control region of mitochondrial DNA (mtDNA) is becoming more commonly used in forensic applications to differentiate among individuals in a population. Two hypervariable regions (HV1 and HV2) are often sequenced following amplification of the mtDNA via the polymerase chain reaction (PCR). More rapid screening assays would reduce both the effort and the expenses of comparing two samples. A methodology has been developed that first uses restriction endonuclease digestion of the PCR-amplified mtDNA using RsaI and Mn/I and then capillary electrophoresis (CE) to separate and size the PCR-RFLP fragments. This rapid procedure offers an alternative method for screening of polymorphisms in amplified mtDNA samples. In addition, the presence of a T→C transition at position 16189, which gives rise to the so-called “C-stretch” in HV1, may be predicted from the presence of nonspecific PCR products in the CE results.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Chichester : Wiley-Blackwell
    ISSN: 0268-2575
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Thermostable lipase from Thermomyces lanuginosus was immobilized in untreated microporous membranes. Melted tallow pumped through the membrane did not wash the enzyme out. From 0.4 to 0.9% of the soluble activity remained after immobilization with half-lives of 1-2 months or more at 50°C. Membranes can be acid/base washed and reloaded with enzyme with no adverse effects. Buffer was required for a long half-life, and recycling the buffer improved the mass transfer of glycerol out of the immobilized lipase reactor. Immobilized activity was unaffected when the pH of the aqueous product changed from 5.5 to 6.5.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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