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  • 1
    Electronic Resource
    Electronic Resource
    Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant dnase in batch culture with increased sialic acid (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 589-595 
    Details
    ISSN: 0006-3592
    Keywords: CH0 cells ; sialidase activity ; recombinant DNase ; sialic acid ; antisense DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Under some cell culture conditions, recombinant glycoprotein therapeutics expressed in Chinese hamster ovary (CHO) cells lose sialic acid during the course of the culture (Sliwkowski et al., 1992; Munzert et al., 1996). A soluble sialidase of CHO cell origin degrades the expressed recombinant protein and has been shown to be released into the culture fluid as the viability of the cells decreases. To reduce the levels of the sialidase and to prevent desialylation of recombinant protein, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA. Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5′ 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values. To test if this level of sialidase would lead to increased sialic acid content of an expressed recombinant protein, the 474 antisense clone was employed as a host for expression of human DNase as a model glycoprotein. The sialic acid content of the DNase produced in the antisense cultures was compared with material made in the wild-type parental cell line. About 20-37% increase in sialic acid content, or 0.6-1.1 mole of additional sialic acid out of a total of 3.0 mole on the product, was found on the DNase made in the antisense cell lines. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 589-595, 1998.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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    Paper (German National Licenses)
  • 2
    Electronic Resource
    Electronic Resource
    Capillary electrophoresis/mass spectrometry determination of inorganic ions using an ion spray-sheath flow interface (1993)
    Weinheim : Wiley-Blackwell
    Electrophoresis 14 (1993), S. 531-539 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The determination of inorganic cations and anions by capillary electrophoresis/mass spectrometry (CE/MS) is reported using an ion spray-sheath flow interface coupling. A twelve-component synthetic mixture of cations which included the positive ions of K, Ba, Ca, Mn, Cd, Co, Pb, Cr, Ni, Zn, Ag, and Cu was loaded into the capillary column at levels ranging from 30 to 300 pg, separated by CE, and detected by indirect UV and in the full-scan (m/z 35-450) positive ion CE/MS mode using an aqueous buffer containing 30 mM creatinine and 8 mM α-hydroxyisobutyric acid, pH 4.8. Creatinine forms adducts with the cations which are observed in the gas phase and requires rather high (120 electron volts) declustering energy to dissociate. This produces a reduction in charge state to form the free, singly charged, inorganic cations which are observed in the mass spectra. CE/MS analysis of an aqueous acidic extract of used aircraft engine oil revealed high levels of lead as well as lower levels of chromium and nickel. CE-indirect UV analysis of a synthetic mixture containing 300 pg each of 11 inorganic ions, which included the anions of Br, Cl, NO2, NO3, S2O3, N3, SCN, SO4, SeO4, oxalate, and MoO4, is shown. The running buffer which affected this separation contained 5 mM ammonium dichromate, 10 mM ammonium acetate, and 20 mM diethylenetriamine at pH 9.3. Although indirect UV detection revealed good separation of these anions, CE/MS analysis of this mixture was complicated by interfering ion current signals from the cluster ions formed by the interaction between the additives and the analytes. Thus only three of these singly charged anions, e.g. Br, SCN, and HSeO4, could be satisfactorily detected in this mixture by CE/MS.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150140181
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  • 3
    Electronic Resource
    Electronic Resource
    Chemical analysis of environmental samples collected in Iraq: Analysis for the presence of chemical warfare agents (1995)
    Chichester : Wiley-Blackwell
    Journal of Chemical Technology AND Biotechnology 64 (1995), S. 115-128 
    Details
    ISSN: 0268-2575
    Keywords: Chemical warfare agents ; Chemical Weapons Convention ; Iraq ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nineteen samples from the United Nations Special Commission 65 on Iraq (UNSCOM 65) were analyzed for chemical warfare (CW) related compounds using a variety of spectroscopic and chromatographic techniques including multinuclear NMR, GC (phosphorus, sulfur and atomic emission detection), GC/MS (electron impact and chemical ionization), tandem MS, HPLC/ion chromatography, HPLC/thermospray/MS, FTIR, ICP and GFAA. The samples consisted of one piece of cloth, one piece of wood, six waters, six soils, two vegetation samples and two mortar shell crosscut sections. No intact CW agents were detected; however, diethyl phosphoric acid was unambiguously identified in three of the water samples and ethyl phosphoric acid was tentatively identified, at lower levels, in one of the water samples. Diethyl phosphoric acid and ethyl phosphoric acid are degradation products of munitions-grade Tabun (GA), an organophosphorus nerve agent. However, these compounds are also degradation products of the Chemical Weapons Convention (CWC) scheduled compound Amiton as well as many commercially available pesticides.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jctb.280640203
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  • 4
    Electronic Resource
    Electronic Resource
    Polymerase chain reaction fingerprinting in fungi using single primers specific to minisatellites and simple repetitive DNA sequences: Strain variation in Cryptococcus neoformans (1995)
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 1648-1656 
    Details
    ISSN: 0173-0835
    Keywords: Polymerase chain reaction ; Fingerprinting ; DNA ; Fungi ; Minisatellites ; Simple repetitive sequences ; Cryptococcus neoformans ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Minisatellites and simple repetitive DNA sequence motifs are used as conventional oligonucleotide probes in DNA-hybridization-based fingerprinting. The same oligonucleotides can be used as single primers in the polymerase chain reaction (PCR) to generate individual PCR fingerprints. In this study, the simple repetitive sequences, (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, and a minisatellite core sequence derived from the wild-type phage M13 (5′ GAGGGTGGCGGTTCT 3′) were used as specific, single primers to amplify hypervariable repetitive DNA sequences during PCR analysis. The potential applications of this technique are demonstrated with clinical isolates of the human pathogenic yeest, Cryptococcus neoformans. PCR fingerprint patterns have remained stable after long-term in vitro passage (〉2½ years to date). Hybridization of the primers to blots of electrophoretically separated chromosomes demonstrated that the target sequences recognized by most of the primers are dispersed through the entire yeast genome. Sequence analysis of the cloned bands obtained by PCR fingerprinting indicated that if the same or extremely similar, inversely oriented tandem repeats are located close to each other, when only one repeat-specific primer is used in the PCR, the region between these repeats is amplified. PCR fingerprinting has a wide range of current and potential applications to fungi, such as clarifying taxonomic questions, facilitating epidemiological studies and improving the diagnosis of mycotic diseases.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501601273
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  • 5
    Electronic Resource
    Electronic Resource
    Identification of pathogenic yeasts of the imperfect genus Candida by polymerase chain reaction fingerprinting (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1548-1559 
    Details
    ISSN: 0173-0835
    Keywords: Polymerase chain reaction ; Fingerprinting ; Pathogenic yeasts ; Minisatellite ; Simple repetitive sequences ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: With the increase in the number of immunocompromised hosts, the number of fungal pathogens has increased markedly. Identification and classification, especially of yeast species and strains, is often difficult when based solely on phenotypic characteristics. Since it became clear that different fungal pathogens require specific treatment strategies, there is a need for simple, rapid and reliable methods to identify fungal isolates. Polymerase chain reaction (PCR) fingerprinting was successfully applied here to identify yeast isolates Microsatellite [(GTG)5; (GACA)4] and minisatellite [(5′GAGGGTGGCGGTTCT 3′), derived from the core-sequence of the phage M13] specific primers were used as single primers in the PCR to amplify hypervariable interrepeat DNA sequences from over 200 European, American and Australian clinical isolates within the genus Candida. Each species, represented by its type strain, could be identified by a specific multilocus pattern, allowing for the assignment of all the isolates to the appropriate species. Intra-species variation in the multilocus profiles was about 20% compared to inter-species variation, which was up to 80%. Anamorph-teleomorph pairs could be identified by highly homologous PCR fingerprint patterns. PCR fingerprinting was more discriminatory when compared with routinely used biochemical tests (Vitek YBC and API ID 32C). PCR fingerprinting has proven to be a powerful tool for the identification of medically important yeasts. It is rapid, sensitive, reliable, highly reproducible, stable in vitro and in vivo, and applicable to large-scale experiments. Potential applications include: yeast taxonomy, epidemiology, environmental surveys, and improvement of the diagnosis of mycotic diseases.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180911
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  • 6
    Electronic Resource
    Electronic Resource
    Micellar electrokinetic chromatography of the anti-human immunodeficiency virus (HIV) agent michellamine B with absorption and laser-induced fluorescence detection (1994)
    Weinheim : Wiley-Blackwell
    Electrophoresis 15 (1994), S. 1310-1315 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Micellar electrokinetic chromatography (MEKC) was applied to the separation of the anti-HIV agents, michellamines A and B, and two other structurally related monomers found in the extract of the Ancistrocladus plants. Using buffers containing either 10 mM sodium phosphate (pH 7.0), 50 mM sodium de-oxycholate and 10-20% acetonitrile or 5 mM sodium phosphate (pH 7.0), 20 mM sodium dodecyl sulfate and 25% acetonitrile allowed baseline separations of the four components in the mixture in less than 10 min. The MEKC methods gave sharper peaks and better resolution compared to high-performance liquid chromatography. For MEKC separation of the plant extracts, UV absorption detection provided adequate sensitivity; however, higher sensitivity could be achieved with UV laser-induced fluorescence detection (LIF). Using the sodium dodecyl sulfate-containing buffer and LIF, the limit of detection for michellamine B was ∼ 2 ng/mL. The sensitivity was degraded ∼100-fold when using the deoxycholate buffer because of high background fluorescence. Preliminary results show that MEKC with LIF is feasible for the sensitive detection of michellamine B in serum.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.11501501199
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