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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 7 (1990), S. 16-31 
    ISSN: 0887-3585
    Keywords: BPTI ; dithiothreitol ; DTT-sensitive mutants ; protein folding ; random mutagenesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 “DTT-sensitive” mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the naive wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated a most sites without completely preventing folding Because this genetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 1151-1160 
    ISSN: 0006-3592
    Keywords: fluorescence ; monitoring ; methane ; fermentation ; NAD(P)H ; F420 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: On-line in situ fluorescence measurements of the methanogenic fermentation were conducted with reactors receiving either glucose or a mixture of volatile fatty acids as the substrate. The reactors were perturbed from steady-state conditions in order to assess the response of fluorescencemonitoring probes. Two fluorescence-monitoring probes were evaluated over a period of 8 months; they performed in a consistent manner, and their response was not significantly affected by the changes in pH and redox potential encountered during routine reactor operation. A commercially available probe, designed to measure NAD(P)H, demonstrated particular promise for detecting imbalance caused by the entry of air, inhibitor addition and was capable of distinguishing between different substrates. This fluorescence-monitoring probe detected imbalance more rapidly than other on-line measurements such as pH, Eh, or gas production, or off-line measurements such as volatile fatty acid concentration or gas composition. An experimental fluorescence-monitoring probe, designed to measure coenzyme F420, also showed some promise in this regard. The response of the fluorescence-monitoring probes also revealed details of the metabolic routes in the reactors and the probes represent a useful research tool. For example, a failure to observe the characteristic response of the NAD(P)H-monitoring probe to formate addition during the metabolism of acetate, propionate, or glucose strongly suggests that any formate liberated during their catabolism is degraded via a different route to exogenously added formate.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 35-45 
    ISSN: 0006-3592
    Keywords: biofilm ; attached growth ; respirometry ; parameter estimation ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Currently, no fast and accurate methods exist for measuring extant biokinetic parameters for biofilm systems. This article presents a new approach to measure extant biokinetic parameters of biofilms and examines the numerical feasibility of such a method. A completely mixed attached growth bioreactor is subjected to a pulse of substrate, and oxygen consumption is monitored by on-line measurement of dissolved oxygen concentration in the bulk liquid. The oxygen concentration profile is then fit with a mechanistic mathematical model for the biofilm to estimate biokinetic parameters. In this study a transient biofilm model is developed and solved to generate dissolved oxygen profiles in the bulk liquid. Sensitivity analysis of the model reveals that the dissolved oxygen profiles are sufficiently sensitive to the biokinetic parameters - the maximum specific growth rate coefficient (⁁μ) and the half-saturation coefficient (Ks) - to support parameter estimation if accurate estimates of other model parameters can be obtained. Monte Carlo simulations are conducted with the model to add typical measurement error to the generated dissolved oxygen profiles. Even with measurement error in the dissolved oxygen profile, a pair of biokinetic parameters is always retrievable. The geometric mean of the parameter estimates from the Monte Carlo simulations prove to be an accurate estimator for the true biokinetic values. Higher precision is obtained for ⁁μ estimates than for Ks estimates. In summary, this theoretical analysis reveals that an on-line respirometric assay holds promise for measuring extant biofilm kinetic parameters. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 35-45, 1998.
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 11 (1993), S. 35-44 
    ISSN: 0263-6484
    Keywords: Tunicamycin ; acetylated low density lipoprotein ; macrophages ; endocytosis ; proteolysis and chloroquine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of tunicamycin (TM) on the metabolism of acetylated low-density lipoprotein (AcLDL) was examined to determine whether N-linked glycosylation is required for the proper function of the AcLDL pathway. Proteolytic degradation of [125I]-AcLDL was increased twofold in the presence of TM. This did not occur via an increase in total lysosomal enzyme activity or extracellular proteolysis; rather, the rate of uptake of [125I]-AcLDL was increased. The enhanced degradation of AcLDL did not lead to a commensurate increase in the rate of synthesis of cholesteryl oleate. Conversely, the rate of cholesterol esterification was reduced in the presence of TM. The uptake of [125I]-AcLDL was more sensitive to inhibition by chloroquine in TM-treated cells. However, the presence of TM did not affect the ability of chloroquine to inhibit constitutive recycling of AcLDL binding sites. These results suggest that N-linked glycosylation may be involved in the regulation of AcLDL metabolism in J774 cells.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Normal human skin fibroblast primary cell lines secrete over 50 proteins into culture medium. These have been mapped previously using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and this technique has now been used to investigate extracellular protein secretion by human osteoblasts in vitro. We report the mapping of a number of consistent markers specific to the osteoblast. In particular, one protein chain with posttranslational modifications was found to be unique to the osteoblast extracellular protein map. The absence of the N- and O-glycoforms of collagenase from the osteoblast profile in this study concurs with findings reported using the immunoprecipitation functional assay and Northern blot analysis. The use of 2-D PAGE in phenotypic assessment provides a more complete analysis than the standard range of single-parameter tests for osteobiasts. Mapping of extracellular and cellular proteins in addition to bone matrix protein analysis will allow a comprehensive analysis of normal osteoblast function. This technique may also be applied to the study of osteoblasts in relation to bone disease and in assessing the phenotypic shift within a normal osteoblast culture.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 14 (1993), S. 644-649 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to map the extracellular secretory activity of normal osteoblasts. The proteins osteonectin, bone sialoprotein, and both the C-telopeptide of collagen I together with collagen I have now been positionally identified. In addition the secretory differences which exist between normal and Pagetic osteoblasts have been mapped with the Pagetic osteoblasts shown to consistently secrete an altered 30 kDa C-telopeptide of collagen type I. The use of the diphosphonate Pamidronate in the treatment of Paget's disease of bone has beneficial effects with suppression of the bone isoenzyme marker alkaline phosphatase. It has been reported that diphosphonates directly inhibit human osteoblast secretory function as well as osteoclast metabolism. The effects of Pamidronate on the secretory activity of normal and Pagetic osteoblast cultures was also investigated. The extracellular protein secretion of normal and Pagetic osteoblasts was not affected by Pamidronate treatment as assessed by 2-D PAGE. This technique allows a comprehensive multiparameter assessment of extracellular secretory activity in normal and diseased states. The findings show that the Pagetic osteoblasts cultured in vitro are functionally abnormal and they support the hypothesis that the underlying problem in Paget's disease is characterised by disorder of osteoblast and osteoclast interactions.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 1553-1558 
    ISSN: 0173-0835
    Keywords: Chemiluminescence ; Probes ; Detection ; Dioxetanes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The conditions for hybridization and detection of enzyme-labeled probes have been optimized in our laboratory for use with oligonucleotides coupled to alkaline phosphatase. We have examined several enzyme-linked probes which are complimentary to commonly used variable number of tandem repeats (VNTR) loci to determine the feasibility of using chemiluminescence for routine application in forensic DNA analysis. It was found that a chemiluminescent detection system employing an alkaline phosphatase activated dioxetane in the presence of chemiluminescent enhancers provides a high degree of sensitivity in hybridization protocols with a significant savings in overall filter processing time. The chemiluminescent system achieved equal or greater sensitivity than observed for 32P-labeled probes in much shorter development times. Furthermore, a new chemiluminescent substrate, Lumi-Phos® Plus, has recently been investigated and found to further decrease the filter development time for forensic assays.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 322-329 
    ISSN: 0887-3585
    Keywords: unfolded proteins ; mutations ; BPTI ; gel electrophoresis ; disulfide-formation kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The effects of amino acid replacements on the hydrodynamic volume of reduced and unfolded bovine pancreatic trypsin inhibitor (BPTI) have been examined by gel electrophoresis. The electrophoretic mobilities of the reduced forms of 46 BPTI variants were compared at room temperature in the absence of denaturants. The single substitutions examined include many different types of replacements at sites throughout the polypeptide, and, collectively, alter 22 of the 58 residues of the wild-type protein. The only substitutions found to alter the electrophoretic mobility of the reduced protein by more than ∼3% are those that change the net charge of the protein. For nine mutants, the rates of disulfide formation in the reduced protein were also examined and found to be very similar to that of the wild-type protein. These results suggest that any structure that may be present in the reduced protein is either relatively insensitive to amino acid replacements or does not greatly influence the averaged properties of the polypeptide chain. © 1993 Wiley-Liss, Inc.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 0887-3585
    Keywords: electrospray ionization mass spectrometry ; noncovalent complexes ; protease ; integrase ; nucleocapsid protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Mass spectrometry (MS) with electrospray ionization (ESI) has shown utility for studying noncovalent protein complexes, as it offers advantages in sensitivity, speed, and mass accuracy. The stoichiometry of the binding partners can be easily deduced from the molecular weight measurement. In many examples of protein complexes, the gas phase-based measurement is consistent with the expected solution phase binding characteristics. This quality suggests the utility of ESI-MS for investigating solution phase molecular interactions. Complexes composed of proteins from the human immunodeficiency virus (HIV) have been studied using ESI-MS. Multiply charged protein dimers from HIV integrase catalytic core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA 1:1 stoichiometry complex was also studied by ESI-MS. No protein-RNA complex was observed in the absence of zinc, consistent with the role of the zinc finger motifs for RNA binding. Proteins Suppl. 2:28-37, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We report a simple method, designated “spot transfer”, where known proteins are excised and eluted from a two-dimensional (2-D) gel run on one gel system to transfer identification of the proteins to a different 2-D gel system by comigration with a comparable sample. In one experiment, 8 of 16 proteins eluted from an isoelectric focusing (IEF) 2-D gel, of the format described for the Celis human keratinocyte database (Celis, J. E. et al., Electrophoresis 1993, 14, 1091-1198), were found to comigrate with proteins in a human T lymphoma (JURKAT) whole cell lysate run using the Millipore Investigator 2-D system. The method should have general utility in allowing the exchange of protein identifications between investigators and could be used to standardize gel loci used in 2-D gel protein databases.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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