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  • Biochemistry and Biotechnology  (59)
  • 1995-1999  (59)
  • 1996  (59)
  • 1
    Electronic Resource
    Electronic Resource
    The role of pH and membrane porosity in preparative electrophoresis (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 771-775 
    Details
    ISSN: 0173-0835
    Keywords: Preparative electrophoresis ; Large-scale electrophoresis ; Protein purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The Gradiflow is a preparative electrophoresis apparatus, allowing fractionation based on a combination of size and charge of proteins in their native (unreduced) form. The prepative fractionation of two proteins of similar size and isoelectric point is demonstrated using the Gradiflow. A separation membrance of appropriate pore size was chosen and then fractionation was “fine tuned” by selecting an appropriate buffer pH to accentuate charge differences between the proteins of interest. Complete separation of mg quantities of bovine serum albumin and ovalbumin was achieved within 40 minPresented at the Second Annual Meeting of the Australian Electrophoresis Society, Sydney, April 29-30, 1995..
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170425
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  • 2
    Electronic Resource
    Electronic Resource
    Characterisation of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid compositional analysis (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 580-587 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Matrix assisted laser desorption mass spectrometry ; Peptide finger-printing ; Amino acid analysis ; Myocardial proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170329
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  • 3
    Electronic Resource
    Electronic Resource
    Fluid shear stress induction of the transcriptional activator c-fos in human and bovine endothelial cells, HeLa, and Chinese hamster ovary cells (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 383-390 
    Details
    ISSN: 0006-3592
    Keywords: c-fos protein ; endothelium ; hemodynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The c-fos protein belongs to a family of transcriptional cofactors that can complex with proteins of the Jun family and activate mRNA transcription from gene promoters containing an activator protein 1 (AP-1) binding element. The shear stress inducibility of the c-fos protein was studied in human and animal cell lines of vastly different origins. Primary human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC, passage 2-14), HeLa cells, and Chinese hamster ovary (CHO) cells were subjected to steady laminar shear stress using a parallel plate flow apparatus. After 1 h of flow exposure at 25 dyn/cm2, the c-fos levels in nuclei of shear stress HUVEC, BAEC, HeLa, and CHO were 5.4 ± 2.0 (n = 3), 2.25 ± 1.38 (n = 6), 2.14 ± 0.07 (n = 8), 1.92 ± 0.58 (n = 2) times higher, respectively, than in matched stationary controls. Flow exposure at 4 dyn/cm2 caused no enhancement of c-fos levels in any of the cell lines tested, but caused significant reduction in c-fos expression in the HeLa cells. The c-fos induction by shear stress could be blocked by pharmacological agents. For example, the flow induction of the c-fos protein levels was blocked by 50% with the preincubation of HUVEC with a protein kinase C inhibitor, H7 (10 μM) and blocked completely in HeLa cells preincubated with the phospholipase C inhibitor, neomycin (5 mM). The minimum time of shear stress exposure required to induce the c-fos protein expression in HeLa cells was found to be as low as 1 min. By Northern analysis, the c-fos mRNA levels were found to be elevated in BAEC, CHO, and HeLa cells exposed to 25 dyn/cm2 for 30 min. These studies indicate that c-fos induction is a consistent genetic response in a variety of mammalian cells that may alter cellular phenotype in mechanical environments. © 1996 John Wiley & Sons, Inc.
    Additional Material: 7 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    Osteoblast migration on poly(α-hydroxy esters) (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 443-451 
    Details
    ISSN: 0006-3592
    Keywords: osteoblast ; migration ; poly(αhydroxy esters) ; poly(DL-lactic-co-glycolic acid) ; PLGA ; biodegradable polymers ; tissue engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We investigated the migration of rat calvaria osteoblast populations on poly(α-hydroxy ester) films for up to 14 days to determine effects of substrate composition and culture conditions on the migratory characteristics of osteoblasts. Initial osteoblast culture conditions included cell colonies formed by seeding a high (84,000 cells/cm2) or low (42,000 cells/cm2) density of isolated osteoblasts on the polymer films, and bone tissue cultures formed by plating bone chips directly on the substrates. High density osteoblast colonies cultured and allowed to migrate and proliferate radially on 85:15 poly(DL-lactic-co-glycolic acid) (PLGA) films, 75:25 PLGA films, and tissue culture polystyrene controls demonstrated that the copolymer ratio in the polymer films did not affect the rate of increase in substrate surface area (or culture area) covered by the growing cell colony. However, the rate of increase in culture area was dependent on the initial osteoblast seeding density. Initial cell colonies formed with a lower osteoblast seeding density on 75:25 PLGA resulted in a lower rate of increase in culture area, specifically 4.9 ± 0.3 mm2/day, versus 14.1 ± 0.7 mm2/day for colonies seeded with a higher density of cells on the same polymer films. The proliferation rate for osteoblasts in the high and low density seeded osteoblast colonies did not differ, whereas the proliferation rate for the osteoblasts arising from the bone chips was lower than either of these isolated cell colonies. Confocal and light microscopy revealed that the osteoblast migration occurred as a monolayer of individual osteoblasts and not a calcified tissue front. These results demonstrated that cell seeding conditions strongly affect the rates of osteoblast migration and proliferation on biodegradable poly(α-hydroxy esters). © 1996 John Wiley & Sons, Inc.
    Additional Material: 10 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    Expression of active spinach glycolate oxidase in Aspergillus nidulans (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 341-346 
    Details
    ISSN: 0006-3592
    Keywords: glycolate oxidase ; Aspergillus nidulans ; heterologous expression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity. © 1996 John Wiley & Sons, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    Immobilization of Candida rugosa lipase to nylon fibers using its carbohydrate groups as the chemical link (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 327-341 
    Details
    ISSN: 0006-3592
    Keywords: Candida rugosa ; immobilization ; olive oil hydrolysis ; phenylglycidate ; glutaraldehyde ; adipic dihydrazide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The aim of this study was to evaluate the immobilization of lipase from Candida rugosa on a nylon support by methods used to attach biomolecules to solid supports through their carbohydrate moieties. The carbohydrate groups were converted to dialdehydes by treatment with sodium periodate. The length of exposure and the periodate amount were optimized to the point where almost total activity retention was obtained. Tests of the immobilized enzyme showed the expressed activity to be significantly higher than the activity obtained with the unimmobilized enzyme. The use of reverse micelles as a way of delivering water to the enzyme was tested and found to give significantly higher activities. The immobilized enzyme activity was also tested with other substrates, one of which was a chiral ester. The immobilized enzyme was found to have high stereoselective efficiency and activity toward racemic methyl methoxyphenyl glycidate, a chiral intermediate used in the manufacture of the drug diltiazem. © 1996 John Wiley & Sons, Inc.
    Additional Material: 15 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    Activation and regeneration of whole cell biocatalysts: Initial and periodic induction behavior in starved Escherichia coli after immobilization in thin synthetic films (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 360-370 
    Details
    ISSN: 0006-3592
    Keywords: induction ; Escherichia coli ; biofilms ; immobilization ; protein synthesis in starved bacteria ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Activation and regeneration of whole cell biocatalytic activity via initial and subsequent induction of the lacZ gene was investigated in starved Escherichia coli using a novel synthetic biofilm. Stationary-phase bacteria were entrapped in 10-80 μm thick multi-layer films, where a copolymer of acrylic and vinyl acetate was the immobilization matrix. The E. coli were placed in a defined starvation medium containing essentially no nitrogen or carbon source and induced initially using lactose or isopropylthiogalactoside (IPTG). Subsequent inductions were performed with IPTG. Comparison studies with suspended bacteria showed that when IPTG was the initial inducing agent, induction kinetics are linear for both immobilized and suspended cells. After induction with lactose, however, a lag time is noted for suspended cells, but not for E. coli in the biofilm. Biocatalytic activity was successfully regenerated by re-inducing starved suspended cells 1-3 days after an initial induction with lactose. This regeneration was demonstrated in the synthesis of additional active β-galactosidase. However, immobilized cells could be re-induced for at least 17 days after the initial induction, and viability in the synthetic biofilms remained greater than 90%, demonstrating that periodic induction is a valuable method for extending the life of whole cell biocatalysts. © 1996 John Wiley & Sons, Inc.
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  • 8
    Electronic Resource
    Electronic Resource
    Enhanced function of cultured epithelium by genetic modification: Cell-based synthesis and delivery of growth factors (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 15-23 
    Details
    ISSN: 0006-3592
    Keywords: tissue engineering ; gene therapy ; artificial skin ; wound-healing growth factors ; growth factor delivery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Skin substitutes, containing cultured keratinocytes of the epidermis (autologous or allogeneic cells), have been used in the treatment of severe burns and other defects of the skin such as chronic ulcers. Our goal is to enhance the functions of the cells used in these skin substitutes by genetic modification. We propose to develop a genetically modified skin graft which would function as a cell-based vehicle for the local synthesis and delivery of wound-healing growth factors. Using retroviral-mediated gene transfer, we have introduced stable copies of the genes encoding platelet-derived growth factor (PDGF-A) or insulin-like growth factor-1 (IGF-1) into cultured human diploid keratinocytes. After stable integration of these genes, the cells secreted significant levels of these growth factors, 744 ng and 502 ng/107 cells/24 h for PDGF-A and IGF-1, respectively. The modified cells were grown to confluence, detached as a multicell-layered epithelial sheet, and transplanted to athymic mice.Seven days after transplantation, grafts secreting PDGF-A or IGF-1 differentiated into a stratified epithelium comparable to unmodified cells. Most importantly, the newly synthesized connective tissue layer subjacent to the PDGF-A-modified grafts was significantly thicker and showed an increase in cellularity, vascularity, type I collagen, and fibronectin deposition when compared to control grafts of unmodified cells or grafts expressing IGF-1.These results demonstrated that the function of the cells of a skin substitute can be enhanced by genetic modification and show that PDGF-A secretion from these cells can mediate changes to the cellular, vascular, and extracellular matrix composition of the adjacent dermal tissue. Moreover, these results suggest that a cell-based method for growth factor synthesis and delivery may be a useful approach to promoting tissue repair. © 1996 John Wiley & Sons, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    A self-regulating cell culture analog device to mimic animal and human toxicological responses (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 45-60 
    Details
    ISSN: 0006-3592
    Keywords: In vitro toxicology ; physiologically based pharmacokinetic models ; cell culture analog ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The overall goal of this project is the development of a new methodology for translating advances in molecular level understanding of toxicological responses into a predictive tool for dose response in whole animals and humans exposed to single compounds or mixtures of compounds. The methodology incorporates a mechanistic cellular level model into a PBPK (physiologically based pharmacokinetic) model which simultaneously guides the development of an in vitro cell culture analog (CCA) to the PBPK. Where the PBPK specifies an organ, (e.g., liver) the in vitro or CCA system contains a compartment with the appropriate cell or cell population (e.g., hepatocytes for the liver). The CCA has significant advantages over other in vitro systems and PBPK systems used independently for evaluating metabolic responses to drugs or potentially toxic chemicals where the exchange of metabolites between organs is likely to be important. The CCA system is superior to a PBPK because an a priori description of complete metabolism is not required and secondary, unexpected interactions can be detected. The CCA system, unlike other in vitro systems, gives a dynamic response that realistically simulates in vivo interactions between organs. Furthermore, the CCA allows dosing on the same basis as animal tests (e.g., milligrams per kilogram of body mass equivalent). Because the construction of a CCA is guided by a PBPK, this approach allows extrapolation to low doses and across species, including extrapolation to humans. We have constructed a prototype system and have conducted proof-of-concept experiments using naphthalene as a test chemical. These experiments clearly demonstrate the ability to generate a reactive metabolite in one compartment and detect its effects (on LDH release and glutathione depletion) in a second compartment. However, this prototype device would be expensive to replicate and requires nearly constant supervision from a trained investigator. For this concept to replace animals an inexpensive, self-regulating device is needed. An initial design to accomplish this goal is described as well as the corresponding model using naphthalene as a test compound. © 1996 John Wiley & Sons, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    Poly(organo phosphazene) nanoparticles surface modified with poly(ethylene oxide) (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 89-95 
    Details
    ISSN: 0006-3592
    Keywords: poly(organo phosphazenes) ; nanoparticles ; poly(ethylene oxide) ; biodegradable materials ; surface modification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of biodegradable derivatives of poly(organo phosphazenes) for the preparation of nanoparticles and their surface modification with the novel poly(ethylene oxide) derivative of poly(organo phosphazene) has been assessed using a range of in vitro characterization methods. The nanoparticles were produced by the precipitation solvent evaporation method from the derivative co-substituted with phenylalanine and glycine ethyl ester side groups. A reduction in particle size to less than 200 nm was achieved by an increase in pH of the preparation medium. The formation (and colloidal stability) of these nanoparticles seems to be controlled by two opposite effects: attractive hydrophobic interactions between phenylalanine ester groups and electrostatic repulsions arising from the carboxyl groups formed due to (partial) hydrolysis of the ester bond(s) at the high pH of the preparation medium. The poly[(glycine ethyl ester)phosphazene] derivative containing 5000-Da poly(ethylene oxide) as 5% of the side groups was used for the surface modification of nanoparticles. Adsorbed onto the particles, the polymer produced a thick coating layer of approximately 35 nm. The coated nanoparticles exhibited reduced surface negative potential and improved colloidal stability toward electrolyte-induced flocculation, relative to the uncoated system. However, the steric stabilization provided was less effective than that of a Poloxamine 908 coating. This difference in effectiveness of the steric stabilization might indicate that, although both the stabilizing polymers possess a 5000-Da poly(ethylene oxide) moiety, there is a difference in the arrangements of these poly(ethylene oxide) chains at the particle surface. © 1996 John Wiley & Sons, Inc.
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