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The interaction of a bioengineered serpin (LEX032) with human leukocyte proteinase 3 (PR 3) has been investigated. LEX032 was found to be a time-dependent inhibitor of PR 3, forming a highly-stable enzyme-inhibitor complex (Ki 12 nM).
Emphysema is a slowly progressive degenerative lung disease involving fragmentation and depletion of elastic fibers, loss of lung elastance, and architectural destruction with ectasia, tortuosity, and loss of bronchioles irrespective of localization or morphological type. Occurring under physiological conditions, predominantly in geriatrics, matrix laxity and destructive parenchymal lesions are indicative of a pathological loss of tissue tensile strength attributable to bioengineering or structural fatigue in repetitively stressed tissues. The occurrence of severe premature emphysema in inherited connective tissue diseases and under some experimental and iatrogenic conditions is supportive evidence. Experiments advocating unrestrained proteolysis as a cause or pathogenic factor are invalid, being based on a false premise and assumed causality.
The T-cell receptor (TCR) is a heterodimeric cell-surface protein consisting of two chains, alpha and beta, each of which is composed of a variable (V) and a constant (C) domain. Crystals of the isolated V(alpha) domain of the murine TCR 2C were grown by serendipity from a solution containing the extracellular domains of the intact TCR 2C and CD3 gamma epsilon-chains. The V(alpha) crystal structure shows how crystal packing can substitute for another V(alpha) domain in a different fashion from that observed in V(alpha)/V(alpha) homodimer and V(alpha)/V(beta) heterodimer structures. Significant conformational changes occur in the CDR3 and beta(3)beta(4) loops that normally form part of the dimer interface. The monomeric V(alpha) domain provides the unique opportunity to study the effect of dimerization on the conformation of the unliganded complementarity-determining regions (CDR) of a TCR. This structure of an individual V(alpha) module has implications for stability and bioengineering of isolated antibody and immunoglobulin domains.
Microbes growing on mixtures of substrates in a chemostat exhibit different substrate utilization patterns, depending on the dilution rate and feed concentrations. For instance, when supplied with high feed concentrations of a binary mixture, both substrates are consumed at low dilution rates, but only one of the substrates is consumed at high dilution rates. The goal of this work is to explain the onset of such transitions, which play a very significant role in ecology and bioengineering. In previous work, we formulated a mathematical model of mixed-substrate growth in batch cultures. We use the extension of this model to continuous cultures as the framework for understanding substrate utilization patterns in continuous cultures. Our explanation rests upon the existence of two special types of dilution rates predicted by the model. The first is the so-called critical dilution rate at which the growth rate becomes zero, leading to cell washout. The existence of the critical dilution rate obtains from the simplest models of microbial growth, and is rooted in the fact that growth is inherently autocatalytic. The second type of special dilution rate, a unique feature of our model, stems from the recognition that synthesis of the enzymes catalysing the uptake of substrates is also autocatalytic. Hence, associated with each substrate is a transition dilution rate at which the synthesis rate of the transport enzyme becomes zero. We show that: (1) the substrate utilization patterns in continuous cultures are completely determined by the relative magnitudes of the critical and transition dilution rates; and (2) the critical and transition dilution rates are in turn determined by the feed concentrations. This allows us to construct an operating diagram, which yields the substrate utilization pattern for any given dilution rate and feed concentrations. The theory explains most of the mixed-substrate phenomena summarized in a recent review article by Egli (1995, Adv. Microbiol. Ecol. 14, 305-386).
As intracellular gene delivery pathways are highly complex combinations of multiple potentially rate-limiting cellular and molecular processes, approaches to the design of synthetic delivery vectors focusing on any single barrier individually will likely be suboptimal. We offer here an "integrative systems" approach to vector characterization and design, combining quantitative experiment and computational modeling studies of vector uptake and trafficking kinetics. This model is validated using data for delivery of a green fluorescent protein (GFP)-encoding plasmid by means of Lipofectamine, permitting specification of model parameter values. The model is then used to make a priori predictions on the effect of polymer length in polyplex vectors, with additional parameter values determined from previous independent experimental studies of plasmid release. Comparison with data on GFP expression via these polyplex vectors shows that the model successfully predicts an experimentally observed biphasic dependence of expression efficiency on polymer length and quantifies the contributions of competing effects yielding the optimal intermediate polymer length. Finally, we use the model to predict potential effects of incorporating nuclear localization sequences in these kinds of synthetic vectors, and find that the degree of benefit from these will depend on the values of other key system properties including the vector unpackaging rate constant. Thus, we demonstrate the usefulness of a bioengineering, integrative-systems modeling approach to improved vector design and analysis.
Biomaterial polymers have been proposed as scaffolds for cell assembly in vascular bioengineering. We describe here a new method for the neovascularization of polyurethane meshes from explants of rat aorta. Aortic rings embedded in collagen-permeated polyurethane meshes and cultured in medium supplemented with fetal bovine serum and vascular endothelial growth factor generated florid microvascular outgrowths that efficiently vascularized the available spaces between polyurethane fibers. The neovessels could be identified in the live cultures by phase-contrast microscopy, and in formalin-fixed preparations by the ABC peroxidase procedure, using the endothelial-specific Griffonia isolectin B4. The aortic outgrowths were successfully labeled with the intravital fluorescent dyes Calcein AM or SPDiOC(18), which are nontoxic and can be used for tracking studies. This study shows that artificial biomaterial meshes can be colonized ex vivo with histotypic microvascular networks, and provides the proof of concept for the future development of stably vascularized devices for in vivo implantation.
Biopolymers as biomaterials and matrices in tissue engineering offer important options in control of structure, morphology and chemistry as reasonable substitutes or mimics of extracellular matrix systems. These features also provide for control of material functions such as mechanical properties in gel, fiber and porous scaffold formats. The inherent biodegradability of biopolymers is important to help regulate the rate and extent of cell and tissue remodeling in vitro or in vivo. The ability to genetically redesign these polymer systems to bioengineer appropriate features to regulate cell responses and interactions is another important feature that offers both fundamental insight into chemistry-structure-function relationships as well as direct utility as biomaterials. Biopolymer matrices for biomaterials and tissue engineering can directly influence the functional attributes of tissues formed on these materials and suggest they will continue play an increasingly important role in the field.
The reconstruction of tissues ex vivo and production of cells capable of maintaining a stable performance for extended time periods in sufficient quantity for synthetic or therapeutic purposes are primary objectives of tissue engineering. The ability to characterize and manipulate the cellular microenvironment is critical for successful implementation of such cell-based bioengineered systems. As a result, knowledge of fundamental biomimetics, transport phenomena, and reaction engineering concepts is essential to system design and development. Once the requirements of a specific tissue microenvironment are understood, the biomimetic system specifications can be identified and a design implemented. Utilization of novel membrane systems that are engineered to possess unique transport and reactive features is one successful approach presented here. The limited availability of tissue or cells for these systems dictates the need for microscale reactors. A capstone illustration based on cellular therapy for type 1 diabetes mellitus via encapsulation techniques is presented as a representative example of this approach, to stress the importance of integrated systems.
Microbiologists have always rely on microscopy to examine microorganisms. When microscopy, either optical or electron-based, is coupled to quantitative image analysis, the spectrum of potential applications is widened: counting, sizing, shape characterization, physiology assessment, analysis of visual texture, motility studies are now easily available for obtaining information on biomass. In this chapter the main tools used for cell visualization as well as the basic steps of image treatment are presented. General shape descriptors can be used to characterize the cell morphology, but special descriptors have been defined for filamentous microorganisms. Physiology assessment is often based on the use of fluorescent dyes. The quantitative analysis of visual texture is still limited in bioengineering but the characterization of the surface of microbial colonies may open new prospects, especially for cultures on solid substrates. In many occasions, the number of parameters extracted from images is so large that data-mining tools, such as Principal Components Analysis, are useful for summarizing the key pieces of information.
The Xenopus oocyte is a widely used model cell for studies of signal transduction mechanisms. Advances in microanalytical methods have made it feasible to perform rapid, localized collection of cytoplasm from individual Xenopus oocytes. Analytes contained in the cytoplasmic sample are separated by electrophoresis in a capillary and simultaneously transferred to a detection region. The development of bioengineered cells as sensitive detectors of intracellular components made quantitative measurements of native signaling molecules within the electrophoresed sample possible. Local determination of the second messenger inositol 1,4,5-trisphosphate is described to illustrate the methods for the sampling, electrophoresis, detection, and quantification of signaling molecules in single oocytes.
Patients suffering from a variety of urologic diseases may be treated with transplanted tissues and organs. However, there is a shortage of donor tissues and organs, which is worsening yearly owing to the ageing population. Scientists in the field of regenerative medicine and tissue engineering are applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured urologic tissues. This chapter reviews recent advances that have occurred in the regeneration of urologic organs and describes how these applications may offer novel therapies for patients with urologic disease.
The temperature/pressure dependency of marine prokaryotes and fungi, in terms of their growth behaviour as well as their potential to produce new metabolites or enzymes, is evaluated. Advanced shake-flask cultivations and controlled bioreactor cultivations following the batch-type, fed-batch-type and/or continuous-type procedures are summarized. After a summary of the fermentation data available so far, values on maximal biomass, specific growth rates, and (sub)optimal production yields are presented. The application of mesophilic microbes, especially bioactive metabolites, to intensify bioprocess engineering studies, is the goal. Cold-active enzymes and thermostable enzymes are the targets of experiments with psychrophilic and hyperthermophilic enzymes. A special challenge to bioengineers is also provided by barophilic strains originating from depths of, say, nearly 11000 m, or from hydrothermal vents.
The development of new surgical techniques for bladder substitution and continent urinary diversion has extended interest in urodynamics of the upper urinary tract. From a subdiscipline attracting mainly scientists and bioengineers, renal pelvic kinetics and ureteral peristalsis have evolved as important factors in routine clinical urology. The observed changes in peristaltic pattern during high diuresis, obstruction and urinary reflux have influenced management of stone disease and neurogenic bladder. The demonstration that high intravesical pressure is reflected to the kidney not only when the ureteric orifice is incompetent, but also during high diuresis, established the necessity for low pressures in neobladders. Much further clarification of urinary transport from the renal tubules to the bladder should be achievable by refined techniques of fluoroscopy, isotopic renography and manometry.
Threonine is an essential amino acid for mammals and birds and an adequate supply is necessary for growth and maintenance. Its production has become the aim of metabolic bioengineering and genetic manipulations. We propose in this paper a rational approach for increasing threonine production in an E. coli strain based on metabolic control theory. We have derived a way to measure the control coefficients of threonine pathway in vivo. The method consists in modelling the results of presteady-state experiments. The in vivo concentrations and activities of the enzymes can then be measured and introduced into the model, so that the in vivo steady-state of the pathway can be evaluated. With such a model it is possible to calculate the theoretical values of the control coefficients of the threonine synthesis flux in vivo.
The development of new radiopharmaceuticals is the basis of the continuing growth of nuclear medicine. Chemical interactions of electron clouds in their three-dimensional conformations bring together, in the process of molecular recognition, the reaction of antibody and antigen, receptor and ligand, enzyme and substrate, hormone and response site. This convergence enables the computer design of molecules such as ligands to fit computer-displayed conformational models showing active centres, positive and negative charges and other interactions. Indeed, given a particular molecule, a complementary binding structure can be devised. The hybridoma approach to monoclonal antibody production is being superceded by the bacterial bioengineer. The gene for the hypervariable region from the spleen cells of immunized mouse can be coupled with the myeloma gene. The polymerase chain reaction can duplicate the DNA a million times over in 20 min and the result transfected into a bacterial plasmid to produce the antibody. These scientific problems are soluble in principle and are being solved. However, so much damage to this developing biological field is being done by regulatory authorities that one must ask who should or can regulate the regulators. These problems have to be overcome in order to provide the new radiopharmaceuticals that are the food and wine of nuclear medicine.
Pulse oximetry is based on a relatively new concept, using the pulsatile variations in optical density of tissues in the red and infrared wavelengths to compute arterial oxygen saturation without need for calibration. The method was invented in 1972 by Takuo Aoyagi, a bioengineer, while he was working on an ear densitometer for recording dye dilution curves. Susumu Nakajima, a surgeon, and his associates first tested the device in patients, reporting it in 1975. A competing device was introduced and also tested and described in Japan. William New and Jack Lloyd recognized the potential importance of pulse oximetry and developed interest among anesthesiologists and others concerned with critical care in the United States. Success brought patent litigation and much competition.
The major rubella envelope protein, E1, and a segment of it, comprising amino acids 207-353, have been separately expressed as fusion proteins with the IgG binding region of Staphylococcus aureus protein A in Escherichia coli. The proteins carry E1-specific antigenicity recognized by monoclonal antibodies raised against whole virus confirming that correct glycosylation is not required for antigenicity. The use of these bioengineered antigens in immunoassays for diagnosis of rubella infection and for immunization in experimental animals is described.
In order to elucidate the role of HPV-16 in the development of genital cancer, NIH3T3 cells were transfected by HPV-16 whole genome and its two early genes, E6-E7. Besides ordinary calcium phosphate/DNA coprecipitation technique, a newly designed recombinant retrovirus containing the HPV-16 genome or subgenomes was used to infect cells for transfer of the target genes. The transforming activities have been demonstrated to be most efficient when a bioengineering technique of this kind is used. HPV-16 DNA was proved to have transforming potential for NIH3T3 cells, and the DNA of HPV-16 was proved to undergo multisite integration into transformed cells and nude mice tumour cells. The E6-E7 open reading frames are sufficient for transforming NIH3T3 cells independently in vitro, which implies that E6-E7 open reading frames are transforming genes or even viral oncogenes of HPV-16. The RNA transcribed by the E6-E7 of HPV-16 was expressed in transformed cells and in tumour cells of nude mice. The use of a recombinant retrovirus for gene transfer in this study is much more efficient than that of calcium phosphate/DNA coprecipitation. The lack of a tissue-culture system suitable for HPV replication in vitro makes HPV gene recombination into a specially engineered retrovirus for viral-mediated gene transfer of particular significance for the possible application of viral carcinogenesis, both in vitro and in vivo, for basic and clinical research.