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PubMed2000

[Tissue engineering with mesenchymal stem cells for cartilage and bone regeneration].

Tissue engineering offers the possibility to fabricate living substitutes for tissues and organs by combining histogenic cells and biocompatible carrier materials. Pluripotent mesenchymal stem cells are isolated and subcultured ex vivo and then their histogenic differentiation is induced by external factors. The fabrication of bone and cartilage constructs, their combinations and gene therapeutic approaches are demonstrated. Advantages and disadvantages of these methods are described by in vitro and in vitro testing. The proof of histotypical function after implantation in vivo is essential. The use of autologous cells and tissue engineering methods offers the possibility to overcome the disadvantages of classical tissue reconstruction--donor site morbidity of autologous grafts, immunogenicity of allogenic grafts and loosening of alloplastic implants. Furthermore, tissue engineering widens the spectrum of surgical indications in bone and cartilage reconstruction.

Bone Regeneration↗
PubMed1999

Chemical engineering of enzymes: altered catalytic activity, predictable selectivity and exceptional stability of the semisynthetic peroxidase seleno-subtilisin.

The increasing demand for enzymes as highly selective, mild, and environmentally benign catalysts is often limited by the lack of an enzyme with the desired catalytic activity or substrate selectivity and by their instability in biotechnological processes. The previous answers to these problems comprised genetically engineered enzymes and several classes of enzyme mimics. Here we describe the potential of chemical enzyme engineering: native enzymes can be modified by merely chemical means and basic equipment yielding so-called semisynthetic enzymes. Thus, the high substrate selectivity of the enzymatic peptide framework is combined with the catalytic versatility of a synthetic active site. We illustrate the potential of chemically engineered enzymes with the conception of the semisynthetic peroxidase seleno-subtilisin. First, the serine endoprotease subtilisin was crystallized and cross-linked with glutaraldehyde to give cross-linked enzyme crystals which were found to be insoluble in water or organic solvents and highly stable. Second, serine 221 in the active site (Enz-OH) was chemically converted into an oxidized derivative of selenocystein (Enz-SeO2H). As a consequence, the former proteolytic enzyme gained peroxidase activity and catalyzed the selective reduction of hydroperoxides. Due to the identical binding sites of the semisynthetic peroxidase and the protease, the substrate selectivity of seleno-subtilisin was predictable in view of the well-known selectivity of subtilisin.

Binding Sites↗
PubMed2006

Influence of fuel properties, nitrogen oxides, and exhaust treatment by an oxidation catalytic converter on the mutagenicity of diesel engine emissions.

Particle emissions of diesel engines (DEP) content polycyclic aromatic hydrocarbons (PAH) these compounds cause a strong mutagenicity of solvent extracts of DEP. We investigated the influence of fuel properties, nitrogen oxides (NO( x )), and an oxidation catalytic converter (OCC) on the mutagenic effects of DEP. The engine was fuelled with common diesel fuel (DF), low-sulphur diesel fuel (LSDF), rapeseed oil methyl ester (RME), and soybean oil methyl ester (SME) and run at five different load modes in two series with and without installation of an OCC in the exhaust pipe. Particles from the cooled and diluted exhaust were sampled onto glass fibre filters and extracted with dichloromethane in a soxhlet apparatus. The mutagenicity of the extracts was tested using the Salmonella typhimurium/mammalian microsome assay with tester strains TA98 and TA100. Without OCC the number of revertant colonies was lower in extracts of LSDF than in extracts of DF. The lowest numbers of revertant colonies were induced by the plant oil derived fuels. In three load modes, operation with the OCC led to a reduction of the mutagenicity. However, direct mutagenic effects under heavy duty conditions (load mode A) were significantly increased for RME (TA98, TA100) and SME (TA98). A consistent but not significant increase in direct mutagenicity was observed for DF and LSDF at load mode A, and for DF at idling (load mode E) when emissions were treated with the OCC. These results raise concern over the use of oxidation catalytic converters with diesel engines. We hypothesise that the OCC increases formation of direct acting mutagens under certain conditions by the reaction of NO( x ) with PAH resulting in the formation of nitrated-PAH. Most of these compounds are powerful direct acting mutagens.

Air Pollutants↗
PubMed2003

Quantitative on-line high-resolution NMR spectroscopy in process engineering applications.

In many technical processes, complex multicomponent mixtures have to be handled, for example, in reaction or separation equipment. High-resolution NMR spectroscopy is an excellent tool to study these mixtures and gain insight in their behavior in the processes. For on-line studies under process conditions, flow NMR probes can be used in a wide range of temperature and pressure. A major challenge in engineering applications of NMR spectroscopy is the need for quantitative evaluation. Flow rates, recovery times, and other parameters of the on-line NMR experiments have to be optimized for this purpose. Since it is generally prohibitive to use deuterated solvents in engineering applications, suitable techniques for field homogenization and solvent signal suppression are needed. Two examples for the application of on-line NMR spectroscopic experiments in process engineering are presented, studies on chemical equilibria and reaction kinetics of the technically important system formaldehyde-water-methanol and investigations on reactive gas absorption of CO(2) in aqueous solutions of monoethanolamine.

Journal Article↗
PubMed2006

Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A.

The display of a protease, carboxypeptidase Y (CPY) or procarboxypeptidase Y (proCPY), which is the vacuolar protease, on the yeast-cell surface was successfully performed using yeast-cell-surface engineering for the first time. Through that we could confirm the processing of vacuolar proteases containing proteinase A (PrA) and proteinase B (PrB) which are related to the maturation of proCPY, using a novel cell-surface engineering technique. Various protease-knockout strains of Saccharomyces cerevisiae with the CPY-displaying system were constructed to evaluate the operation of the activation process of CPY. The display of CPY (CPY-agg, which is a fusion protein of CPY with C-terminal half of alpha-agglutinin) on the cell surface was confirmed by immunofluorescence staining. The activity of the CPY-agg was determined after the conversion of proCPY to active CPY by treatment of whole cells with proteinase K. In the proCPY-displaying CPY-knockout strain and PrB-knockout strain, CPY was displayed as an active (mature) form, but in the proCPY-displaying PrA-knockout strain, CPY was present as an inactive form (proCPY). These facts indicate that PrA had been already activated before its transport to the vacuole and that active mature PrA might convert proCPY to CPY before the transport of proCPY to the vacuole. From these results, it was suggested that by using the yeast-cell-surface engineering at the location of the initial step, the autocatalytic activation from proPrA to PrA might occur before the vacuolar branch separates from the main secretory pathway.

Antigens, Surface↗
PubMed1997

Regulatory Oversight of Genetically Engineered Microorganisms: Has Regulation Inhibited Innovation?

/ Using detailed interviews with company representatives and researchers in the field, this paper examines the factors that might account for the slow pace of development of genetically engineered microorganisms (GEMs) intended for environmental release. We specifically analyzed the role of the regulatory system in shaping innovation. We identified at least two cases where industry decided to discontinue the development of a genetically engineered microbial product because of concerns over regulatory oversight. However, most often industry decisions to continue or halt development of GEMs were based on an evaluation of the particular product's efficacy and potential for profitability. Thus the inability of GEMs to perform up to expectations in the field, rather than the regulatory constraints, appears to be the factor responsible for the slow pace of development. KEY WORDS: Genetically engineered microorganisms; Biotechnology; Regulation of biotechnology; Innovation; Environmental release

Journal Article↗
PubMed2026

Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding.

This review synthesizes the molecular mechanisms of cold tolerance in pepper, integrating multi-omics data,genome editing, and precision breeding strategies to accelerate the development of cold-resilient cultivars. Cold stress is a significant environmental factor that affects the growth, productivity, and fruit quality of Capsicum annuum by impairing membrane integrity photosynthesis and cellular redox homeostasis. Although pepper has several endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, its cold tolerance remains limited due to low transcriptional activation of key regulators, functional redundancy among cold-responsive genes, and the polygenicity of cold tolerance. These complexities, combined with low genetic diversity and linkage drag, have hindered the improvement of cold-resistant cultivars through conventional breeding. This review brings together the recent progress in understanding the molecular mechanisms of cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Precision Breeding 2.0 is a new innovation that combines the integration of multi-omics-based target identification with next-generation genome-editing techniques, allowing precise and multiplex engineering of complex and interconnected regulatory networks instead of single genes. We cover new approaches such as engineering the DREB/CBF pathway, allele-specific editing and targeted disruption of negative regulators to enhance the pathway(s) involved in cold response. Moreover, we propose a roadmap for integration of transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to accelerate the identification, validation, and deployment of superior alleles to boost cold tolerance. This review provides a foundation for developing climate-resilient pepper cultivars by connecting functional genomics with precision genome engineering approaches to maintain productivity under variable environmental conditions.

Capsicum↗
PubMed2000

Control engineering and electromyographic kinesiology analyses of normal human gait.

In this study, we analyzed the electrical activity patterns of the antagonistic bi-articular and mono-articular muscles of both legs during normal gait cycles, in terms of electromyographic (EMG) kinesiology and control engineering. For control engineering analyses, we utilized a mechanical two-joint link model equipped with antagonistic pairs of bi-articular and mono-articular muscles. It was confirmed that the coordinated activity pattern, in which the bi-articular muscles of the rectus femoris (Rf) and the medial hamstrings (Mh) showed criss-cross EMG patterns, and the mono-articular muscles of the gluteus maximus and the vastus medialis showed sustained activities during the early stance phase in the gait cycle, contributed to the output force control and the output force direction control. Reversal of Rf and Mh activities was responsible for changes in the output force direction during the heel contact period. The results obtained here strongly highlight the importance of and necessity for control engineering evaluation of coordinated muscle activities of bi-articular and mono-articular antagonistic muscles for analyses not only of gait but also of sports injuries.

Adolescent↗
PubMed2005

A comparative risk assessment of genetically engineered, mutagenic, and conventional wheat production systems.

Wheat (Triticum aestivum L.) varieties produced using modern biotechnologies, such as genetic engineering and mutagenic techniques, have lagged behind other crop species, but are now being developed and, in the case of mutagenic wheat, commercially grown around the world. Because these wheat varieties have emerged recently, there is a unique opportunity to assess comparatively the potential environmental risks (human health, ecological, and livestock risks) associated with genetically engineered, mutagenic, and conventional wheat production systems. Replacement of traditional herbicides with glyphosate in a glyphosate-tolerant (genetically engineered) wheat system or imazamox in an imidazolinone-tolerant (mutagenic) wheat system may alter environmental risks associated with weed management. Additionally, because both systems rely on plants that express novel proteins, the proteins and plants themselves may impose risks. The purpose of our study was to examine comparatively the multiple aspects of risk associated with different wheat production systems in the US and Canada using the risk assessment paradigm. Specifically, we used tier 1 quantitative and qualitative risk assessment methods to compare specific environmental risks associated with the different wheat production systems. Both glyphosate and imazamox present lower human health and ecological risks than many other herbicides associated with conventional wheat production systems evaluated in this study. The differences in risks were most pronounced when comparing glyphosate and imazamox to herbicides currently with substantial market share. Current weight-of-evidence suggests that the transgenic CP4 EPSPS protein present in glyphosate-tolerant wheat poses negligible risk to humans, livestock, and wildlife. Risk for mutated AHAS protein in imidazolinone-tolerant wheat most likely would be low, but there are not sufficient effect and exposure data to adequately characterize risk. Environmental risks for herbicides were more amenable to quantitative assessments than for the transgenic CP4 EPSPS protein and the mutated AHAS protein.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1984

Knowledge engineering: a daily activity on a hospital ward.

Two common barriers against the development and diffusion of expert systems in medicine are the difficulty of design and the low level of acceptance. An original experience which entails potential solutions of these issues is reported on: the task of knowledge engineering is performed by residents and medical students on a psychiatry ward. They developed a computer program for the psychopharmacological treatment of depression, using a sophisticated knowledge acquisition system, EMYCIN. The knowledge engineering sessions are analysed in detail and a structured method is proposed. A transcript of a sample run of the resulting program is presented along with an evaluation of its performance, acceptance, educational potential, and amount of endeavour required. The impact of the knowledge engineering process itself is then assessed both from the residents and the medical students standpoint. Finally, the possibility of generalizing the experiment is examined.

Antidepressive Agents↗
PubMed1993

Noise level of dental handpieces and laboratory engines.

Impaired hearing in dental practitioners has been the subject of various investigations. Although individual age and susceptibility can play a significant role, the intensity of the noise, distance from the noise source, and total duration of the noise are the important factors in hearing. In this study the noise levels of different handpieces and laboratory engines were evaluated. Handpiece noise measurements were made while instruments were running free and during operation with various cutting tools. These measurements were performed with two kinds of noise-measuring equipment: (1) a precision sound level meter and (2) a noise recording and analyzing circuit. The results indicated that (1) there were significant differences among the noise levels of the various dental engines used, (2) the low-speed, angled-design handpiece has the lowest noise level, whereas the laboratory engine has the highest, (3) the noise level increases during cutting was compared with noncutting, and (4) there was no significant difference between the two methods of measurement.

Dental High-Speed Equipment↗
PubMed1983

Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny.

Cloned DNA sequences encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase have been inserted into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the "rooty" locus. Transformation of tobacco stem segments with the engineered bacterial strains produced attenuated crown gall tumors that were capable of regeneration into intact, normal tobacco plants. The yeast gene and entire transferred DNA (T-DNA) were present in the regenerated plants in multiple copies, and nopaline was found in all tissues. The plants were fertile, and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA. Expression of nopaline in the germinated seedlings derived from one regenerated plant was variable and did not correlate with the levels of T-DNA present in the seedlings. Preliminary evidence indicates that nopaline in progeny of other similarly engineered plants is more uniform. The disarming of pTiT37 by insertions at the "rooty" locus thus appears to produce a useful gene vector for higher plants.

Alcohol Oxidoreductases↗
PubMed1989

Electrochemical engineering approach to the irrigation of tooth canals under the influence of a vibrating file.

Doubt exists concerning the irrigation of secondary tooth canals when the vibration of a file introduced into the main root canal is superimposed on flow-through irrigation of the main canal. Depending on the type of commercial equipment used, the vibrations of the file are sonic or ultrasonic. We use the methodology of an electrochemical engineering approach in a study of the intensity of the irrigation of the secondary canals under the influence of sonic and ultrasonic vibrations of a file located at various heights in the main canal. The tooth canals are simulated by small electrochemical cells containing electrodes located at the end of cavities simulating the secondary canals. An electrochemical method is used to measure the mass transfer coefficients at these electrodes, a classical method in electrochemical engineering laboratories. The values of the mass transfer coefficients are representative of the importance of the irrigation of the electrodes. Thus they give a quantitative idea of the vibrations on the irrigation of secondary tooth canals. The work demonstrates the usefulness of the electrochemical engineering approach in contributing to the solution of a biomedical problem.

Electrochemistry↗
PubMed1996

Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques.

The engineering of living tissues in vivo requires new concepts in cell culture technology. In contrast to conventional cell cultures, the development of tissues depends on a three-dimensional arrangement of cells and the formation or synthesis of an appropriate extracellular matrix. Special emphasis is given to the major role of the extracellular matrix and cell differentiation in an artificial tissue. New technical approaches of in vitro tissue engineering are compared to the natural development of tissues in vivo. Current methods using resorbable biomaterials, tissue encapsulation and perfusion culture are discussed. Major consideration is given to scaffold structures of biomaterials that define a three-dimensional shape of a tissue or guide matrix formation. The different goals of tissue engineering such as in vitro models and transplant production are taken into account in the described techniques. Practical concepts comprising cell multiplication and differentiation in subsequent steps for future clinical applications are outlined.

Animals↗
PubMed1996

Tissue engineering scaffolds using superstructures.

Here, scaffolds as cell and tissue carriers are approached from an engineering point of view, emphasizing material superstructuring in the design of supports. Superstructure engineering provides optimal spatial and nutritional conditions for cell maintenance by the arrangement of structural elements (e.g. pores or fibres) so as to vary the order of cell to cell contact. This approach is illustrated in the design of several scaffolds: knitted fabrics as three-dimensional superstructures for optimized osteosynthesis implants, a new injectable open porous implant system, an angiopolar non-degradable ceramic cell carrier, and an injectable or microsurgically implantable entangled carrier system. The implications for tissue engineering are discussed.

Anisotropy↗
PubMed1993

A comparative study of the use of primary Chinese hamster liver cultures and genetically engineered immortal V79 Chinese hamster cell lines expressing rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus assays.

Liver microsome preparations (S9 mix) have been extensively used for in vitro genotoxicity studies to provide the capacity for the activation of indirect genotoxins. However, the use of S9 preparations with mammalian cell cultures has raised considerable toxicity problems which limit their use to exposure times which are only a small fraction of the cell cycle. In addition, false negative results may be obtained if reactive metabolites are unable to penetrate the cell membrane or have short half-lives. The generation and detection of a promutagen within a single cell would therefore be advantageous. To this end, we have studied the bioactivation of a panel of promutagens (benzo[a]pyrene, cyclophosphamide, 2-aminoanthracene and sterigmatocystin) in low passage Chinese hamster fibroblasts of hepatic origin (LiC2 cells) and in a series of V79 Chinese hamster cell lines genetically engineered to express rat liver cytochrome P450 cDNAs. These include strains XEM2 (expresses CYP1A1), SD1 (CYP2B1) and strains XEMd-MZ and XEMd-NH which express CYP1A2. The end point selected for study was the induction of micronuclei. The protocol incorporated a cytochalasin B-induced cytokinesis block and the enumeration of micronuclei in the resulting binucleate cells which have undergone one nuclear division following the induction of chromosome damage. Micronuclei containing whole chromosomes and chromosome fragments were distinguished by the use of CREST antibody specific for kinetochore protein as a measure for the presence of centromeres. Micronuclei were induced by the test agents in low passage liver fibroblasts and in immortal V79 cultures only in the presence of Aroclor-induced S9 preparations. The data obtained from micronucleus assays of the genetically engineered V79 cell lines demonstrated the utility of each strain for the optimal detection and quantification of the activity of the individual test compounds. Kinetochore antibody demonstrated differences in the kinetics of induction of micronuclei containing chromosome fragments and whole chromosomes with chemicals such as benzo[a]pyrene. As part of this cytogenetic study, we also conducted karyotypic analyses and spindle fidelity assays of the V79 cell lines to investigate the presence of chromosomal instabilities which may arise as a consequence of the genetic engineering procedure. Such studies represent an important quality control step in the validation of the suitability of each cell line prior to their use in genotoxicity studies.

Animals↗
PubMed1985

Acute toxicity of gasoline and ethanol automobile engine exhaust gases.

A comparative inhalation exposure study was performed to investigate the potential health effect of gasoline and ethanol engine exhaust fumes. Wistar rats housed in inhalation chambers were exposed to test atmospheres of various concentrations of carbon monoxide (CO) and gasoline and ethanol exhaust fumes diluted with air. CO level, temperature, relative humidity and flow rate were monitored continually to control the gas concentration and the environment. The dilution method gave a concentration within 1.0% of the target. The LC50s for 3-h exposures were determined for the 3 test atmospheres. The results demonstrated that the acute toxicity, in terms of LC50, of the gasoline-fuelled engine was significantly higher than that of the ethanol-fuelled engine.

Animals↗
PubMed1993

Natural genetic engineering of the bacterial genome.

The term 'natural genetic engineering' means viewing genetic change as a coordinated cell biological process, the reorganization of discrete genomic modules, resulting in the formation of new DNA structures. Examples of natural genetic engineering continue to accumulate, and the concept can be used to integrate observations which demonstrate the similarity between in vitro genetic engineering and the action of in vivo agents of genetic change.

DNA, Bacterial↗

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