Influence of reaction and diffusion on spatial organization of mitochondria and effectiveness factors in skeletal muscle cell design

A mathematical model is developed to analyze the influence of chemical reaction and diffusion processes on the intracellular organization of mitochondria in skeletal muscle cells. The mathematical modeling approach uses a reaction-diffusion analysis of oxygen, ATP, and ADP involved in energy metabolism and mitochondrial function as governed by oxygen supply, volume fraction of mitochondria, and rates of reaction. Superimposed upon and coupled to the continuum species material balances is a cellular automata (CA) approach governing mitochondrial life cycles in response to the metabolic state of the cell. The effectiveness factor (η), defined as the ratio of reaction rate in the system with finite rates of diffusion to those in the absence of any diffusion limitation is used to assess diffusional constraints in muscle cells. The model shows the dramatic effects that the governing parameters have on the mitochondrial cycle of life and death and how these effects lead to changes in the distribution patterns of mitochondria observed experimentally. The model results showed good agreement with experimental results on mitochondrial distributions in mammalian muscle fibers. The η increases as the mitochondrial population is redistributed toward the fiber periphery in response to a decreased availability of oxygen. Modification of the CA parameters so that the mitochondrial lifecycle is more sensitive to the oxygen concentration caused larger mitochondrial shifts to the edge of the cell with smaller changes in oxygen concentration, and thus also lead to increased values of η. The present study shows that variation in oxygen supply, muscle activity and mitochondrial ATP supply influence the η and are the important parameters that can cause diffusion limitations. In order to prevent diffusion constraints, the cell resorts to shifts in their mitochondrial population towards the cell periphery, thus increasing η.     

Production of 2,3-butanediol from D-xylose by Klebsiella oxytoca ATCC 8724

It is known that 2,3-butanediol is a potentially valuable chemical feedstock that can be produced from the sugars present in hemicellulose and celluose hydrolysates. Klebsiella oxytoca is able to ferment most pentoses, hexoses, and disaccharides. Butanediol appears to be a primary metabolite, excreted as a product of energy methabolism. The theoretical maximum yield of butanediol from monosaccharides is 0.50 g/g. This article describes the effects of pH, xylose concentration, and the oxygen transfer rate on the bioconversion of D-xylose to 2,3-butanediol. Product inhibition by butanediol is also examined. The most important variable affecting the kinetics of this system appears to be the oxygen transfer rate. A higher oxygen supply favors the formation of cell mass at the expense of butanediol. Decreasing the oxygen supply rate increases the butanediol yield, but decreases the overall conversion rate due to a lower cell concentration.     

Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems

Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models.     

Optimization of L-phenylalanine production of Corynebacterium glutamicum under product feedback inhibition by elevated oxygen transfer rate.

Production feedback inhibition both on cell growth and on product formation of phenylalanine fermentation might be alleviated by elevated oxygen supply. Batch fermentations by a high phenylalanine producing strain Corynebacterium glutamicum CCRC 18335 at various initial phenylalanine concentrations (P(0)) ranging from 0 to 20 g/L and different oxygen transfer rate coefficients (K(L)a) ranging from 23 to 76 h(-1) were studied. The fermentation parameters with respect to P(0) were strongly dependent on K(L)a. Cell yield favored higher K(L)a and lower P(0). Product yield with respect to varying phenylalanine concentration was evaluated by the relative oxygen availability (ROA). The optimal ROA for phenylalanine formation was strongly dependent on the product concentration. While P(0) was low, the product inhibition was less significant and the maximum product yield occurred while ROA was at 0.5-0.6. While P(0) was high, the product inhibition was significant and the maximum product yield occurred while ROA was at 0.8-0.9. These results suggest that the product feedback inhibition of phenylalanine fermentation processes can be alleviated by a gradual increase in oxygen supply rate while the increasing product concentration is taken into account. The strategy is demonstrated in a fed-batch culture with elevated oxygen supply. The final phenylalanine concentration was 23.2 g/L, which was 45% better than that of the fed-batch fermentation without elevated oxygen supply. Likewise, the maximum productivity was improved by 42% at 0.37 g/(L x h).     

The influence of a granulocytic inhibitor(s) on hematopoiesis in an in vivo culture system.

The in vivo diffusion chamber (DC) technique for mouse marrow culture was used to determine the effect of a granulocyte inhibitor on the proliferation of the pluripotent stem cell(CFU-s) and the granulocyte progenitor cell (CFU-c). Granulocyte conditioned medium was injected intraperitoneally into mice bearing DCs during the initial 48 hr of culture. The early injections of inhibitor resulted in a significantly reduced number of granulocytic progeny formed within the DCs while there was no growth inhibition of mouse fibroblasts cultured under identical conditions. The reduced cell production was due in part to a significant reduction in the self-renewal rate of the CFU-c while no apparent direct effect was observed upon the growth of the CFU-s within the same cultures. These data suggest that the granulocytic inhibitor(s) acted to reduce the proliferation within the CFU-c population and thereby diminished the amplification potential inherent in the initial cell inoculum.     

THE INFLUENCE OF A GRANULOCYTIC INHIBITOR(S) ON HEMATOPOIESIS IN AN IN VIVO CULTURE SYSTEM

The in vivo diffusion chamber (DC) technique for mouse marrow culture was used to determine the effect of a granulocyte inhibitor on the proliferation of the pluri-potent stem cell (CFU-s) and the granulocyte progenitor cell (CFU-c). Granulocyte conditioned medium was injected intraperitoneally into mice bearing DCs during the initial 48 hr of culture. The early injection of inhibitor resulted in a significantly reduced number of granulocytic progeny formed within the DCs while there was no growth inhibition of mouse fibroblasts cultured under identical conditions. The reduced cell production was due in part to a significant reduction in the self-renewal rate of the CFU-c while no apparent direct effect was observed upon the growth of the CFU-s within the same cultures. These data suggest that the granulocytic inhibitor(s) acted to reduce proliferation within the CFU-c population and thereby diminished the amplification potential inherent in the initial cell inoculum.     

Granulocyte and macrophage proliferation after injection of antitumor active and inactive strains of Corynebacterium parvum

Summary Corparvax, a strain of Corynebacterium parvum with strong antitumor activity, had a greater and more prolonged effect of increasing the production of granulocytes and macrophages than did a weak antitumor strain, CN5888. Following the injection of Coparvax to mice, there was a prompt and sustained increase in serum granulocyte/macrophage colony-stimulating activity, an increase in the number of spleen granulocyte/macrophage progenitor cells, an increased rate of proliferation of the bone marrow granulocyte/macrophage progenitor cells and an increase in the number of blood granulocytes and monocytes. The time courses of the increased rates of proliferation of granulocyte/macrophage progenitor cells following the injection of Coparvax were different in the bone marrow and the spleen, suggesting that local microenvironmental factors were also important.If immunostimulants such as C. parvum are to be used in chemoimmunotherapy programs, the kinetics of the increased proliferative rate of the granulocyte/macrophage progenitor cells may be important, since the more rapidly proliferating cells will be more affected by cell cycle-active chemotherapeutic agents.with the technical assistance of Beverly M. Dunne and L. Atherton     

Comparison of substrate utilization and growth kinetics between immobilized and suspended Pseudomonas cells

A methodology is described for measurement if immobilized and suspended cell growth and substrate utilization kinetics parameters. Substrate utilization and growth kinetics were compared between immobilized and suspended cells for toluene degrading Pseudomonas strains K3-2 and 2,4-dichlorophenoxyacetic acid (2,4-D) degrading strain DBO131(pR0101), respectively. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between the immobilized and suspended Pseudomonas cells to determine the effect of immobilization on cellular growth and substrate utilization. Factors influencing the experimental design included calculated oxygen flux rates, primary carbon substrate flux rates, and shear stresses on the immobilize cell. Statistical interpretation of the cellular reaction rate parameters indicates that only the growth kinetics of the toluene system were significantly altered upon immobilization. Substrate utilization kinetics remained unchanged upon immobilization. The substrate growth associated half-saturation constant (K(g)) for the toluene system increased by 30-fold and the maximum specific growth rate (mu(max)) decreased by 2-fold upon immobilization. Implication of these results for experimental determination of cellular kinetic parameters and for immobilization cell bioreactors design are discussed. (c) 1993 John Wiley & Sons, Inc.     

凋亡素2配体对放射线诱导95-D细胞凋亡的影响的研究

目的:探讨凋亡素2配体(Apo-2L)对放射线诱导肺癌95-D细胞凋亡的影响。方法:MTT法检测不同浓度凋亡素2配体在体外对肺癌95-D细胞的抑制率,将细胞分为4组,对照组、凋亡素2配体组、单纯照射组、凋亡素2配体+放射照射组,流式细胞仪检测各组凋亡率及细胞周期。结果:凋亡素2配体对95-D细胞的体外抑制作用明显,随着药物浓度的增大及时间的延长,抑制率明显增高(P0.05)。流式细胞术显示凋亡素2配体与放射线联用能够使95-D细胞的凋亡率提高,与单用凋亡素2配体组及单纯放疗组相比,凋亡率差异显著(P0.05)。结论:凋亡素2配体在体外具有抑制95-D细胞增殖的作用并能够促进细胞的凋亡,同时凋亡素2配体联合放射线可以明显提高肺癌95-D细胞的凋亡率。     

Effect of temperature on the myoglobin-facilitated transport of oxygen in skeletal muscle.

An analysis of thermal effects on the facilitative transport of oxygen in skeletal muscle fibers is presented. Steady-state mass and energy transport balances are written and solved analytically or numerically using a finite-difference procedure. It is shown that no significant spatial thermal gradients exist due to internal reactions or bulk conduction effects across a muscle fiber. At typical muscle conditions, it is predicted that increased global temperature reduces the fraction of oxygenated myoglobin, increases local oxygen concentrations, and increases the percentage of oxygen flux attributed to oxy-myoglobin. The maximum supportable oxygen consumption rate, mO2max, is defined as the highest consumption rate sustainable without developing anoxic regions at the center of the fiber. By considering only temperature sensitive effects within fibers, mO2max is found to increase slightly with temperature at low temperatures. This increase is due to thermal effects on the diffusion coefficients as opposed to effects associated with the kinetics of the myoglobin-oxygen reaction. If the simulations include the temperature effect associated with oxygen solubility in blood plasma, mO2max decreases with temperature. A sensitivity analysis was performed by varying the values of relevant parameters. The maximum consumption rate was least affected by parameters associated with the kinetic and equilibrium constants and most affected by the diffusion coefficients and the concentration of myoglobin.     

Response of multipotent (CFU) and granulocyte (diffusion chamber assay) progenitor cells and differentiating cells of murine haematopoietic tissues to a perturbation of the steady state

Regulation of haematopoiesis was investigated by studying the response of haematopoietic tissues of mice to a perturbation of the steady state by vinblastine (VLB). Progenitor cells were quantified ly limiting dilution analysis of diffusion chamber cultures of haematopoietic cells and by the spleen colony technique. The diffusion chamber technique appears to assay granulocyte progenitor cells and those multipotent progenitor cells that become committed to granulopoiesis during chamber culture. The spleen colony technique probably assays multipotent progenitor cells. Decaying oscillatory responses to VLB were observed for progenitor cells as well as for differentiating cells in bone marrow. The period lengths of the diffusion chamber progenitor cell oscillations might indicate that these were induced by humoral feedback signal(s) from nonproliferative granulocytes. The oscillations of the multipotent progenitor cells of bone marrow were less pronounced and were earlier damped than those of the granulocyte progenitor cells. This may support the hypotesis that multipotent progenitor cells are regulated by more efficient mechanisms, which may depend on short range cell-cell interactions rather than long range humoral regulators.     

Simian immunodeficiency virus inhibits bone marrow hematopoietic progenitor cell growth.

The pathogenic mechanisms underlying the depressed hematopoietic functions seen in human immunodeficiency virus-infected individuals were explored in rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac). Bone marrow hematopoietic progenitor cell colony formation, both granulocyte/macrophage (CFU-GM) and erythrocyte (BFU-E), was shown to be decreased in number in SIVmac-infected rhesus monkeys. SIVmac was readily isolated from bone marrow cells of infected monkeys and was shown to be harbored in macrophages rather than T lymphocytes. The in vitro infection of normal bone marrow cells by SIVmac inhibited colony formation. A striking in vivo correlation between increased SIVmac load in bone marrow cells and decreased hematopoietic progenitor cell colony growth was also shown. Finally, inhibition of SIVmac replication in bone marrow macrophages resulted in increased progenitor cell colony growth from bone marrow cells. These results suggest that the infection of bone marrow macrophages by the acquired immunodeficiency syndrome (AIDS) virus may contribute to depressed bone marrow hematopoietic progenitor cell growth. Moreover, inhibition of AIDS virus replication in these macrophages might induce significant improvement in hematopoietic function.     

Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome.

The clinical radiosensitizer bromodeoxyuridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.     

Model-based estimation of myeloid hematopoietic progenitor cells in ex vivo cultures for cell and gene therapies

Ex vivo production of hematopoietic progenitor cells has potential applications for cell therapy to alleviate cytopenias associated with chemotherapy and for gene therapy. In both therapies, progenitor and stem cells are considered crucial factors for therapeutic success. Assays for progenitor cells, however, take 2 weeks to complete, which is similar to the length of a typical culture. Therefore, a real-time estimation of the percentage or number of progenitor cells, based on rapid measurements, would be useful for optimization of feeding and harvest decisions. In this study, metabolic activity assays and flow cytometric analysis were used to estimate the content of progenitor cells. The measured metabolic activities are a collective contribution from all types of cells. Cells in granulomonocytic cultures have been lumped into six cell types and metabolic rates have been modeled as a linear function of cell composition and growth rate and as a nonlinear function of cell density. Data from 24 experiments were utilized to determine the model parameters in a calibration step. These data include flow cytometric analysis of more mature hematopoietic cells, progenitor cell colony assays, total cell content, and metabolite concentrations, and cover a wide range of cell composition, cell density, and growth rate. After calibration, the model is able to deliver good predictions of progenitor cell content for cultures with higher percentages of progenitor cells, as well as the peak progenitor cell content, based only on parameters that can be rapidly measured. With the aid of those predictions a harvest strategy was developed that will allow optimizing the harvest time based on the culture kinetics of each patient or donor inoculum, rather than using retrospective analysis to determine a uniform harvest time.     

How suspension cultures of Catharanthus roseus respond to oxygen limitation: small-scale tests with applications to large-scale cultures

The effect of oxygen supply on the growth of suspension cultures of Catharanthus roseus in Erlenmeyer flasks was investigated. Below a critical oxygen supply rate the culture could not survive. By increasing the oxygen supply, a point is reached where the culture survives but no growth is possible. At higher oxygen supply rates there is a regime where both growth rate and the maximum biomass concentration increase with oxygen supply. Eventually there comes a point where no further increase in biomass is achieved, probably due to the depletion of the sugars; however, the growth rate continues to increase with oxygen supply until a maximum growth rate is obtained. The ratio of fresh to dry weight at maximum fresh weight increased with shaker table speed of rotation accompanied by a greater rate of sugar depletion.     

Planktonic-cell yield of a pseudomonad biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10(9) cells ml(-1), suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Kinetic aspects of the bioconversion of L-tyrosine into L-DOPA by cells of Mucuna pruriensL. Entrapped in different matrices

Plant cells of Mucuna pruriens L. entrapped In calcium alginate, calcium pectinate, agarose, or gelatine were able to convert L-tyrosine to L-DOPA, which was released Into the medium. Michaelis-Menten kinetics could be applied on the entrapped cells, based on the measurement of initial rates of L-DOPA production. The calculated apparent affinity constants were comparable with the affinity constants obtained with enzyme preparations. Comparison of the apparent maximum rate of bioconversion of the entrapped cells and the maximum rate of bioconversion of a derived cell homogenate indicated that the systems were not operating optimally. Measurement of the effective diffusion coefficients of L-tyrosine pointed out that this substrate could diffuse freely into the matrices. From the initial rates of bioconversion and the effective diffusion coefficients, the observable modulus was calculated for each system. The obtained values confirmed that the diffusional supply rate of L-tyrosine was not the limiting factor. For oxygen, which was utilized for byconversion as well as for cell respiration, the calculated observable moduli was directed toward strong oxygen transfer limitations. The values found for the oxygen consumption indicated that the entrapped cells remained partly or totally viable in the four matrices tested. Based on the highest viability and the highest rates of bioconversion, it was concluded that alginate-entrapped cells of M. pruriens formed the most suitable biocatalytic system for the production of L-DOPA from L-tyrosinre.     

Fermentative ability of Zymomonas mobilis under various oxygen supply conditions in batch culture

The performance of Zymomonas mobilis under various oxygen supply conditions in batch culture was quantitatively investigated. Although both the cell growth rate and ethanol productivity decreased with increases in the oxygen supply, the production of by-products such as acetaldehyde and acetic acid increased. The ethanol productivity was more sensitive to oxygen supply than the growth rate. Oxygen supply also affected the morphology of the cells and an increase in oxygen supply resulted in the elongation of the cells. It was also found that both metabolic activity and fermentation balance were largely affected by changes in the dissolved oxygen concentration during the steady state in oxygen concentration.     

Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome

The clinical radiosensitiser bromodeoxyridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.     

Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot (Daucus carota L.) cell cultures

A bioreactor control system was used to investigate the effects of two dissolved oxygen concentrations (10% and 100%) on the growth and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled without the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing oxygen resulted in a lower growth rate and in a delay in sugar uptake kinetics. Nonetheless, varying levels of oxygen were observed to have no effect on the final dry biomass. The higher alcohol dehydrogenase activity obtained under reduced oxygen conditions suggests that proliferating cultures adapted to the hypoxic environment by inducing alcoholic fermentation. Cell differentiation was highly sensitive to reduced oxygen since under this condition, the somatic embryo production was inhibited by about 75%. Sugar uptake and embryo formation were also delayed.Abbreviations ADH alcohol dehydrogenase - 2,4-D 2,4-dichlorophenoxyacetic acid - DO2 dissolved oxygen - SE somatic embryos - Tris tris(hydroxymethyl)-aminoethane