Current topics in cellular regulation. Vol. 30, 1989

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Insulin was identified as the main component for keeping the specific growth rate constant. The specific growth rate offers a powerful tool for evaluating animal cell cultures and to find growth limiting substances. Unable to display preview. Download preview PDF. Skip to main content. Advertisement Hide. This process is experimental and the keywords may be updated as the learning algorithm improves. This is a preview of subscription content, log in to check access.

Jo, E. Step-fortifications of nutrients in mammalian cell culture. CrossRef Google Scholar. Indeed, a major reason for cell inhibition by intracellular ethanol is reduced water activity, and intracellular acetic acid combined with ethanol will then further reduce the water activity, possibly below the critical level for cell growth [29]. The biophysical mechanisms behind the effect of ethanol and n-butanol on the acetic acid diffusion rate were elucidated using molecular dynamics simulations of a model membrane designed to resemble the lipid composition of S.

To the best of our knowledge, this is the first time biophysical mechanisms governing the effect of ethanol and n-butanol have been elucidated using a complex model membrane with physiological concentrations of the lipids, including IPC, a yeast sphingolipid proven to strongly influence membrane properties [10]. In previous studies, membranes with only one or two different lipid species have mainly been used [23] [30] [31].

The complex membrane composition used in the present work enabled us to better approximate the effect of alcohols on the heterogeneous cell membrane of S. Our simulations showed that alcohols preferably partition into the head group region of the lipid bilayer, a fact also demonstrated in simpler membrane systems [23] [31]. Secondly, n-butanol partitions deeper into the membrane than ethanol, as a direct effect of the longer n-butanol molecule, thereby disrupting more of the van der Waals forces that connect the membrane lipids.

Studies on model membranes with only one lipid component confirm that n-butanol partitions into the membrane to a greater extent than ethanol [23] [33]. The partitioning of alcohols into the head group region of the lipid bilayer pushed the lipids apart, causing an increase in the membrane area. A direct effect of increased lateral area is decreased membrane thickness, as the lipid tails are given more space to adopt less straight conformations.

We observed a reduction in membrane thickness with both alcohols, but not of the same magnitude as the increase in membrane area. The increase in area per lipid and the decrease in membrane thickness have been confirmed for ethanol in simpler membrane systems [30] [35]. Increased membrane fluidity has been reported as an effect of alcohols in yeast and bacteria [22] [36] [37] , and is a natural consequence of increased membrane area and reduced membrane thickness.

In our simulations we determined the lipid order, i. Previous studies using molecular dynamics simulations have also shown that relatively high alcohol concentrations are required to perturb membrane fluidity [23] [31]. It has also been found that microorganisms exposed to alcohol regulate the lipid profile of the cell membrane so as to alter membrane fluidity [22] , and high temperature increasing membrane fluidity, combined with lignocellulose derived inhibitors intensified cell inhibition by ethanol [38]. The biophysical mechanisms governing the effects of alcohols on the cell membrane were mainly increased membrane area and, to a smaller extent, reduced membrane thickness and reduced lipid order.

These changes in physiochemical membrane properties probably caused the increase in acetic acid diffusion rate that we observed experimentally in S. To obtain further evidence that the changes in physiochemical membrane properties caused by alcohols increase acetic acid membrane diffusion, we determined the number of water molecules in the membrane interior of our simulated model membrane.

This increased polarity deeper in the membrane, caused by water, probably facilitates the diffusion of acetic acid through the membrane.

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Previous experiments in a mutant of S. Furthermore, our work shows that the diffusion rate of acetic acid can be greatly influenced by compounds that partition into the cell membrane, such as alcohols. It also highlights the need for a holistic view of the process and knowledge concerning the parameters influencing the physiochemical properties of the membrane, to support conscious choices in process design and strain engineering.

For example, when selecting the appropriate lignocellulosic raw material, it should be borne in mind that softwood has a lower acetyl content than hardwood and annual plants [11] , and is perhaps more suitable for alcohol production, while hardwood and annual plants, with a high acetyl content, are better suited as raw materials for products that do not affect the physiochemical properties of the membrane leading to an increased acetic acid diffusion rate.

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Ethanol and n-butanol severely influenced the rate of acetic acid diffusion in S. The increase in diffusion rate was explained biophysically by alcohol partitioning into the head group region of the lipid bilayer, thereby causing a considerable increase in the area per lipid, together with reduced membrane thickness and lipid order. The increased acetic acid diffusion rate led to reduced specific growth rates and prolonged lag phase of S. The findings of this study demonstrate the successful use of a complex model membrane, including glycerophospholipids, sphingolipids, and sterols, in molecular dynamics simulations, to provide molecular details of the effect of alcohols on the membrane.

It further demonstrates that the diffusion rate of acetic acid can be strongly affected by compounds that partition into the cell membrane, and highlights the need for considering interaction effects between compounds in the design of microbial processes. Vitamin solution and trace element solution were prepared as described previously [42]. Potassium hydrogen phthalate buffer mM was used to maintain the culture at pH 5.

However, mixing medium and stock solutions each with a pH of 5 did not result in a medium of pH 5 when using highly concentrated stock solutions. Therefore, to ensure the correct pH, potassium hydrogen phthalate buffer was prepared at pH 4, mixed with mineral medium and supplements at pH 5, and the mixtures were then separately adjusted to pH 5 using KOH. Exponentially growing cells with an optical density OD at nm of 2 were harvested and used to inoculate cultures for acetic acid diffusion rate determination, or to inoculate microscale cultures to determine the maximum specific growth rate in the presence of ethanol, n-butanol and acetic acid.

Cell cultures for acetic acid diffusion measurements. Cells were resuspended and concentrated times in 50 mM ice-cold potassium hydrogen phthalate buffer at pH 5, and then stored on ice. Acetic acid diffusion was measured by mixing a small amount of [ 14 C] acetic acid with a larger fraction of non-labeled acetic acid. This resulted in total acetic acid concentrations of 0.

The concentration of acetic acid in the stop solution corresponded to the concentration of acetic acid used in the specific diffusion assay. The filters were then washed with 10 mL stop solution and placed in vials with 10 mL Emulsifier-Safe TM scintillation liquid Perkin Elmer, Groningen, the Netherlands and shaken thoroughly. Acetic acid diffusion kinetics was measured using a final amount of 0. To ensure the correct pH, all buffer solutions containing ethanol and n-butanol were separately adjusted to pH 5. The stop solution with the cells was then rapidly filtered and treated as described above for the determination of acetic acid diffusion.

Analysis of intracellular acetic acid concentration. The amount of intracellular acetic acid was determined by measuring the radioactive decay of [ 14 C] acetic acid using a liquid scintillation counter Perkin Elmer, Wallac Guardian The number of scintillation counts was correlated to the concentration of acetic acid by preparing standards of the acetic acid mixture added directly to the scintillation liquid.

Samples were corrected for acetic acid adsorption on the filter and cell surface by subtracting the radioactivity of blanks prepared with the acetic acid mixture and cells added directly to the stop solution, and rapidly processed according to similar procedure as for the samples. The measured radioactive decay was linear in the concentration range evaluated, and no quenching effects from the sample matrix were observed. Microscale cultures to evaluate the effect of ethanol, n-butanol and acetic acid on cell growth.

Cells were concentrated to OD 4 by removing half of the supernatant and resuspending the cells in the remaining supernatant. Microscale cultures of 1 mL were then inoculated at OD 0. To minimize evaporation, the plates were covered with a gas-permeable sealing foil with an evaporation-reducing layer m2p labs.

Optical density values from the BioLector were converted to OD values using a standard curve. The same membrane model was used as in our previous study [10]. The bilayer was neutralized by 64 sodium ions. The membrane was solvated with water and alcohol molecules using the Packmol program [43]. Lipids, water and alcohols were described with the Stockholm lipids [44] , TIP3P [45] , and general Amber force fields [46] , respectively.

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The atomic partial charges on the alcohols were determined using the AM1-BCC method [47] with the Antechamber program [48]. The membranes were simulated with molecular dynamics employing Gromacs software [49] ersion 4. The membranes were simulated for ns; the last ns were used for analysis, and snapshots were collected every 10 ps.

Pressure was maintained at 1 atm using a Parinello-Rahman barostat [51] with a 10 ps coupling constant, controlling the pressure along the membrane normal independently of the membrane plane. Electrostatic interactions were treated with particle-mesh Ewald summation [54] using a 1 nm real-space cut-off.

Van der Waals interactions were cut off at 1 nm, but a long-range continuum correction was added.


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The membrane area and the uncertainty in this were calculated from the size of the simulation box. The bilayer thickness was defined as the average distance between the peak density of the phosphate groups in the two leaflets. Lipid tail order was calculated from the average deuterium order parameter, which estimates the orientation of each carbon in the fatty acyl chain, using a standard formula [55].

The uncertainties of the thickness and order parameters were estimated with block averaging.

The number of water molecules in the membrane interior was determined from the intercept of the density of the water oxygen atoms and the density of the IPC carbon tail atoms: the membrane interior was defined as the area where the tail density was greater than the water density. The uncertainty in the intercept, and thus the uncertainty in the number of water molecules in the interior, was estimated by bootstrap samples of the densities.

Volume 35. Current Topics in Cellular Regulation

The area of the bulk water in the simulation was determined from the intercept of the density of the water oxygen atoms and the density of the IPC phosphate moiety: the area of the bulk water was determined to be the area of the simulation box where the water density was greater than the phosphate density. The number of alcohols in the bulk and the membrane was estimated from this intercept and the average area of the membrane. The uncertainties in the number of alcohols in either the bulk or the membrane as well as the uncertainty in the location of the peak density of the POPI glycerol moiety, alcohol hydroxyl and alcohol terminal methyl group were determined by a bootstrap procedure, similar to the uncertainty of the number of interior water molecules.

References L. Jarboe, L. Royce, and P. Liu, "Understanding biocatalyst inhibition by carboxylic acids", Frontiers in Microbiology , vol. Giannattasio, N. Guaragnella, M. Marra, "Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid", Frontiers in Microbiology , vol. Casal, H. Cardoso, and C. Leao, "Mechanisms regulating the transport of acetic acid in Saccharomyces cerevisiae", Microbiology , vol.

Mollapour, and P. Orij, S. Brul, and G.

Series: Current Topics in Cellular Regulation

Russell, "Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling", Journal of Applied Bacteriology , vol. Fernandes, N. Mira, R. Vargas, I. Canelhas, and I. Lindahl, S. Genheden, L. Eriksson, L. Olsson, and M. Bettiga, "Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii", Biotechnology and Bioengineering , vol.

Taherzadeh, R. Eklund, L. Gustafsson, C. Niklasson, and G. Lindberg, A. Santos, H. Riezman, L. Gorter de Vries, S. Grijseels, M. Swinnen, M. Nevoigt, J. Daran, J. Pronk, and A. Sauer, D. Porro, D. Mattanovich, and P. Branduardi, "Microbial production of organic acids: expanding the markets", Trends in Biotechnology , vol. Mira, M. Taylor, L. Nagy, and T. Chapter References. Subject Index.


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  6. Praise for the Series: "Timely High standard of Writing It is to be highly recommended. This informative publication brings together knowledge of various aspects of cellular regulation. Current Topics in Cellular Regulation reviews the progress being made in those specialized areas of study that have undergone substantial development.

    It also publishes provocative new theories and concepts and serves as a forum for the discussion of general principles. Researchers in cellular regulation as well as biochemists, molecular and cell biologists, microbiologists, biophysicists, physiologists, nutritionists, and pathologists will find Current Topics in Cellular Regulation a useful source of up-to-date information. High standard of writing Biochemists whose major interests include enzymology, control of metabolic pathways, or organelle and membrane synthesis should find this book thought-provoking and well worth their time.

    It is strongly recommended. Libraries serving the biological research community should give this volume high priority. We are always looking for ways to improve customer experience on Elsevier. We would like to ask you for a moment of your time to fill in a short questionnaire, at the end of your visit. If you decide to participate, a new browser tab will open so you can complete the survey after you have completed your visit to this website. Thanks in advance for your time. Skip to content. Search for books, journals or webpages All Pages Books Journals.