Hydrodynamic damage to animal cells.

Animal cells are affected by hydrodynamic forces that occur in culture vessel, transfer piping, and recovery operations such as microfiltration. Depending on the type, intensity, and duration of the force, and the specifics of the cell, the force may induce various kinds of responses in the subject cells. Both biochemical and physiological responses are observed, including apoptosis and purely mechanical destruction of the cell. This review examines the kinds of hydrodynamic forces encountered in bioprocessing equipment and the impact of those forces on cells. Methods are given for quantifying the magnitude of the specific forces, and the response thresholds are noted for the common types of cells cultured in free suspension, supported on microcarriers, and anchored to stationary surfaces.     

Measurement of biocolloid collision efficiencies for granular activated carbon by use of a two-layer filtration model

Point-of-use filters containing granular activated carbon (GAC) are an effective method for removing certain chemicals from water, but their ability to remove bacteria and viruses has been relatively untested. Collision efficiencies (alpha) were determined using clean-bed filtration theory for two bacteria (Raoutella terrigena 33257 and Escherichia coli 25922), a bacteriophage (MS2), and latex microspheres for four GAC samples. These GAC samples had particle size distributions that were bimodal, but only a single particle diameter can be used in the filtration equation. Therefore, consistent with previous reports, we used a particle diameter based on the smallest diameter of the particles (derived from the projected areas of 10% of the smallest particles). The bacterial collision efficiencies calculated using the filtration model were high (0.8 < or = alpha < or = 4.9), indicating that GAC was an effective capture material. Collision efficiencies greater than unity reflect an underestimation of the collision frequency, likely as a result of particle roughness and wide GAC size distributions. The collision efficiencies for microspheres (0.7 < or = alpha < or = 3.5) were similar to those obtained for bacteria, suggesting that the microspheres were a reasonable surrogate for the bacteria. The bacteriophage collision efficiencies ranged from > or = 0.2 to < or = 0.4. The predicted levels of removal for 1-cm-thick carbon beds ranged from 0.8 to 3 log for the bacteria and from 0.3 to 1.0 log for the phage. These tests demonstrated that GAC can be an effective material for removal of bacteria and phage and that GAC particle size is a more important factor than relative stickiness for effective particle removal.     

Mitotic spindle morphogenesis in animal cells.

Assembly of the mitotic spindle is an interesting example of morphogenesis at the cellular level. The temporal control of this major event involves the periodic activation of the cyclin-cdc2 kinase complex. In this review, I report recent results that have shed some light on the temporal regulation of centrosome duplication, microtubule nucleation and microtubule dynamics. Reorganization of highly dynamic microtubules into a bipolar spindle probably requires kinesin and dynein-like motors and their role is discussed in an hypothetical model that may be applicable to all mitotic spindles.     

Fluid-mechanical damage of animal cells in bioreactors.

The fluid-mechanical and some biological aspects of damage to animal cells in bioreactors due to agitation and/or aeration are attracting renewed attention. In microcarrier bioreactors, cell damage is due to forces generated by the interaction of microcarrier beads with each other and also with small turbulent eddies. For freely suspended cells grown in mixed bioreactors, cell damage is most frequently due to bubble breakup or fast-draining liquid films around rearranging gas-liquid interfaces.     

The turnover of tRNAs microinjected into animal cells.

Red cell-mediated microinjection has been used to study tRNA turnover in SV3T3 mouse cells and TC7 cells, an African green monkey kidney line. The turnover of endogenous tRNA, measured by labeling with 3H-methionine, was first-order with half-lives of approximately one day in SV3T3 and two days in TC7 cells. 32PtRNA isolated from E. coli or TC7 cells turned over at the same rate as endogenous tRNA when injected into either SV3T3 or TC7 cells. This demonstrates that cellular processes, not properties inherent to tRNAs, are responsible for the difference in tRNA turnover observed between SV3T3 and TC7 cells. These results further indicate that the mechanism of tRNA turnover in mammaliam cells does not distinguish prokaryotic from eukaryotic tRNAs. In contrast to unmodified tRNA, glyoxalated tRNA was rapidly degraded upon injection. Thus altered tRNA's, like altered proteins, are turned over more rapidly in animal cells.     

Initiator RNA of nascent DNA from animal cells.

Nascent DNA synthesized by intact cells has been examined for the presence of RNA that may function as a primer in the discontinuous synthesis of DNA. A low molecular weight fraction that contains nascent DNA was isolated from a human lymphoblastoid cell line in logarithmic growth. After labeling the 5′ ends with bacteriophage T4 polynucleotide kinase and [γ-³²P]ATP, and digestion of the DNA with DNAase, a DNAase-resistant oligonucleotide was isolated. This fragment consisted of approximately 9 ribonucleotide residues, with 5′ terminal purines ($\text{A}\text{G}\text{=}\text{3·5}\text{1}$), plus one to three 3′ terminal deoxynucleotides resulting from incomplete removal by DNAase. Approximately 10% of short nascent DNA chains contained the nonanucleotide molecule. An additional 20% of the nascent DNA contained ribooligomers shorter than 9 residues, with 5′ termini substantially increased in pyrimidines, which may result from degradation of the nonanucleotide. These results extend previous studies that demonstrated a similar ribooligonucleotide present at the 5′ end of most or all short nascent DNA chains synthesized in broken cell systems. Together with the results obtained by Reichard and co-workers (Reichard et al., 1974) with polyoma virus, the data support a mechanism by which a short initiator RNA serves as primer for discontinuously synthesized DNA in animal cells.     

DNA-topoisomerases of animal cells

The properties and functions of the DNA topoisomerases are reviewed on the basis of the literature and the author's own data. The techniques of isolation and characterization of the covalent complexes of the topoisomerases with DNA are described.     

Quantum efficiencies of bacteriorhodopsin photochemical reactions.

Determination of quantum efficiencies of bacteriorhodopsin (bR) photoreactions is an essential step toward a full understanding of its light-driven proton-pumping mechanism. The bR molecules can be photoconverted into and from a K state, which is stable at 110 K. I measured the absorption spectra of pure bR, and the photoequilibrium states of bR and K generated with 420, 460, 500, 510, 520, 540, 560, 570, 580, 590, and 600 nm illumination at 110 K. The fraction of the K population in the photoequilibrium state, fk, is determined by AbR and AK the absorbances of the bR and K states at the excitation wavelengths, and also by phi 1 and phi 2, the quantum efficiencies for the bR to K and K to bR photoconversion: fK = phi 1 AbR/(phi 1AbR + phi 2Ak). By assuming that the ratio phi 1/phi 2 is the same at two different but close wavelengths, for example 570 and 580 nm, the value of phi 1/phi 2 at 570 and 580 nm was determined to be 0.55 +/- 0.02, and the spectrum of the K state was obtained with the peak absorbance at 607 nm. The values of phi 1/phi 2 at the other excitation wavelengths were then evaluated using the known K spectrum, and show almost no dependence on the excitation wavelength within the main band. The result phi 1/phi 2 = 0.55 +/- 0.02 disagrees with those of many other groups. The advantages of this method over others are its minimal assumptions and its straightforward procedure.     

The efficiencies of the component steps of oxidative phosphorylation. II. Experimental determination of the efficiencies in mitochondria and examination of the equivalence of membrane potential and pH gradient in phosphorylation

In the accompanying article (T.E. Gunter and B.D. Jensen, 1986 Arch. Biochem. Biophys. 248, 289-304), a method is described for measuring the efficiencies of individual steps of the process of oxidative phosphorylation. The results of applying this method to the case of state 3 phosphorylation in rat liver mitochondria are reported here. The rate of energy use (or power use) at the gradient generation, leakage, and phosphorylation steps are reported as efficiencies and energy use factors in tabular form. The limits of the degrees of coupling of the gradient generation and phosphorylation steps are also determined and under the current conditions of measurement these degrees of coupling are found to be quite close to unity. The data can be used to show that the only sets of the stoichiometric parameters noH (the charge/2e- ratio in this case from succinate to oxygen), nPH (the H+/ATP ratio), and nTH (number of protons translocated during substrate-product transport) which are simultaneously consistent with both the laws of thermodynamics and with the current data are 8, 3, 1, and 6, 3, 0. The The efficiency of the phosphorylation step which is independent of noH and nTH averages 80% for the control data analyzed. If noH is 8 (succinate to oxygen), the average value of the efficiency of generation of the electrochemical proton gradient is approximately 91 percent. Since very little power (energy) would then be left over to be coupled in parallel to phosphorylation through some other means of coupling, this would place the electrochemical proton gradient in the direct path of power flow and identify it as "an" intermediate in the process. This would suggest that any other intermediate should be considered as being "in series" with the electrochemical proton gradient. The agents butyrate and propionate have been employed to permit investigation over a range of pH gradient and membrane potential. Both butyrate and propionate decrease the efficiency of generation of the electrochemical proton gradient and increase proton leakage. In addition, butyrate activates electron transport whereas propionate inhibits it. By using butyrate to modify the values of pH gradient and membrane potential, it can be shown that the ratio of the efficiency with which the pH gradient is used in phosphorylation to that with which the membrane potential is used is 1.08 +/- 0.38.     

Endoglycosidase H-sensitive glycopeptides in eleven different animal cells.

We have examined the distribution of mannose-labelled glycopeptides in eleven different animal cells grown in vitro. All of the cells examined contained endoglycosidase H-sensitive species of high and low molecular weight, associated with the cell material and with the cell surface; however, the distribution between the two pools was different, suggesting a 'sorting out' of glycoproteins. Another conclusion from our studies is that the oligosaccharide processing known to occur during or after membrane glycoprotein translation is incomplete in a high percentage of mannose-containing N-linked oligosaccharides of cell surfaces. There was no consistent correlation between the relative amounts of endoglycosidase H-sensitive and -resistant glycopeptides and whether the cells were normal, virus-transformed or tumour-derived.     

Concentrating mammalian cells I. large-scale animal cell culture

Concentrating mammalian cells in the large-scale cell culture serves different purposes: the retention of animal cells in bioreactor systems to increase the usable cell density and, therefore, the unit productivity; the concentration of mammalian cells for their subsequent reuse. This review describes the present status of both types of concentration, compares the different application possibilities with their advantages/disadvantages, and gives a short future outlook for this field of biotechnology.