Effects of cell adhesion to the substratum on the growth of chick embryo fibroblasts

Chick embryo fibroblasts were plated on Petri dishes that had not been treated for use in tissue culture (bacteriological dishes). On these dishes the cells grow at the same exponential rate as cells plated on tissue culture dishes, but their growth becomes inhibited sooner after plating, and therefore at a lower cell number per dish. The inhibition of cell growth on bacteriological dishes is correlated with the formation of cell clumps. Clump formation is reversible by mechanical transfer of the clumps to a tissue culture dish: the cells migrate out of the clumps, form a monolayer, and cell growth resumes.Clump formation was studied by time-lapse cinematography, and was found to be due to reduced adhesion of the cells to the bacteriological dish surface. This reduced adhesiveness of the substratum is due to a lower number of negatively-charged residues on the bacteriological dish surface, which can be measured by the binding of crystal violet. The number of negatively-charged residues, and therefore the adhesiveness of the substratum can be altered by treatment of the dishes with sulfuric acid. Serum components of the medium were found to affect cell adhesion to the bacteriological dishes, consequently altering the efficiency of cell attachment, the extent of cell growth and the pattern of clump formation.The cells in clumps were compared with those in confluent monolayers on tissue culture dishes. Growth-inhibited cells on both types of dish were found to be equally viable. Cells in clumps on bacteriological dishes were found to be inhibited in the G1 phase of the cell cycle, as are cells in density-inhibited monolayers. Infection by the oncogenic virus, Rous sarcoma virus, can release the cells from growth-inhibition on both types of dish. Cell-induced alterations of the medium are not involved in the growth inhibition of cells on bacteriological dishes.     

The influence of 1.2 microT, 60 Hz magnetic fields on melatonin- and tamoxifen-induced inhibition of MCF-7 cell growth

We independently examined the findings of Harland and Liburdy, who reported that 1.2 microT(rms), 60 Hz magnetic fields could significantly reduce the inhibitory action of physiological levels of melatonin (10(-9) M) and of pharmacological levels of tamoxifen (10(-7) M) on the growth of MCF-7 human breast cancer cells in vitro. We used two testing protocols. In the melatonin study, the cell numbers per dish on day 7 of treatment were determined using a hemocytometer assay. In the tamoxifen study we used an expanded protocol, employing an alternative cell counting assay to characterize the cell numbers per dish on days 4, 5, 6, and 7. In both the melatonin and tamoxifen studies, cells were plated on 35 mm dishes and placed in each of two exposure chambers inside 5% CO(2) incubators. One exposure chamber was energized to produce 1.2 microT(rms), 60 Hz magnetic fields and the other chamber was not energized. Treatment was continuous until assays were performed. Cells were harvested at selected times, and enumerated without knowledge of treatment. In the melatonin study, the experiment was repeated three times, whereas in the tamoxifen study, each experiment was repeated nine times. In the melatonin study, cell numbers per dish were significantly reduced (by 16.7%) in the melatonin treated cultures after 7 days of incubation compared to control cultures, whereas in the presence of 1.2 microT(rms), 60 Hz magnetic fields, the melatonin treated cultures had the same cell populations as the control cultures. In the tamoxifen study, tamoxifen reduced the cell growth by 18.6 and 25% on days 6 and 7, respectively, in the chamber not energized, while in 1.2 microT(rms), 60 Hz fields, tamoxifen reduced the cell growth only by 8.7 and 13.1%, respectively. These results are consistent with those reported by Harland and Liburdy. A critical element of this successful replication effort was the constructive communication established and maintained with the original investigators. Bioelectromagnetics 22:122-128, 2001. Published 2001 Wiley-Liss, Inc.     

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument‐caused deviations in enzyme kinetics between two MTP‐readers, we comprehensively quantified temperature distribution in 96‐well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96‐well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature‐induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.     

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells.

A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4 x 10(5) cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 microgram DNA or 5 micrograms protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous for other purposes as well where the availability of cells or test substances are limiting factors for large test series.     

The yeast mitochondrial transport proteins: new sequences and consensus residues, lack of direct relation between consensus residues and transmembrane helices, expression patterns of the transport protein genes, and protein-protein interactions with other proteins

Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa subunit with six transmembrane helices. The subunit possesses a sequence motif highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35 yeast (S. cerevisiae) MTP genes were resequenced since the masses of their proteins deviate significantly from the typical 30 kDa. We now find these four proteins to have 545, 285, 902, and 502 residues, respectively. Together with only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of some of the five residues that are absolutely conserved among the 12 MTPs with identified transport function and 17 other MTPs. We do now find these five consensus residues also in the new sequences of YNL083W and YFR045W. Additional analyses of the 35 yeast MTPs show that the location of transmembrane helix sequences do not correlate with the general consensus residues of the MTP family; protein segments connecting the six transmembrane helices and facing the intermembrane space are not uniformly short (about 20 residues) or long (about 40 residues) when facing the matrix; most MTPs have at least one transmembrane helix for which the sum of the negative hydropathy values of all residues yields a very small negative value, suggesting a membrane location bordering polar faces of other transmembrane helices or a non-transmembrane location. The extra residues of the three large MTPs are hydrophilic and at the N-terminal. The 200-residue N-terminal segment of YNL083W has four putative Ca2+-binding sites. The 500-residue N-terminal segment of YPR021C shows sequence similarity to enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is expressed solely during sporulation, while the expressions of YFR045W, YPR021C, and YDR470C are induced by various stress situations. These results also show that the 35 MTP genes are expressed under a rather diverse set of metabolic conditions that may help identify the function of the proteins. Interestingly, yeast two-hybrid screens, that will also be useful in identifying the function of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with non-MTPs.     

Action of 50 Hz magnetic fields on neurite outgrowth in pheochromocytoma cells

This study tests the capacity of 50 Hz magnetic and electric fields to stimulate neurite outgrowth in PC-12D cells, a cell line which originated from a pheochromocytoma in rat adrenal medulla. The cells were plated on collagen-coated, plastic petri dishes and exposed to sinusoidal 50 Hz magnetic fields for 22 h in a 5% CO₂ incubator at 37°C. Two 1,000 turn coils, 20 cm in diameter, were assembled in a Helmholtz configuration to generate a magnetic field in a vertical orientation, thereby inducing a companion electric field in the dish with intensity proportional to radius. A magnetic-field shield housed the control samples in the same incubator. Total cells and number of cells with neurites at least as long as one cell diameter or having a growth cone were counted within a radius of 0.3 cm of the dish center and within an annulus of 1.7–1.8 cm radii in 60 mm dishes, at 3.6 cm radius in 100 mm dishes, and between 1.9 and 2.1 cm radii in the outer well of organ culture dishes, which are physically separated into two concentric wells. Sham exposure demonstrated no difference in percentage of cells with neurites between the exposed and control locations in the incubator. Exposures were done at 4.0. 8.9, 22, 29, 40, 120, 236, and 400 milliGauss (mG). At dish radii of 1.7–1.8 cm in the 60 mm dishes these magnetic flux densities induced electric fields of 1.1, 2.5, 5.9, 8.1, 11, 33, 65, and 110 μV/m, respectively, while within a radius of 0.3 cm, the induced electric fields were less than 0.2, 0.4, 1.0, 1.5, 1.9, 6.0, 11, and 19 μV/m, respectively. For other dishes, the larger radii produced proportionally larger induced electric fields. At each field strength, there were two control dishes and four to nine exposed dishes: 100 or more cells were counted at each location on the dishes. The results demonstrate that magnetic fields stimulate neurite outgrowth in a flux-density-dependent manner between 22 and 40 mG, reaching an apparent stimulation plateau between 40 and 400 mG; no effects were seen at 8.9 mG or lower. There was no apparent neurite stimulation due to the electric field. Although relatively low intensity (?22mG) magnetic fields alone can stimulate a morphological response in a cell which is normally stimulated by nerve growth factor molecules binding to membrane receptors, the chemical basis of this response is unknown. © 1993 Wiley-Liss. Inc.     

Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates

A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling volumes (600–1,200 μL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing.     

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells

Summary A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4×10⁵ cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling, with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 μg DNA or 5 μg protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous, for other purposes as well where the availability of cells or test substances are limiting factors for large test series.     

Microvinification--how small can we go?

High-throughput methodologies to screen large numbers of microorganisms necessitate the use of small-scale culture vessels. In this context, an increasing number of researchers are turning to microtiter plate (MTP) formats to conduct experiments. MTPs are now widely used as a culturing vessel for phenotypic screening of aerobic laboratory cultures, and their suitability has been assessed for a range of applications. The work presented here extends these previous studies by assessing the metabolic footprint of MTP fermentation. A comparison of Chardonnay grape juice fermentation in MTPs with fermentations performed in air-locked (self-induced anaerobic) and cotton-plugged (aerobic) flasks was made. Maximum growth rates and biomass accumulation of yeast cultures grown in MTPs were indistinguishable from self-induced anaerobic flask cultures. Metabolic profiles measured differed depending on the metabolite. While glycerol and acetate accumulation mirrored that of self-induced anaerobic cultures, ethanol accumulation in MTP ferments was limited by the increased propensity of this volatile metabolite for evaporation in microlitre-scale culture format. The data illustrates that microplate cultures can be used as a replacement for self-induced anaerobic flasks in some instances and provide a useful and economical platform for the screening of industrial strains and culture media.     

Simultaneous confidence regions for closed tests,including Holm‐, Hochberg‐, and Hommel‐related procedures

The derivation of simultaneous confidence regions for some multiple‐testing procedures (MTPs) of practical interest has remained an unsolved problem. This is the case, for example, for Hochberg's step‐up MTP and Hommel's more powerful MTP that is neither a step‐up nor a step‐down procedure. It is shown in this article how the direct approach used previously by the author to construct confidence regions for certain closed‐testing procedures (CTPs) can be extended to a rather general setup. The general results are then applied to a situation with one‐sided inferences and CTPs belonging to a class studied by Wei Liu. This class consists of CTPs based on ordered marginal p‐values. It includes Holm's, Hochberg's, and Hommel's MTPs. A property of the confidence regions derived for these three MTPs is that no confidence assertions sharper than rejection assertions can be made unless all null hypotheses are rejected. Briefly, this is related to the fact that these MTPs are quite powerful. The class of CTPs considered includes, however, also MTPs related to Holm's, Hochberg's, and Hommel's MTPs that are less powerful but are such that confidence assertions sharper than rejection assertions are possible even if not all null hypotheses are rejected. One may thus choose and prespecify such an MTP, though this is at the cost of less rejection power.     

Bonferroni parallel gatekeeping - transparent generalizations, adjusted p -values, and short direct proofs

In the two-step version (Dmitrienko, Tamhane, Wang and Chen, 2006) of the Bonferroni parallel-gatekeeping multiple-testing procedure (MTP): (a) a family F1 of null hypotheses H is used as a gatekeeper for another family F2 in that no H in F2 can be rejected unless at least one H is rejected in F1; (b) a Bonferroni MTP is used for F1 at local multiple-level alpha in the first step; and (c) Holm's (1979) step-down MTP is used in the second step for F2 at a local multiple level that depends on the rejections made in the first step. It is shown in this article that this two-step procedure can be generalized in that any MTP with multiple-level control and available multiplicity-adjusted p -values can be used instead of Holm's MTP in the second step. A further generalization related to what Dmitrienko, Molenberghs, Chuang-Stein and Offen (2005) called modified Bonferroni parallel gatekeeping is also given where in case all H s in F2 are rejected, additional rejections in F1 can be made in a third step at local multiple-level alpha through any MTP that is more powerful than the initial Bonferroni MTP, e.g. Holm's MTP. The proofs that these two generalized Bonferroni parallel-gatekeeping MTPs have multiple-level alpha are short and direct, without closed-testing arguments. Multiplicity-adjusted p -values can easily be calculated for these MTPs. The extensions to several successive gatekeeper families are straightforward. An illustration is given.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Alternative Confidence Regions for Bonferroni‐Based Closed‐Testing Procedures that are not Alpha‐Exhaustive

This article complements the results in Guilbaud (Biometrical Journal 2008; 50 :678–692). Simultaneous confidence regions were derived in that article that correspond to any given multiple testing procedure (MTP) in a fairly large class of consonant closed‐testing procedures based on marginal p‐values and weighted Bonferroni tests for intersection hypotheses. This class includes Holm's MTP, the fixed‐sequence MTP, gatekeeping MTPs, fallback MTPs, multi‐stage fallback MTPs, and recently proposed MTPs specified through a graphical representation and associated rejection algorithm. More general confidence regions are proposed in this article. These regions are such that for certain underlying MTPs which are not alpha‐exhaustive, they lead to confidence assertions that may be sharper than rejection assertions for some rejected null hypotheses H when not all Hs are rejected, which is not the case with the previously proposed regions. In fact, various alternative confidence regions may be available for such an underlying MTP. These results are shown through an extension of the previous direct arguments (without invoking the partitioning principle), and under the same general setup; so for instance, estimated quantities and marginal confidence regions are not restricted to be of any particular kinds/dimensions. The relation with corresponding confidence regions of Strassburger and Bretz (Statistics in Medicine 2008; 27 :4914–4927) is described. The results are illustrated with fallback and parallel‐gatekeeping MTPs.     

Multiparameter analysis of human epithelial tumor cell lines by laser scanning cytometry

Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Methods for sample preparation, event triggering, and the performance of multiple LSC measurements on disaggregated fixed human cells were developed using normal lymphocytes and two human breast cancer cell lines, JC-1939 and MCF-7, as test populations. Optimal conditions for individual cell identification by LSC were found to depend on several factors, including deposited cell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Sparsely deposited cells exhibited the least cell overlap and the brightest immunofluorescent staining. Major advantages of using DNA probes over a cytoplasmic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relatively sharply demarcated nuclear region. The DNA-binding dye LDS-751 was found to be suboptimal for quantitative DNA measurements but useful as a triggering measurement that permits the performance of simultaneous fluorescein isothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements on each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multiple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence measurements on fixed epithelial cells by LSC based on event triggering using the DNA-binding dye LDS 751. Although not ideal for quantitative measurements of cell DNA content, the large Stokes shift of this dye permits the performance of three or more additional fluorescence measurements on each cell.     

Multiparameter analysis of progeny of individual cells by laser scanning cytometry

BACKGROUND: Effectiveness of antitumor drugs to suppress unrestricted proliferation of cancer cells is commonly measured by cell clonogenicity assays. Assays of clonogenicity are also used in studies of stem/progenitor cells and in analysis of carcinogenic transformation. The conventional assays are limited to providing information about frequency of colonies (cloning efficiency) and do not reveal the qualitative (phenotype) attributes of individual colonies that may yield clues on mechanisms by which cell proliferation was affected by the studied agent. METHODS: Laser scanning cytometry (LSC) was adapted to identify and characterize size and phenotype of colonies of MCF-7 cells growing in microscope slide chambers, untreated and treated with the cytotoxic ribonuclease, onconase (Onc). Individual colonies were located and data representing each colony were segmented based on >650-nm fluorescence excited by a He-Ne laser of the cells whose protein was stained with BODIPY 630/650-X. The DNA of the cells was stained with propidium iodide (red fluorescence) whereas specific proteins (estrogen receptor [ER] or tumor suppressor p53) were detected immunocytochemically (green fluorescence), each excited by an Ar ion laser. RESULTS: A plethora of attributes of individual colonies were measured, such as (a) morphometric features (area, circumference, area/circumference ratio, DNA or protein content per area ratio), (b) number of cells (nuclei), (c) DNA content, (d) protein content and protein/DNA ratio, and (e) expression of ER or p53 per colony, per total protein, per nucleus or per DNA, within a colony. Also cell cycle distribution within individual colonies and heterogeneity of colonies with respect to all the measured features could be assessed. The colonies growing in the presence of Onc had many of the above attributes different than the colonies from the untreated cultures. CONCLUSIONS: Analysis of the features of cell colonies by LSC provides a wealth of information about the progeny of individual cells. Changes in colony size and phenotype, reflecting altered cell shape, cell size, colony protein/DNA ratio, and expression of individual proteins, may reveal mechanisms by which drugs suppress the proliferative capacity of the cells. This may include inducing growth imbalance and differentiation and modulating expression of the genes that may be associated with cell cycle, apoptosis, or differentiation in a progeny of individual cells. Extensions of LSC may make it applicable for automatic analysis of cloning efficiency and multiparameter analysis of cell colonies in soft agar. Such analyses may be useful in studies of the mechanisms and effectiveness of antitumor drugs, in the field of carcinogenesis, and for analyzing primary cultures and assessing tumor prognosis and drug sensitivity. The assay can also be adapted to analysis of microbial colonies.     

Acquisition of triacylglycerol transfer activity by microsomal triglyceride transfer protein during evolution

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of neutral-lipid-rich apolipoprotein B (apoB) lipoproteins. Previously we reported that the Drosophila MTP transfers phospholipids but does not transfer triglycerides. In contrast, human MTP transfers both lipids. To explore the acquisition of triglyceride transfer activity by MTP, we evaluated amino acid sequences, protein structures, and the biochemical and cellular properties of various MTP orthologues obtained from species that diverged during evolution. All MTP orthologues shared similar secondary and tertiary structures, associated with protein disulfide isomerase, localized to the endoplasmic reticulum, and supported apoB secretion. While vertebrate MTPs transferred triglyceride, invertebrate MTPs lacked this activity. Thus, triglyceride transfer activity was acquired during the transition from invertebrates to vertebrates. Within vertebrates, fish, amphibians, and birds displayed 27%, 40%, and 100% triglyceride transfer activity compared to mammals. We conclude that MTP triglyceride transfer activity first appeared in fish and speculate that the acquisition of triglyceride transfer activity by MTP provided for a significant advantage in the evolution of larger and more complex organisms.     

Spreading of human fibroblasts in serum-free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin)

We have tested the effect of dithiothreitol (DTT) treatment on the initial spreading of human fibroblasts in serum-free medium in tissue culture dishes. Cell spreading was inhibited following treatment of these cells with 10 mM DTT. Inhibition occurred when the cells were treated at 37°C but not at 4° and was reversible metabolically but not by the addition of sulfhydryl oxidizing reagents. The inhibition was overcome when DTT-treated human fibroblasts were plated on cold insoluble globulin (plasma fibronectin)—coated dishes. Under these conditions spreading appeared to be completely normal, including the formation of focal adhesions. Analysis of the fibronectin concentrations in the human fibroblasts following DTT treatment indicated that there was little decrease in the absolute level of activity as determined in a biological assay for BHK cell spreading on culture dishes. Analysis of the fibronectin distribution on the DTT-treated human fibroblasts by indirect immunofluorescence using a specific anti-CIG antiserum revealed that fibronectin was no longer deposited onto the culture dish surfaces. Even when the DTT-treated human fibroblasts spread in the presence of fetal calf serum, the cell fibronectin remained for the most part in a perinuclear location. These results indicate that DTT treatment of human fibroblasts prevents the normal translocation of fibronectin from a perinuclear location to the surface of the culture dish. This study further supports our hypothesis that the initial spreading in serum-free medium of fibroblasts from cell strains depends upon secretion of fibronectin onto the culture dish surface.     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Large scale in vitro experiment system for 2 GHz exposure

A beam formed radiofrequency (RF) exposure-incubator employing a horn antenna, a dielectric lens, and a culture case in an anechoic chamber is developed for large scale in vitro studies. The combination of an open type RF exposure source and a culture case through which RF is transmitted realizes a uniform electric field (+/-1.5 dB) in a 300 x 300 mm area that accommodates 49 35 mm diameter culture dishes. This large culture dish area enables simultaneous RF exposure of a large number of cells or various cell lines. The RF exposure source operates at 2142.5 MHz corresponding to the middle frequency of the downlink band of the International Mobile Telecommunication 2000 (IMT-2000) cellular system. The dielectric lens, which has a gain of 7 dB, focuses RF energy in the direction of the culture case and provides a uniform electric field. The culture case is sealed and connected to the main unit for environmental control, located outside the anechoic chamber, via ducts. The temperature at the center of the tray, which contains the culture dishes in the culture room, is maintained at 37.0 +/- 0.2 degrees C by air circulation. In addition, the appropriate CO2 density and humidity supplied to the culture case realizes stable long-term culture conditions. Specific absorption rate (SAR) dosimetry is performed using an electric field measurement technique and the Finite Difference Time Domain (FDTD) calculation method. The results indicate that the mean SAR of the culture fluid at the bottom of the 49 (7 x 7 array) culture dishes used in the in vitro experiments is 0.175 W/kg for an antenna input power of 1 W and the standard deviation of the SAR distribution is 59%. When only 25 culture dishes (5 x 5 array) are evaluated, the mean SAR is 0.139 W/kg for the same antenna input power and the standard deviation of the SAR distribution is 47%. The proliferation of the H4 cell line in 72 h in a pair of RF exposure-incubators reveals that the culture conditions are equivalent to those of a common CO2 incubator.