Life Cycle Analysis of Mammalian Cells: II. Cells from the Chinese Hamster Ovary Grown in Suspension Culture

A method for life cycle analysis in mammalian cells which utilizes the collection function has been applied to the Chinese hamster ovary grown in suspension. The following durations were found for the various parts of the life cycle: S, 4.13 hours; G1, 4.71 hours; G2, 2.81 hours; mitosis, 0.81 hours. The cell has a total generation time of 12.4 hours as opposed to 20.1 hours for the S3 HeLa cell. However, the relative lengths of each phase of the life cycle are identical within experimental uncertainty in the two cells.     

Life Cycle Analysis of Mammalian Cells: III. The Inhibition of Division in Chinese Hamster Cells by Puromycin and Actinomycin

Analysis of the effects of actinomycin and puromycin on the G₂ and mitotic parts of the life cycle in Chinese hamster ovary cells grown in suspension and synchronized by thymidine treatment has been carried out. Rates of division of partially synchronized cell populations were measured in the presence and absence of the drugs, and various controls were performed to test for absence of complex side effects. Actinomycin produces a block 1.9 hr before completion of division, while puromycin produces a block almost coinciding with the initiation of mitosis. Evidence is presented that the puromycin block may be a double one, inhibiting one kind of protein synthesis that virtually coincides with the beginning of mitosis and another that occurs about 8 min earlier. The data are interpreted in terms of the time interval between messenger formation and its associated protein synthesis in this region of the life cycle. The various events studied have been provisionally mapped in the G₂ and mitotic periods of the life cycle.     

Application of Methods for Mammalian Chromosome Spreads to the Cells of Cestodes

Tapeworm cells obtained by physical maceration between ground-glass surfaces are incubated for 3 hr in Hanks' balanced salt solution (BSS) supplemented with colchicine to a concentration of 10-⁴ M. After washing in BSS, the cells are incubated for 10 min in 1/4 strength BSS then centrifuged 10 min. Fixation of the intact button of cells (or alternatively, by squirting the cells directly into the fixative) in Carnoy's alcohol-chloroform-acetic acid (6;3:1) for 30 min follows, and cells, dispersed and washed in the fixative, are flattened by dropping the suspension on clean, water-wet slides which are then air-dried and stained with Giemsa diluted 1 ml;47 ml with distilled water to which 2 ml of buffer—M/15 KH₂PO₄, 32 ml, mixed with M/15 Na₂HPO₄, 68 ml—is added. After staining 15 min and washing in distilled water, slides are air-dried and mounted with resin. Well separated and well stained chromosomes have resulted.     

Mitochondrial Events in the Life and Death of Animal Cells: A Brief Overview

Traditionally, mitochondria have been viewed as the powerhouse of the cell, i.e., the site of theoxidative phosphorylation machinery involved in ATP production. Consequently, much of theresearch conducted on mitochondria over the past 4 decades has focused on elucidating both thosemolecular events involved in ATP synthesis by oxidative phosphorylation and those involved inthe biogenesis of the oxidative phosphorylation machinery. While monumental achievements havebeen made, and continue to be made, in the study of these remarkable but extremely complexprocesses essential for the life of most animal cells, it has been only in recent years that a largebody of biological and biomedical scientists have come to recognize that mitochondria participatein other important processes. Two of these are cell death and aging which, not surprisingly, are relatedprocesses both involving, in part, the oxidative phosphorylation machinery. This new awareness hassparked a new and growing area of mitochondrial research, that has become of great interest to awide variety of scientists ranging from those involved in elucidating the role of mitochondria incell death and aging to those interested in either suppressing or facilitating these processes as itrelates to identifying new therapies or drugs for human disease. It is the purpose of this briefintroductory review to provide an overview of those mitochondrial events involved in the life anddeath of animal cells and to indicate how these events might relate to the human aging process.Much more is known, much remains controversial, and even more remains to be learned as indicatedin the excellent set of minireviews that follow.     

A Simple Method for Purification of Vestibular Hair Cells and Non-Sensory Cells,and Application for Proteomic Analysis

Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.     

A Methodological Framework for the Reconstruction of Contiguous Regions of Ancestral Genomes and Its Application to Mammalian Genomes

The reconstruction of ancestral genome architectures and gene orders from homologies between extant species is a long-standing problem, considered by both cytogeneticists and bioinformaticians. A comparison of the two approaches was recently investigated and discussed in a series of papers, sometimes with diverging points of view regarding the performance of these two approaches. We describe a general methodological framework for reconstructing ancestral genome segments from conserved syntenies in extant genomes. We show that this problem, from a computational point of view, is naturally related to physical mapping of chromosomes and benefits from using combinatorial tools developed in this scope. We develop this framework into a new reconstruction method considering conserved gene clusters with similar gene content, mimicking principles used in most cytogenetic studies, although on a different kind of data. We implement and apply it to datasets of mammalian genomes. We perform intensive theoretical and experimental comparisons with other bioinformatics methods for ancestral genome segments reconstruction. We show that the method that we propose is stable and reliable: it gives convergent results using several kinds of data at different levels of resolution, and all predicted ancestral regions are well supported. The results come eventually very close to cytogenetics studies. It suggests that the comparison of methods for ancestral genome reconstruction should include the algorithmic aspects of the methods as well as the disciplinary differences in data aquisition.     

Analysis of Cell Flow and Cell Loss Following X-Irradiation Using Sequential Investigation of the Total Number of Cells In the Various Parts of the Cell Cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. the generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G₂ blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. the experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Analysis of the Cell Cycle and a Method Employing Synchronized Cells for Study of Protein Expression at Various Stages of the Cell Cycle

Study of protein expression during the cell cycle requires preparation of pure fractions of cells at various phases of the cell cycle. This was achieved by the development of methods for cell synchronization. Successful cell synchronization requires knowledge of the duration of all phases of the cell cycle. So, in the present review these interrelated problems are considered together. The first part of this review deals with basic methods employed for analysis of duration of cell cycle phases. The second summarizes data on treatments used for cell synchronization. Methods for calculation of percent of cells at various stages of the cell cycle in fractions of synchronized cells are considered in the third part. The fourth part of this review deals with a method of study of protein expression during the cell cycle by means of immunoblotting of synchronized cell fractions. In the Appendix, basic principles are illustrated with practical examples of analysis of the cell cycle, synchronization, and study of expression of some proteins at various stages of the cell cycle using synchronized XL2 (Xenopus laevis) cells.     

On the Metabolic Relationship between the Calvin Cycle and the Tricarboxylic Acid Cycle: IV. A Plant Survey for Anomalous Acetyl Coenzyme A

Leaves of 10 randomly selected plants representing eight dicotyledonous families were exposed to ¹⁴CO₂ for a 10-minute period in the light. Citrate and alanine were isolated, purified isotopically, and degraded to obtain the ¹⁴C-isotope distribution of corresponding carbon atoms, i.e. citrate (C-1,2) and alanine (C-2,3). The cited carbon atoms of alanine were equally labeled as is typical of a 3-carbon intermediate derived from photosynthetic 3-phosphoglycerate. The carbon atoms of citrate, equivalent to acetyl-CoA, were unequally labeled. The citrate (C-1,2) isotope ratio ranged from 1.20 to 1.78 for the various plants compared to a ratio of unity in the uniformly labeled control. The results infer that 3-phosphoglycerate produced in the chloroplast is not the singular precursor of mitochondrial citrate.     

A Generalized Simplest Equation Method and Its Application to the Boussinesq-Burgers Equation

In this paper, a generalized simplest equation method is proposed to seek exact solutions of nonlinear evolution equations (NLEEs). In the method, we chose a solution expression with a variable coefficient and a variable coefficient ordinary differential auxiliary equation. This method can yield a Bäcklund transformation between NLEEs and a related constraint equation. By dealing with the constraint equation, we can derive infinite number of exact solutions for NLEEs. These solutions include the traveling wave solutions, non-traveling wave solutions, multi-soliton solutions, rational solutions, and other types of solutions. As applications, we obtained wide classes of exact solutions for the Boussinesq-Burgers equation by using the generalized simplest equation method.     

A Method for Numerical Simulation of Single Limb Ground Contact Events: Application to Heel-Toe Running

The objective of this work was to develop a method to simulate single-limb ground contact events, which may be applied to study musculoskeletal injuries associated with such movements. To achieve this objective, a three-dimensional musculoskeletal model was developed consisting of the equations of motion for the musculoskeletal system, and models for the muscle force generation and ground contact elements. An optimization framework and a weighted least-squares objective function were presented that generated muscle stimulation patterns that optimally reproduced subject-specific movement data. Experimental data were collected from a single subject to provide initial conditions for the simulation and tracking data for the optimization. As an example application, a simulation of the stance phase of running was generated. The results showed that the average difference between the simulation and subject's ground reaction force and joint angle data was less than two inter-trial standard deviations. Further, there was good agreement between the model's muscle excitation patterns and experimentally collected electromyography data. These results give confidence in the model to examine musculoskeletal loading during a variety of landing movements and to study the effects of various factors associated with injury. Limitations were examined and areas of improvement for the model were presented.     

A Study of Residue Correlation within Protein Sequences and Its Application to Sequence Classification

We investigate methods of estimating residue correlation within protein sequences. We begin by using mutual information (MI) of adjacent residues, and improve our methodology by defining the mutual information vector (MIV) to estimate long range correlations between nonadjacent residues. We also consider correlation based on residue hydropathy rather than protein-specific interactions. Finally, in experiments of family classification tests, the modeling power of MIV was shown to be significantly better than the classic MI method, reaching the level where proteins can be classified without alignment information.     

A Method for the Electrophoretic Analysis of Proteins and RNAs of Single Cells*

SYNOPSIS A method is described for the electrophoretic analysis of proteins or RNAs from individual amebae. The method is based on fluorographic autoradiography of semi-micro polyacrylamide gels in which [³⁵S]methionine or [³H]uridine materials from single cells have been subjected to electrophoresis. The method is more sensitive and provides better resolution than previous methods for single cells. It is suitable, also, for quantitation of the separated components.     

A Conserved DNA Damage Response Pathway Responsible for Coupling the Cell Division Cycle to the Circadian and Metabolic Cycles

The circadian clock drives endogenous oscillations of cellular and physiological processes with a periodicity of approximately 24 h. Progression of the cell division cycle (CDC) has been found to be coupled to the circadian clock, and it has been postulated that gating of the CDC by the circadian cycle may have evolved to protect DNA from the mutagenic effects of ultraviolet light. When grown under nutrient-limiting conditions in a chemostat, prototrophic strains of budding yeast, Saccharomyces cerevisiae, adopt a robust metabolic cycle of ultradian dimensions that temporally compartmentalizes essential cellular events. The CDC is gated by this yeast metabolic cycle (YMC), with DNA replication strictly segregated away from the oxidative phase when cells are actively respiring. Mutants impaired in such gating allow DNA replication to take place during the respiratory phase of the YMC and have been found to suffer significantly elevated rates of spontaneous mutation. Analogous to the circadian cycle, the YMC also employs the conserved DNA checkpoint kinase Rad53/Chk2 to facilitate coupling with the CDC. These studies highlight an evolutionarily conserved mechanism that seems to confine cell division to particular temporal windows to prevent DNA damage. We hypothesize that DNA damage itself might constitute a “zeitgeber”, or time giver, for both the circadian cycle and the metabolic cycle. We discuss these findings in the context of a unifying theme underlying the circadian and metabolic cycles, and explore the relevance of cell cycle gating to human diseases including cancer.     

Analysis and Characterization of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU)-resistant Euglena: I. Growth, Metabolic and Ultrastructural Modifications during Adaptation to Different Doses of DCMU

Cultures of Euglena gracilis Klebs strain Z Pringsheim were grown photoorganotrophically in the presence of different concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the range of 0.05 to 250 micromolar. Cultures were serially transferred and various metabolic parameters were followed for 10 weeks. A process of adaptation occurred which was divided operationally into three phases. A phase of ultrastructural disorganization occurred, succeeded by a recovery phase; their intensity and duration were functions of the dose of DCMU. A stable adaptation phase then ensued. This phase was observed in all cultures except that exposed to the highest DCMU concentration. Adapted cells from all of the DCMU cultures contained twice the protein and half the paramylon of the control cells and thus utilized the carbon source to accumulate cellular reserves with only half the efficiency of controls. DCMU affected cellular metabolism as well as photosynthesis.     

A Method for DNA Extraction from the Desert Cyanobacterium Chroococcidiopsis and Its Application to Identification of ftsZ

A method was developed for extraction of DNA from Chroococcidiopsis that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of Chroococcidiopsis to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of Chroococcidiopsis cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of Chroococcidiopsis, and a 570-bp fragment of the gene ftsZ was cloned and sequenced.     

A novel approach to the problem of geographic allocation of environmental impact in Life Cycle Assessment and Material Flow Analysis

Life Cycle Assessment and Material Flow Analysis, in spite of their invaluable contribution to the investigation of the environmental performance of human-dominated processes, still fall short of properly addressing the issue of the geographic distribution of the potential environmental impacts, which often has wide-reaching environmental as well as political implications. An innovative allocation method based on matrix algebra is introduced here, in order to allow to split the calculated environmental impact indicators into fractions thereof which are geographically attributed to the different world regions. This is done on the basis of: (i) where the analyzed process takes place and (ii) where the directly and indirectly required fossil and nuclear fuels are sourced from (including those for electricity production). The method has been successfully tested on primary aluminium production, as a first case study.