Contributions of Cell Growth and Biochemical Reactions to Nongenetic Variability of Cells

Cell-to-cell variability in the molecular composition of isogenic, steady-state growing cells arises spontaneously from the inherent stochasticity of intracellular biochemical reactions and cell growth. Here, we present a general decomposition of the total variance in the copy number per cell of a particular molecule. It quantifies the individual contributions made by processes associated with cell growth, biochemical reactions, and their control. We decompose the growth contribution further into variance contributions of random partitioning of molecules at cell division, mother-cell heterogeneity, and variation in cell-cycle progression. The contribution made by biochemical reactions is expressed in variance generated by molecule synthesis, degradation, and their regulation. We use this theory to study the influence of different growth and reaction-related processes, such as DNA replication, variable molecule-partitioning probability, and synthesis bursts, on stochastic cell-to-cell variability. Using simulations, we characterize the impact of noise in the generation-time on cell-to-cell variability. This article offers a widely-applicable theory on the influence of biochemical reactions and cellular growth on the phenotypic variability of growing, isogenic cells. The theory aids the design and interpretation of experiments involving single-molecule counting or real-time imaging of fluorescent reporter constructs.     

Differential Responses of Buckwheat Leaf Cells to Growth Substances Stimulating Cell Division

Isolated buckwheat cotyledons form calli, roots or buds whencultured in an appropriate medium. A medium containing high2,4-D (5 mg 1^–1) and low KN (01 mg I^–1), which inducescallus formation, was found to stimulate cell division in thelayer between palisade and spongy parenchyma tissue after 72h. Low 2,4-D and low KN (01 mg I^–1 each), which stimulatesroot formation in buckwheat cotyledons, induces divisions primarilyin spongy parenchyma cells. In a high benzylaminopurine (10^–5M) and a low IAA (10^–6 M) medium, which favours bud induction,cell divisions were localized to the palisade layer. The differentialresponsiveness of leaf cells to various hormone treatments isdiscussed.     

Two Independent Contributions to Step Variability during Over-Ground Human Walking

Human walking exhibits small variations in both step length and step width, some of which may be related to active balance control. Lateral balance is thought to require integrative sensorimotor control through adjustment of step width rather than length, contributing to greater variability in step width. Here we propose that step length variations are largely explained by the typical human preference for step length to increase with walking speed, which itself normally exhibits some slow and spontaneous fluctuation. In contrast, step width variations should have little relation to speed if they are produced more for lateral balance. As a test, we examined hundreds of overground walking steps by healthy young adults (N = 14, age < 40 yrs.). We found that slow fluctuations in self-selected walking speed (2.3% coefficient of variation) could explain most of the variance in step length (59%, P < 0.01). The residual variability not explained by speed was small (1.5% coefficient of variation), suggesting that step length is actually quite precise if not for the slow speed fluctuations. Step width varied over faster time scales and was independent of speed fluctuations, with variance 4.3 times greater than that for step length (P < 0.01) after accounting for the speed effect. That difference was further magnified by walking with eyes closed, which appears detrimental to control of lateral balance. Humans appear to modulate fore-aft foot placement in precise accordance with slow fluctuations in walking speed, whereas the variability of lateral foot placement appears more closely related to balance. Step variability is separable in both direction and time scale into balance- and speed-related components. The separation of factors not related to balance may reveal which aspects of walking are most critical for the nervous system to control.     

Morphological and Biochemical Changes During Programmed Cell Death of Rat Cerebellar Granule Cells

Cultured cerebellar granule cells deprived of depolarizing concentrations of KCl and serum die by programmed cell death. Recently, it was shown that serum removal by itself can lead to oxidative stress and DNA fragmentation in these cells. We have modified the protocol which initiates cell death in such a way that only the effect of KCl withdrawal-induced cell death was observed. We have performed a series of experiments to correlate the structural and biochemical changes in this process of cell death. Significant morphological alterations occur in cell bodies and neurites during a 48-hour period of KCl removal. Cell viability dropped to 53%, 34% or 10% of control levels, respectively, as a result of 1-, 2-, or 3-day KCl removal. A series of experiments was conducted to determine the change of total protein level, protein synthesis rate, RNA synthesis rate, and mitochondrial activity during the first 48 hours of KCl removal. These studies not only provide a picture correlating the morphological and biochemical changes in the process of programmed cell death, but also serve as a reference for future studies of this complex phenomenon.     

A Measure of Biochemical Individual Variability

Once a questionable substance is found in a polluted area, we determine its MLD, LD₅₀ etc. by animal experiments and then taking the species difference into consideration, we provide its MTD, LD₅₀ etc. for the man. However, there exists clearly a wide difference in its tolerance among persona even with the same sex and age belonging to the same race. Our main themes are (i) what is a rational measure of the biochemical individual variability, (ii) what kind of empirical law holds with respect to this measure, and (iii) what is the reason why it holds. This is a summary of my research sponsored by our Agency of Science and Technology [1979a] with some additional results.     

Effect of Growth Regulators and Herbicides on Photosynthetic Partial Reactions in Isolated Leaf Cells

The effect of indoleacetic acid, gibberellic acid, 2,4-dichloro-phenoxyacetic acid and amitrole at various concentration levels ranging from 0.05 to 1.0 mM on the photosynthetic evolution of O₂ and ¹⁴CO₂ fixation by isolated leaf cells was studied. The plant growth regulators enhanced O₂ evolution and ¹⁴CO₂ fixation at low concentrations and were inhibitory beyond a critical level. The amitrole had an inhibitory effect at all the concentration levels used. All the substances exhibited similar patterns of effect on the ferricyanide reduction by isolated chloroplasts and on the electron transport rates of sub-chloro-plast particles containing PS-I and PS-II independently, under non-phosphorylating conditions. As was seen from the response in all the three electron transport systems of the chloroplast studied, the electron transport chain connecting PS-II and PS-I could be considered as a possible site of action at least for the growth regulating substances as it is the only part that is common to all the three reactions. The phosphorylation associated with this part of the electron transport was “inhibited” by the substances even at the lowest concentration used. The stimulation of non-phosphorylating electron flow, with a simultaneous reduction in the rate of ATP synthesis, at low concentration levels indicated that these substances played a possible uncoupling role. The amitrole on the other hand appeared to have a generalized non-specific inhibitory action on all the partial reactions of photosynthesis.     

Random Networks of Catalytic Biochemical Reactions

Formulae for the probabilities of cycles and components are obtained for large random networks by combinatorical considerations and computer experiments. Formulae for the occurrence and distribution of cycles and of components of certain number and size are received for random networks.     

Random Networks of Catalytie Biochemical Reactions

Formulae for the probabilities of components and cycles and of their numerical distribution are obtained for large random networks by computer experiments and combinatorical considerations. Probability considerations are made and formulae for the occurrence and distribution of cycles and of components of certain number, size and structure are received for random networks.     

Close Interactions between Mesenchymal Stem Cells and Neuroblastoma Cell Lines Lead to Tumor Growth Inhibition

Mesenchymal stem cells (MSCs) have attracted much interest in oncology since they exhibit marked tropism for the tumor microenvironment and support or suppress malignant cell growth depending on the tumor model tested. The aim of this study was to investigate the role of MSCs in the control of the growth of neuroblastoma (NB), which is the second most common solid tumor in children. In vivo experiments showed that systemically administered MSCs, under our experimental conditions, did not home to tumor sites and did not affect tumor growth or survival. However, MSCs injected intratumorally in an established subcutaneous NB model reduced tumor growth through inhibition of proliferation and induction of apoptosis of NB cells and prolonged the survival of hMSC-treated mice. The need for contact between MSCs and NB cells was further supported by in vitro experiments. In particular, MSCs were found to be attracted by NB cells, and to affect NB cell proliferation with different results depending on the cell line tested. Moreover, NB cells, after pre-incubation with hMSCs, acquired a more invasive behavior towards CXCL12 and the bone marrow, i.e., the primary site of NB metastases. In conclusion, this study demonstrates that functional cross-talk between MSCs and NB cell lines used in our experiments can occur only within short range interaction. Thus, this report does not support the clinical use of MSCs as vehicles for selective delivery of antitumor drugs at the NB site unless chemotherapy and/or radiotherapy create suitable local conditions for MSCs recruitment.     

Evergreen Foliage Contributions to the Spring Growth of Taxus

Taxus media cv. Hicksii plants were grown one season under a low and high level of nitrogen fertilization. Before growth in the spring the plants were divided into two groups, one of which was defoliated and the other left intact. The growth and spring utilization of the nitrogen and carbohydrate reserves of defoliated plants were compared to the intact plants 0, 2, 4 and 6 weeks after growth started in the spring. The plants were separated into buds (all new growth), roots and stems and analyzed for changes in total nitrogen, basic and non-basic amino acids, hemicelluloses, soluble sugars, organic acids and chlorophyll. The older evergreen needles from plants grown under low nitrogen levels contain 20 % of the carbohydrate and 24% of the nitrogen used in spring growth. The needles from plants grown under high nitrogen levels contained 56% of the carbohydrate and 49% of the nitrogen used in spring growth. Removal of the old needles before spring growth removed this nitrogen and carbohydrate reserve and reduced the total plant chlorophyll content after 6 weeks of growth to 50% of that found in intact plants, with the result that defoliated plants did not show a growth response to nitrogen. Amino acids accumulated in the stems and buds of defoliated plants as carbohydrates became limiting. The defoliated plants removed 25% more available carbohydrates from the roots and stems than intact plants and their buds contained 50% less available carbohydrates. Plants without old needles showed similar growth rates under low and high nitrogen regimes and produced 33% of the dry weight of intact plants grown under high nitrogen levels and 66% of the dry weight of intact plants grown under low nitrogen levels. The old needles of taxus plants contain substantial amounts of reserve nitrogen and carbohydrate and these needles greatly influence the extent and rapidness of growth in the spring. When the needles are removed, the other tissues can supply an adequate amount of nitrogen but the carbohydrate supply becomes limiting for spring growth.     

miR-146a Inhibits Cell Growth,Cell Migration and Induces Apoptosis in Non-Small Cell Lung Cancer Cells

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P<0.05). The patients with high miR-146a expression in their tumors showed longer progression-free survival (25.6 weeks in miR-146a high patients vs. 4.8 weeks in miR-146a low patients, P<0.05). miR-146a is therefore a strong candidate prognostic biomarker in NSCLC. Thus inducing miR-146a might be a therapeutic strategy for NSCLC.     

Application of the Percolation Theory to Random Networks of Biochemical Reactions

Percolation Theory is used to get statements about random networks. It can be seen that the probability distribution that vertices belong to a finite cluster in dependence on the mean catalytic activity C = K/N has an analogy to phase transition of second kind.     

Influence of the Rate of Cell Growth and Cell Density on Interferon Action in Chick Embryo Cells

Chick embryo cells became more sensitive to the action of interferon the longer they remained in culture. This phenomenon was found even before confluency had been reached. The relative insensitivity of newly seeded cells was not due to a loss of receptors. Cells synthesizing deoxyribonucleic acid (DNA) at a high rate were less sensitive to interferon action than cells synthesizing DNA at a low rate, but the inhibition of DNA synthesis had no effect on interferon action. An increase in the number of cells used for seeding resulted in an earlier appearance of increased sensitivity to interferon action. These results are discussed in relation to the induction process in animal cells.     

Prochloron (Prochlorophyta): Biochemical Contributions to the Chlorophyll and RNA Composition

The prochlorophyte Prochloron, a symbiont of the colonial ascidianDidemnum molle, was collected in the Indian Ocean around Giravaru(Maldives) in depths between 1 and 40 m. The chlorophyll a tob ratio of the algal symbionts was higher in colonies livingbetween 1–6 m, compared to that determined for Prochloronfrom a depth of 30 m. This property for chromatic adaptationin correlation with changes in the total content of chlorophyllis dependent upon environmental factors. The association betweenDidemnum and Prochloron is only a facultative symbiosis. Thesize of the colonies, growing near the water surface is large(up to 3 cm), and it gradually decreases to 0.2 cm in a depthof 30 m dim locations. At a depth of 40 m the tunicates do notcontain the algal symbionts. Applying quantitative preparative isolation and sensitive immunologicalas well as biochemical detection techniques we have no evidencefor the existence of poly(A) stretches in RNA species from Prochloron.Moreover, we failed to detect both sn/scRNAs and their proteins,typically associated with them in RNP complexes from eukaryotes.From the data we suggest that mRNA synthesis proceeds in Prochloronin a way similar to prokaryotes. ¹ This contribution is dedicated to Prof. Dr. Dr. B. Schmidton the occasion of his 50th birthday.     

Cell to Cell Variability of Radiation-Induced Foci: Relation between Observed Damage and Energy Deposition

Most studies that aim to understand the interactions between different types of photon radiation and cellular DNA assume homogeneous cell irradiation, with all cells receiving the same amount of energy. The level of DNA damage is therefore generally determined by averaging it over the entire population of exposed cells. However, evaluating the molecular consequences of a stochastic phenomenon such as energy deposition of ionizing radiation by measuring only an average effect may not be sufficient for understanding some aspects of the cellular response to this radiation. The variance among the cells associated with this average effect may also be important for the behaviour of irradiated tissue. In this study, we accurately estimated the distribution of the number of radiation-induced γH2AX foci (RIF) per cell nucleus in a large population of endothelial cells exposed to 3 macroscopic doses of gamma rays from ⁶⁰Co. The number of RIF varied significantly and reproducibly from cell to cell, with its relative standard deviation ranging from 36% to 18% depending on the macroscopic dose delivered. Interestingly, this relative cell-to-cell variability increased as the dose decreased, contrary to the mean RIF count per cell. This result shows that the dose effect, in terms of the number of DNA lesions indicated by RIF is not as simple as a purely proportional relation in which relative SD is constant with dose. To analyse the origins of this observed variability, we calculated the spread of the specific energy distribution for the different target volumes and subvolumes in which RIF can be generated. Variances, standard deviations and relative standard deviations all changed similarly from dose to dose for biological and calculated microdosimetric values. This similarity is an important argument that supports the hypothesis of the conservation of the association between the number of RIF per nucleus and the specific energy per DNA molecule. This comparison allowed us to calculate a volume of 1.6 μm³ for which the spread of the specific energy distribution could explain the entire variability of RIF counts per cell in an exposed cell population. The definition of this volume may allow to use a microdosimetric quantity to predict heterogeneity in DNA damage. Moreover, this value is consistent with the order of magnitude of the volume occupied by the hydrated sugar-phosphate backbone of the DNA molecule, which is the part of the DNA molecule responsible for strand breaks.     

Physiological and Biochemical Changes in Bacterial Cells Exposed to Oxygen

Pure oxygen at 1 atm exerts two opposite effects on Pseudomonas saccharophila: it inhibits sucrose uptake and lipid synthesis, but it enhances the formation of polysaccharides.     

Human Pluripotent Stem Cell-Derived Cardiomyocytes: Response to TTX and Lidocain Reveals Strong Cell to Cell Variability

Stem cell derived cardiomyocytes generated either from human embryonic stem cells (hESC-CMs) or human induced pluripotent stem cells (hiPSC-CMs) hold great promise for the investigation of early developmental processes in human cardiomyogenesis and future cell replacement strategies. We have analyzed electrophysiological properties of hESC-CMs (HES2) and hiPSC-CMs, derived from reprogrammed adult foreskin fibroblasts that have previously been found to be highly similar in terms of gene expression. In contrast to the similarity found in the expression profile we found substantial differences in action potentials (APs) and sodium currents at late stage (day 60) of in vitro differentiation with higher sodium currents in hiPSC-CMs. Sensitivity to lidocain was considerably reduced in hESC-CMs as compared to hiPSC-CMs, and the effect could not be explained by differences in beating frequency. In contrast, sensitivity to tetrodotoxin (TTX) was higher in hESC-CMs suggesting different contributions of TTX-sensitive and TTX-resistant sodium channels to AP generation. These data point to physiological differences that are not necessarily detected by genomics. We conclude that novel pharmacological screening-assays using hiPSC-CMs need to be applied with some caution.