Henri Atlan

Overexpression of the Na+/K+/Cl− cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts

Na+/K+/Cl− cotransporter activity is stimulated in early G1 phase of the cell cycle and this stimulation was shown to be an essential event in fibroblast cell proliferation. In order to elucidate further the role of the Na+/K+/Cl− cotransporter in cell proliferation, we overexpressed the gene encoding the Na+/K+/Cl− cotransporter in mouse fibroblasts, and analyzed cellular phenotypic changes. Mouse Balb/c 3T3 cells were stably transfected with the cDNA of the shark rectal gland Na+/K+/Cl− cotransporter gene (NKCC1), and expressed in a mammalian vector under the cytomegalovirus promoter (Balb/c‐NKCC1 cells). The transfected cells exhibited up to 10‐fold greater bumetanide‐sensitive Rb+ influx compared to the control cells. The Balb/c‐NKCC1 cells have acquired a typical transformation phenotype indicated by: (1) Loss of contact inhibition exhibited by growth to a higher cell density in confluent cultures, and formation of cell foci; (2) proliferation in low serum concentrations; and (3) formation of cell colonies in soft agar. The control cells transfected with the NKCC1 gene inserted in the opposite orientation in the vector retained their normal phenotype. Furthermore, the two specific inhibitors of the Na+/K+/Cl− cotransporter activity; bumetanide and furosemide inhibited the clonogenic efficiency in the NKCC1 transfected cells. These control experiments indicate that the apparent transformation phenotype acquired by the Balb/c‐NKCC1 cells was not merely associated with the process of transfection and selecting for the neomycin‐resistant clones, but rather with the overexpression of the Na+/K+/Cl− cotransporter gene. In order to ascertain that the regulated and normal expression of the Na+/K+/Cl− cotransporter control cell proliferation, the effect of bumetanide a specific inhibitor of the cotransporter, was tested on Balb/c 3T3 cell proliferation, induced by fibroblasts growth factor (FGF) and fetal calf serum (FCS). Bumetanide inhibited synchronized Balb/c 3T3 cell exit from the G0/G1 arrest and entering S‐phase. The inhibition was reversible, as removal of bumetanide completely released cell proliferation. Taken together, these results propose that the NKCC1 gene is involved in the control of normal cell proliferation, while its overexpression results in apparent cell transformation, in a manner similar to some protooncogenes. J. Cell. Physiol. 182:109–118, 2000.

The cellular computer DNA: Program or data

The classical metaphor of the genetic program written in the DNA nucleotidic sequences is reconsidered. Recent works on algorithmic complexity and logical properties of computer programs and data are used to question the explanatory value of that metaphor. Structural properties of strings are looked for which would be necessary to apply to DNA sequences if the metaphor is to be taken literally. The notion of sophistication is used to quantify meaningful complexity and to distinguish it from classical computational complexity. In this context, the distinction between program and data becomes relevant and an alternative metaphor of DNA as data to a parallel computing network embedded in the global geometrical and biochemical structure of the cell is discussed. An intermediate picture of an evolving network emerges as the most likely where the output of the cellular computing network can produce, at a different time scale, changes in the structure of the network itself by means of changes in the DNA activity patterns.

T cells and autoantibodies to human HSP70 in type 1 diabetes in children.

We studied T-cell proliferative responses (stimulation index: SI) and autoantibodies to human HSP60, HSP70 and HSP90 proteins in 25 children (mean age 10.1+/-3.8 years) newly diagnosed with Type 1 diabetes. The control group for T cells included 25 adults and three pediatric donors without Type 1 diabetes. Controls for antibodies included 10 pediatric subjects. The T-cell responses to HSP70 of the test group (mean SI=4.5+/-3.1) were significantly greater than those of the control group (meanSI=1.4+/-0.6; p<0.0001); the incidence of HSP70 responders was (85%) compared to 14% in the control group. All but three of the Type 1 children who responded to HSP70 also responded to HSP60 (85%). The T-cell responses of the Type 1 group to HSP90 (mean SI=1.7+/-1.1) were similar to those of the control group (mean SI=1.5+/-0.7). We mapped HSP70 epitopes recognized by T cells in seven subjects using overlapping peptides of the molecule. Among the Type 1 subjects, IgG seropositivity was 45% to HSP60, 30% to HSP70, and 15% to HSP90. Thus, we conclude that children with newly diagnosed Type 1 diabetes manifest heightened T-cell autoimmunity to HSP70 and HSP60, but not to HSP90.

Theories of immune networks

to Immune Networks.- I.- Natural Id-Anti-Id Networks and the Immunological Homunculus.- Self-Nonself Immunological Tolerance and Idiotype Networks.- II.- Extensive Percolation in Reasonable Idiotypic Networks.- The Concept of Idiotypic Network: Deficient or Premature?.- Dynamical Behavior of Discrete Models of Jernes Network.- Some Reflections on Memory in Shape Space.- III.- Regulation of the Immune Response: A Discrete Mapping Approach.- Simulation of the Immune Cellular Response by Small Neural Networks.- Discrete Time Versus Continuous Time Approach to the Autoimmune Response.- Optimizing the Immune Control of Parasitic Invasion.- Index of Contributors.

Network regulation of autoimmunity: An automaton model

Four classes of regulatory T lymphocytes have been implicated in the control of experimental autoimmune diseases: a pair of helper and suppressor T lymphocytes that recognize the self-antigen (antigen-specific); and a pair of helper and suppressor T lymphocytes that recognize the autoimmune effector lymphocytes (anti-idiotypic). The anti-idiotypic pair of regulators was detected following vaccination against autoimmune disease using autoimmune effector T clones as vaccines. To learn how the anti-idiotypic regulatory lymphocytes might function in concert with the antigen-specific regulatory lymphocytes, we devised a network in which the cell populations could be viewed as interconnected automata. Analysis of this novel network model suggests how self-tolerance may operate, how progressive autoimmune disease may develop, and how T-cell vaccination can control autoimmune disease.

Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells

SummaryThe addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result ofVm increase while theKm for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrinic acid. Replacing chloride in the medium by NO3−, CO3− and CH3COO− resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl−-dependent.

HIV time hierarchy: winning the war while, loosing all the battles

AIDS is the pandemic of our era. A disease that scares us not only because it is fatal but also because its insidious time course makes us all potential carriers long before it hands us our heads in a basket. The strange three stage dynamics of aids is also one of the major puzzles while describing the disease theoretically (Pantaleo et al., N. Engl. J. Med. 328 (1993) 327). Aids starts, like most diseases, in a peak of virus expression [R.M. Zorzenon dos Santos, Immune responses: Getting close to experimental results with cellular automata models, in: D. Stauffer (Ed.), Annual Review of Computational Physics VI, 1999, pp. 159–202; R.M. Zorzenon dos Santos, S.C. Coutinho, On the dynamics of the evolution of HIV infection, cond-mat/0008081], which is practically wiped out by the immune system. However it then remains in the body at a low level of expression until later (some time years later) when there is an outbreak of the disease which terminally cripples the immune system causing death from various common pathogens. In this paper we show, using a microscopic simulation, that the time course of AIDS is determined by the interactions of the virus and the immune cells in the shape space of antigens and that it is the viruss ability to move more rapidly in this space (its high mutability) that causes the time course and eventual “victory” of the disease. These results open the way for further experimental and therapeutic conclusions in the ongoing battle with the HIV epidemic.

Modeling complexity in biology

Biological systems, unlike physical or chemical systems, are characterized by the very inhomogeneous distribution of their components. The immune system, in particular, is notable for self-organizing its structure. Classically, the dynamics of natural systems have been described using differential equations. But, differential equation models fail to account for the emergence of large-scale inhomogeneities and for the influence of inhomogeneity on the overall dynamics of biological systems. Here, we show that a microscopic simulation methodology enables us to model the emergence of large-scale objects and to extend the scope of mathematical modeling in biology. We take a simple example from immunology and illustrate that the methods of classical differential equations and microscopic simulation generate contradictory results. Microscopic simulations generate a more faithful approximation of the reality of the immune system.

Fibroblast growth factor induces a transient net K+ influx carried by the bumetanide-sensitive transporter in quiescent BALB/c 3T3 fibroblasts

The bumetanide-sensitive transport system performed a net efflux of K+ in serum deprived quiescent cells. The addition of partially purified fibroblast growth factor (FGF) to G0/G1 phase 3T3 fibroblasts induced a transient net influx of K+, carried out by the bumetanide-sensitive transport system for 2-6 minutes. The stimulation of the bumetanide-sensitive K+ influx by FGF was followed by stimulation of the ouabain-sensitive K+ influx. In addition, both the bumetanide-sensitive and the ouabain-sensitive K+ influxes were found to be similarly stimulated when the G0/G1 3T3 cells were treated with insulin. These results suggest that growth factors such as FGF and insulin induce a change in the action of the bumetanide-sensitive transporter from performing net K+ efflux along its concentration gradient to an uphill transport pumping of K+ into the cell. We propose, therefore, that the bumetanide-sensitive transporter contributes to the increase in the intracellular K+ (and probable Na+) stimulated by growth factors such as FGF and insulin in early G1 phase of the cell cycle.

RANDOM BOOLEAN NETWORKS

Random boolean networks are defined and used as models of systems composed of many interacting components. Computer simulations showing the organization of these networks in subnets, as well as a temporal organization in limit cycles are reported. The influence of the different boolean laws on this organization is investigated and results concerning the stability of the organization against “surgery” and external noise are presented.