BrdU-Hoechst-Giemsa analysis of DNA replication in synchronized lymphocyte cultures

The combination of a technique of reversible methotrexate (MTX) imposed G₁/S block in cultures of human lymphocytes with the BrdU-Hoechst-Giemsa technique permitted the study of DNA replication patterns in individual chromosomes at different intervals of the S phase in a cell cohort with uniform S + G₂ duration. The procedure did not increase either the frequency of chromosomal breakage or SCE freqeuncy. The technique applied permitted visualization of the banding pattern in over 90% of mitoses. Examination of mitoses following different times of exposure to BrdU revealed a high degree of synchrony in the progression of the cell cohort examined through the S phase.The presence of two distinct late replication patterns of the allocyclic X chromosome was confirmed in studies on lymphocytes from normal human females by this technique. Interindividual and intercellular differences of the replication pattern have been demonstrated. The replicating patterns from one individual were relatively constant.The analysis of the Y chromosome has revealed marked differences of the termination of replication in individual cells. Euchromatic regions have been shown to complete DNA synthesis first, followed by the distal part of the long arm and, finally, by the region of Yq11/Yq12 junction. Lateral asymmetry was localised at this region.This paper was presented as a preliminary communication at the Helsinki Chromosome Conference Aug. 29–31, 1977, and, in its final form, at the 7th International Chromosome Conference, Oxford, Aug. 26–30, 1980.     

Analysis of a neural oscillator

Although the Matsuoka neural oscillator, which was originally proposed as a model of central pattern generators, has widely been used for various robots performing rhythmic movements, its characteristics are not clearly explained even now. This article shows two closed-form relations that express the frequency and amplitude of the generated oscillation as functions of the parameters of the model. Although they are derived based on a rough linear approximation, they accord with the result obtained by a simulation considerably. The obtained relations also give us some nontrivial predictions about the properties of the oscillator.     

Laser flow cytofluorometric analysis of HTC cells synchronized with hydroxyurea, nocodazole and aphidicolin

The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.     

Density gradient analysis of newly replicated DNA from synchronized mouse lymphoma cells

The replication of DNA in synchronous cultures of mouse lymphoma cells was investigated by use of CsCl density gradient centrifugation. We found that the buoyant density of newly replicated DNA depended upon the particular stage of S phase in which synthesis occurred. In early S phase, newly replicated DNA exhibited buoyant densities which were slightly higher, on the average, than that of pre-existing DNA. As S phase progressed, newly replicated DNA shifted to lower buoyant densities, until, near the end of S phase, densities less than pre-existing DNA were observed. These observations are discussed in terms of their possible relevance to base compositional differences between nucleotide sequences made in early as opposed to middle or late S phase.     

Morphometric analysis of B2cAMP-induced reverse transformation in synchronized CHO cells

Summary Synchronized transformed and reverse-transformed (by 10⁻³ M B₂cAMP) CHO-K1 cells, growing adherent to plastic, are characterized by means of geometric and densitometric parameters at the level of both the entire cell and of the nuclei at various time intervals after selective mitotic detachment. Transformed and reverse-transformed cells triple-stained with Feulgen, Napthol Yellow S, and periodic acid-Schiff appeared very similar in terms of integrated optical density (IOD), related to either polysaccharides, protein, or DNA amount. On the other hand, a shift from a polygonal to a spindle-shaped morphology is accompanied by a significant decrease in both form factor and average optical density (AOD) of intact cell and nuclei, which are the most conspicuous measured changes caused by B₂cAMP, in addition to a lengthening of the cell cycle duration. In both control and treated cells, important and parallel cell-cycle-dependent modulations of geometric and densitometric parameters are also observed, for both the cytoplasmic (i.e., cell morphometry) and DNA space (i.e., nuclear morphometry). Specifically, the modulation in nuclear morphometry during G1, S, G2, andM phases confirms previous findings on synchronized HeLa cells. The optical density threshold-dependence of geometric parameters shows that, while becoming fusiform, the cytoplasm of reverse-transformed cells had a particularly low optical density precisely in the polar area. Utilization of such an approach in the development of anobjective morphological classification of all cell lines grown as monolayers “in vitro” is also discussed.     

Antiphonal four-part synchronized chorusing in a Neotropical wren

Plain-tailed wrens (Thryothorus euophrys) live in groups that sing synchronized choruses, the contributions of females and males alternating with each other in cycles, within which each sex sings two of the four parts, the whole achieving near perfect synchrony. As each bird has a repertoire of ca 20 phrases of each type, the synchrony also requires them to choose the same type at the same time as others of their sex. Songs can last up to 2min, during which individuals join in and drop out. This must be one of the most complex singing performances yet described in a non-human animal.     

An analysis of DNA replication in synchronized CHO cells treated with benzo[a]pyrene diol epoxide

Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.     

Phase-response curves and synchronized neural networks

We review the principal assumptions underlying the application of phase-response curves (PRCs) to synchronization in neuronal networks. The PRC measures how much a given synaptic input perturbs spike timing in a neural oscillator. Among other applications, PRCs make explicit predictions about whether a given network of interconnected neurons will synchronize, as is often observed in cortical structures. Regarding the assumptions of the PRC theory, we conclude: (i) The assumption of noise-tolerant cellular oscillations at or near the network frequency holds in some but not all cases. (ii) Reduced models for PRC-based analysis can be formally related to more realistic models. (iii) Spike-rate adaptation limits PRC-based analysis but does not invalidate it. (iv) The dependence of PRCs on synaptic location emphasizes the importance of improving methods of synaptic stimulation. (v) New methods can distinguish between oscillations that derive from mutual connections and those arising from common drive. (vi) It is helpful to assume linear summation of effects of synaptic inputs; experiments with trains of inputs call this assumption into question. (vii) Relatively subtle changes in network structure can invalidate PRC-based predictions. (viii) Heterogeneity in the preferred frequencies of component neurons does not invalidate PRC analysis, but can annihilate synchronous activity.     

Periodic pulsatile stimulation of a nonlinear oscillator

We consider pulsatile periodic stimulation of an integrate-and-fire oscillator, and investigate the possible phase-locked patterns between the intrinsic rhythm and the forcing system. These stimulations are varied according to their period and intensity, corresponding to controllable experimental parameters. Phase transition curves are derived and analyzed under functional iteration. Two different perturbative mechanisms are considered, leading to significant differences in possible behaviors. Bistability can be obtained in one case.The analysis is reduced to the investigation of two-parameter families of discontinuous maps of the circle into itself.     

Coupling of a slow and a fast oscillator can generate bursting

A general mechanism underlying bursting is proposed and described. It consists of two coupled nonlinear oscillators with different frequencies, where the slower oscillator alternatively switches the faster one on and off. This mechanism is shown to work in an extended Bonhoefer-van der Pol oscillator as well as in a modified version of the Hodgkin-Huxley equations.     

Model for a population-based microbial oscillator

Genetic oscillators are a major theme of interest in the emerging field of synthetic biology. Until recently, most work has been carried out using intra-cellular oscillators, but this approach restricts the broader applicability of such systems. Motivated by a desire to develop large-scale, spatially distributed cell-based computational systems, we present an initial design for a population-level oscillator which uses three different bacterial strains. Our system is based on the client-server model familiar to computer science, and uses quorum sensing for communication between nodes. Importantly, it is robust to perturbation and noise. We present the results of extensive in silico simulation tests, which confirm the feasibility of our design.     

Multiple modes of a conditional neural oscillator

We present a model for a conditional bursting neuron consisting of five conductances: Hodgkin-Huxley type time- and voltage-dependent Na⁺ and K⁺ conductances, a calcium activated voltage-dependent K⁺ conductance, a calcium-inhibited time- and voltage-dependent Ca⁺⁺ conductance, and a leakage Cl(_– conductance. With an initial set of parameters (versionS), the model shows a hyperpolarized steady-state membrane potential at which the neuron is silent. Increasingg _Na and decreasingg _Cl, whereg _i , is the maximal conductance for speciesi, produces bursts of action potentials (BursterN). Alternatively, an increase ing _Ca produces a different bursting state (BursterC). The two bursting states differ in the periods and amplitudes of their bursting pacemaker potentials. They show different steady-stateI–V curves under simulated voltage-clamp conditions; in simulations that mimic a steady-stateI–V curve taken under experimental conditions only BursterN shows a negative slope resistance region. ModelC continues to burst in the presence of TTX, while bursting in ModelN is suppressed in TTX. Hybrid models show a smooth transition between the two states.