Morphology and dynamics of chromosome territories in living cells.

Chromosome territories formed by fluorescence-labeled sub-chromosomal foci were analyzed in time-lapse series of 3D confocal data sets of living HeLa and human neuroblastoma cells. The quantitative analysis of the chromosome territory morphology confirmed previous results obtained by visual observation [Zink et al., Hum. Genet. 102 (1998) 241-251] that chromosome territories persisted as stable entities over an observation time >4 h. The changes in morphology with time of single chromosome territories were found to be less pronounced than differences in morphology of different chromosome territories in fixed cells. The analysis of the individual motion of chromosome territories recently showed 'Brownian' diffusion-like motion at very slow rates [Bornfleth et al., Biophys. J. 77 (1999) 2871-2886]. Here, we show that the mutual motion of different chromosome territories was independent and also 'Brownian' diffusion-like.     

Organization of early and late replicating DNA in human chromosome territories

It has been suggested that DNA organized into replication foci during S-phase remains stably aggregated in non-S-phase cells and that these stable aggregates provide fundamental units of nuclear or chromosome architecture [C. Meng and R. Berezney (1991) J. Cell Biol. 115, 95a; E. Sparvoli et al. (1994) J. Cell Sci. 107, 3097-3103; D. A. Jackson and A. Pombo (1998) J. Cell Biol. 140, 1285-1295; D. Zink et al. (1998) Hum. Genet. 112, 241-251]. To test this hypothesis, early and late replicating DNA of human diploid fibroblasts was labeled specifically by incorporating two different thymidine analogs [J. Aten (1992) Histochem. J. 24, 251-259; A. E. Visser (1998) Exp. Cell Res. 243, 398-407], during distinct time segments of S-phase. On mitotic chromosomes the amount and spatial distribution of early and late replicating DNA corresponded to R/G-banding patterns. After labeling cells were grown for several cell cycles. During this growth period individual replication labeled chromosomes were distributed into an environment of unlabeled chromosomes. The nuclear territories of chromosomes 13 and 15 were identified by additional chromosome painting. The distribution of early and late replicating DNA was analyzed for both chromosomes in quiescent (G0) cells or at G1. Early and late replicating DNA occupied distinct foci within chromosome territories, displaying a median overlap of only 5-10%. There was no difference in this regard between G1 and G0 cells. Chromosome 13 and 15 territories displayed a similar structural rearrangement in G1 cells compared to G0 cells resulting in the compaction of the territories. The findings demonstrate that early and late replicating foci are maintained during subsequent cell cycles as distinctly separated units of chromosome organization. These findings are compatible with the hypothesis that DNA organized into replicon clusters remains stably aggregated in non-S-phase cells.     

Quantitative motion analysis of subchromosomal foci in living cells using four-dimensional microscopy

The motion of subchromosomal foci and of whole chromosome territories in live human cell nuclei was investigated in four-dimensional space-time images. Visualization of subchromosomal foci was achieved by incorporating Cy3-dUTP into the nuclear DNA of two different cell types after microinjection. A subsequent segregation of the labeled cell nuclei led to the presence of only a few labeled chromosome territories on a background of nonlabeled chromatin (. Hum. Genet. 102:241-251). This procedure yielded many distinct signals in a given cell nucleus. Motion analysis in four-dimensional space-time images was performed using single-particle tracking and a statistical approach to the detection of a possible directional motion of foci relative to the center of mass of a chromosome territory. The accuracy of the analysis was tested using simulated data sets that closely mirrored the experimental setup and using microparticles of known size. Application of the analysis tools to experimental data showed that mutual diffusion-like movements between foci located on different chromosomes were more pronounced than inside the territories. In the time range observed, movements of individual foci could best be described by a random diffusion process. The statistical test for joint directed motion of several foci inside chromosome territories revealed that foci occasionally switched from random to directional motion inside the territories.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

By autoradiography with ³H-thymidine and ³H-deoxycytidine it is shown that chromosomes 1 and 16 in cultures of embryonic fibroblasts at the termination of the S period synthesise AT- and GC-rich DNA at different rats: in both chromosomes the labelling of AT-bases is more intensive. In leucocyte cultures both nucleotide pairs label equally in these chromosomes. Chromosomes 2, 3, 4–5 and 21–22 are labelled equally in both cultures with respect to AT-and GC-pairs. Fibroblasts and leucocytes differ in the relative intensity of DNA synthesis at the end of the S period: chromosomes 1,16 and 21–22 contain more label in the case of fibroblasts (chromosome 1 solely due to AT-pairs) and chromosome 4–5 in the case of leucocytes. Analysis of distribution of late label along chromosome 1 showed that in fibroblast cultures the pericentromeric regions of both arms are labelled more intensively in respect to both nucleotide pairs than in leucocyte cultures. Both in fibroblast and leucocyte cultures no significant distinctions in the distribution of AT-and GC-pairs along chromosome 2 were established. In fibroblast cultures the pericentromeric regions of both arms of chromosome 3 are labelled more intensively than other regions. In leucocyte cultures the pericentromeric region of the short arm of this chromosome is labelled with the same intensively as in fibroblasts, whereas in the pericentromeric region of the long arm the intensity of incorporation of labelled synthesis precursors decreases. — Analysis of results obtained in the present study together with data of previous studied (Slesinger et al., 1974; Lozovskaya et al., 1976; Lozovskaya et al., 1977) shows that differences between the two types of cells in the intensity of late ³H-thymidine labelling in the C-heterochromatin regions of chromosomes 1 and 16 may be explained both by variation of replication time in leucocytes as compared with fibroblasts and by variation of the content of AT- rich DNA. Differences observed in other chromosomes are probably due to different times of replication of these chromosomes in leucocytes and fibroblasts. — Thus, the process of cell system differentiation involves not only differential activity of the genome (the main mechanism) that is connected with differences in the replication time of chromosomes and of their regions but also variation of the quantity of genetic material.     

An improved polyurethane support system for monitoring growth in plant cell cultures

An improved culture system for plant cells that employs filter paper resting on polyurethane saturated with liquid medium is described. It combines a simplified version of the system outlined by Weber and Lark [1979, Theor Appl Genet 55: 81–86] with the method of growth estimation described by Horsch et al. [1980, Can J Bot 58: 2402–2406]. The growth of plated cells or callus can be conveniently monitored through repeated non-destructive fresh weight measurements of the filter paper and adhering cells, thereby allowing the construction of a complete growth curve over the course of an experiment. Experiments with 3 Nicotiana genotypes (N. plumbaginifolia Viv., N. tabacum L. ‘SC 58’ and N. tabacum ‘WI 38’) and 3 Vitis vinifera L. genotypes (‘Chenin Blanc’, ‘Dogridge’ and ‘White Riesling’) clearly demonstrate higher growth rates of plated cells on polyurethane supports compared with agar. Further experiments with N. plumbaginifolia illustrate the use of polyurethane supports for culturing cells at low pH (4.0) and the recovery of spent medium for monitoring changes in pH. These features will greatly facilitate quantitative studies of mineral nutrition and metal toxicity in cultured cells. Polyurethane supports also allow the incorporation of conditioned medium or feeder cells to support the growth of cells at low densities and facilitate the rapid recovery of variant cells.     

Dynamic behaviors of microtubules in cytosol

Highly anisotropic microtubules (MTs) immersed in cytosol are a central part of the cytoskeleton in eukaryotic cells. The dynamic behaviors of an MT–cytosol system are of major interest in biomechanics community. Such a solid–fluid system is characterized by a Reynolds number of the order 10^?3 and a slip ionic layer formed at the MT–cytosol interface. In view of these unique features, an orthotropic shell-Stokes flow model with a slip boundary condition has been developed to explore the distinctive dynamic behaviors of MTs in cytosol. Three types of motions have been identified, i.e., (a) undamped and damped torsional vibration, (b) damped longitudinal vibration, and (c) overdamped bending and radial motions. The exponentially decaying bending motion given by the present model is found to be in qualitative agreement with the existing experimental observation [Felgner et al., 1996. Flexural rigidity of microtubules measured with the use of optical tweezers, Journal of Cell Science 109, 509–516 ].     

Journal of Cellular Physiology: Volume 228, Number 6, April 2013

Cover: SEM morphology analysis of hMSCs cultured on different biomaterials. Please see article in this issue by Manfrini et al., pages 1229–1237. Cover designed by Lucía Sagredo Sánchez.     

Differential requirements of a mitotic acetyltransferase in somatic and germ line cells

During mitosis different types of cells can have differential requirements for chromosome segregation. We isolated two new alleles of the separation anxiety gene (san). san was previously described in both Drosophila and in humans to be required for centromeric sister chromatid cohesion (Hou et al., 2007; Williams et al., 2003). Our work confirms and expands the observation that san is required in vivo for normal mitosis of different types of somatic cells. In addition, we suggest that san is also important for the correct resolution of chromosomes, implying a more general function of this acetyltransferase. Surprisingly, during oogenesis we cannot detect mitotic defects in germ line cells mutant for san. We hypothesize the female germ line stem cells have differential requirements for mitotic sister chromatid cohesion.     

Sports and hybrids of triploid Hosta ‘Sum and Substance’ reveal chromosome losses and gains in all three apical layers

Ploidy distributions in L1, L2, and L3 apical or meristematic layers of 56 different plants (79 accessions) from vegetative and sexual progeny of the triploid Hosta ‘Sum and Substance’ were determined. Nuclear DNA contents (2C) of each apical layer were measured by flow cytometry with propidium iodide, and inferred ploidies are calculated. During tissue culture, the triploid (L1–L2–L3?=?3–3–3) Hosta ‘Sum and Substance’ exhibited chromosome losses resulting in somaclonal variants such as DNA aneuploids (e.g., 2.7–2.7–2.7) and aneuploidy chimeras (e.g., 3–2.7–2.7). Most interestingly, some chimeras exhibited even an increase in genome size as in plants with 3.5–3–3 configuration. Hybrids of H. ‘Sum and Substance’ show only losses of nuclear DNA compared with the original triploid. This gives rise to fully aneuploid plants and no chimeras. The measurements of Hosta ‘Sum and Substance’ lineages of sports and hybrids indicate that chromosome losses or gains are an important source of new cultivars. The complexity of chromosomal distribution in derivatives from the triploid Hosta ‘Sum and Substance’ is discussed.     

ENDOGENOUS ELECTROMAGNETIC FORCES IN LIVING CELLS: IMPLICATIONS FOR TRANSFER OF REACTION COMPONENTS

The mechanisms of order in living cells and in particular of the space–time order of chemical reactions are largely unknown. Directed transport of components to a reaction region cannot be explained by Brownian motion, which is disordered and increases entropy of the system. Besides transport by the motor proteins along microtubules, the endogenous electromagnetic field can provide translation motion of reaction components. This forced translation motion can have a high degree of certainty to reach the target region.     

Formulas predicting survival in patients with heart transplantation

Kirklin et al. (J Heart Transpl, 7 (1988) 331–336) reported survival data in 132 patients who underwent heart transplantation. Survival was evaluated by using the product-limit method of Kaplan-Meier and maximum likelihood method. In addition, the effect of pulmonary vascular resistance on survival was estimated by using multivariate analysis. A microcomputer program in BASIC for predicting the survival probability after transplantation in patients with heart transplantation is designed. The formula used in this program is derived from the survival data reported by Kirklin et al. (J Heart Transpl, 7 (1988) 331–336). A mathematical model of the ‘probacent’-probability equation and a computer program previously published by the author are employed in this study. Analysis of the computer-assisted predicted values and the data reported by Kirklin et al. (J Heart Transpl, 7 (1988) 331–336) indicates that the program is accurate and reliable with a complete agreement in expressing survival probability as a function of time after heart transplantation. The computer-assisted predictive formula can determine the relationship between the time and the survival probability and may be of value for prognostic evaluation of patients. The computer-assisted mathematical model of the ‘probacent’-probability equation may be proposed as a general approximation method to make useful predictions of probable outcomes in various biomedical phenomena.     

Converted neural cells: induced to a cure?

Many neurodegenerative disorders such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and others often occur as a result of progressive loss of structure or function of neurons. Recently, many groups were able to generate neural cells, either differentiated from induced pluripotent stem cells (iPSCs) or converted from somatic cells. Advances in converted neural cells have opened a new era to ease applications for modeling diseases and screening drugs. In addition, the converted neural cells also hold the promise for cell replacement therapy (Kikuchi et al., 2011; Krencik et al., 2011; Kriks et al., 2011; Nori et al., 2011; Rhee et al., 2011; Schwartz et al., 2012). Here we will mainly discuss most recent progress on using converted functional neural cells to treat neurological diseases and highlight potential clinical challenges and future perspectives.     

A tale of too many centrosomes

Having the correct number of centrosomes is crucial for proper chromosome segregation during cell division and for the prevention of aneuploidy, a hallmark of many cancer cells. Several recent studies (Basto et al., 2008; Kwon et al., 2008; Yang et al., 2008) reveal the importance of mechanisms that protect against the consequences of harboring too many centrosomes.     

The Studies on the Karyotypes and Cytogeography of Taxodium Rich.(Taxodiaceae)

The present paper deals with the karyotypic analysis of Taxodium ascendens Brongn. The somatic chromosomes in root-tip cells of the plant are found to be 2n =22, all with median and submedian constrictions. A character of the karyotype is that the chromosome 10 has a long kinetochore region (Plate 1:1). According to the terminology defined by Levan et al.^[18], the karyotype formula is k(2n)=22=20m+2sm, which is different to Huang et Hsu’s^[8] K(2n)=24=22m+2B(m). The karyotype belongs to “lA” of Stebbins’^[24] karyotypic symmetry and is generally regarded as a relatively primitive one. The species’ chromosome complement is 2n=22=2L+8M₂+12M₁ according to I.R.L.difined by Kuo et al.^[15] based on relative length. The lengths, arm ratios and types of chromosomes of the species are given in Table 1-I. The morphology of the chromosomes and the karyotype, are given in Plate 1:1. In the light of the works of Schlarbaum et al.^[21] and Mehra et al.^[17], K(2n)=22=20m (2SAT)+2sm and 2n=22=2L+6M₂+14M₁ are for T. distichum (L.) Rich. (see Table 1-II), K(2n)=20m+2sm and 2n=22=4L+4M₂+12M₁+2S for T. mucronatum Tenore (see Table 1-III, Plate 1:2), which belong to “lA” and “2A” respectively. The differences between three species in the ratio of the longest to the shortest chromosome, I.R.L. and the proportion of chromosomes with arm ratio >2 show that the karyotype of T. mucronatum is the most advanced and that of T. distichum the most primitive. The present author suggests that the sequence of evolutionary advance be T. distichum, T. ascendens, T. mucronatum. Based on the evidence from the karyotype analyses, ecology and geographical distribution (including fossil), the secondary center of genetic diversity (Fig. 1) and the probable evolu-tionary pattern (Fig. 2) of Taxodium are discussed.     

Flow cytometric analysis of European flat oyster, Ostrea edulis, haemocytes using a monoclonal antibody specific for granulocytes

The importance of haemocytes in mollusc defence mechanisms can be inferred from their functions. They participate in pathogen elimination by phagocytosis (Cheng, 1981; Fisher, 1986). Hydrolytic enzymes and cytotoxic molecules produced by haemocytes contribute to the destruction of pathogenic organisms (Cheng, 1983; Leippe & Renwrantz, 1988; Charlet et al., 1996; Hubert et al., 1996; Roch et al., 1996). Haemocytes may also be involved in immunity modulation by the production of cytokines and neuropeptides (Hughes et al., 1990; Stefano et al., 1991; Ottaviani et al., 1996). As a result, the literature dealing with bivalve haemocyte studies has increased during the last two decades. Most of these publications use microscopy for morphological analysis (Seiler & Morse, 1988; Auffret, 1989; Hine & Wesney, 1994; Giamberini et al., 1996; Carballal et al., 1997; Lopez et al., 1997; Nakayama et al., 1997), and functional analysis (e.g. phagocytosis) (Hinsch & Hunte, 1990; Tripp, 1992; Mourton et al., 1992; Fryer & Bayne, 1996; Mortensen & Glette, 1996). Flow cytometry represents a rapid technique applicable to both morphological and functional studies of cells in suspension. While the measurements based on autofluorescence provide information on cell morphology, the analyses with fluorescent markers including labelled antibodies, offer data on phenotyping and cell functions. As a result, its application has greatly contributed to the investigation of immunocyte functions and differentiation in vertebrates (Stewart et al., 1986; Rothe & Valet, 1988; Ashmore et al., 1989; Koumans-van Diepen et al., 1994; Rombout et al., 1996; Caruso et al., 1997). Some authors studied oyster haemocyte populations by flow cytometry based on cellular autofluorescence (Friedl et al., 1988; Fisher & Ford, 1988; Ford et al., 1994). However, no analysis using specific monoclonal antibodies has been reported to date. In this study, a protocol for studying European flat oyster, Ostrea edulis, haemocytes by flow cytometry using a monoclonal antibody specific for granulocytes and an indirect immunofluorescence technique have been developed. European flat oysters, Ostrea edulis, 7-9 cm in shell length were obtained from shellfish farms in Marenne Oléron bay (Charente Maritime, France) on the French Atlantic coast. All individuals were purchased just before each experiment and processed without any previous treatment.     

Structure and dynamics of interphase chromosomes

During interphase chromosomes decondense, but fluorescent in situ hybridization experiments reveal the existence of distinct territories occupied by individual chromosomes inside the nuclei of most eukaryotic cells. We use computer simulations to show that the existence and stability of territories is a kinetic effect that can be explained without invoking an underlying nuclear scaffold or protein-mediated interactions between DNA sequences. In particular, we show that the experimentally observed territory shapes and spatial distances between marked chromosome sites for human, Drosophila, and budding yeast chromosomes can be reproduced by a parameter-free minimal model of decondensing chromosomes. Our results suggest that the observed interphase structure and dynamics are due to generic polymer effects: confined Brownian motion conserving the local topological state of long chain molecules and segregation of mutually unentangled chains due to topological constraints.     

181 Integrative analysis of gene expression and protein–protein interaction networks in Glioblastoma

Glioblastoma multiforme (GBM) is the most malignant of all the brain tumors with very low median survival time of one year, as per Central Brain Tumor Registry of the USA, 2001. Efforts are ongoing to understand this disease pathogenesis in complete details. Global gene expression changes in GBM pathogenesis have been studied by several groups using microarray technology (e.g. Carro et al., 2010). One of the many approaches to ‘understand the control mechanisms underlying the observed changes in the activity of a biological process’ (Cline et al., 2007) is integration of gene expression and protein–protein interactions (PPI) datasets. Among several examples, aberrant activation of Wnt/β-catenin signaling pathway as well as sonic hedgehog (SHH) signaling pathway is reported in GBMs (Klaus & Birchmeier, 2008). Further, these two pathways are also involved in proliferation and clonogenicity of glioma cancer stem cells (Li et al., 2009), which are thought to play a role in glioma initiation, proliferation, and invasion, and are one of the important points of intervention. Hedgehog–Gli1 signaling is also found to regulate the expression of stemness genes. In this paper, analyses of the relationship between the significant differential expression of these and other genes and the connectivity as well as topological features of a PPI network would be discussed. This way, genes potentially overlooked when relying solely on expression profiles may be identified which can be biologically relevant as possible drug target/s or disease biomarker/s.     

Comparative Studies of the Thermodynamic Stabilities Between Sheared A:G and Watson-Crick A:U(T) Base Pairs in RNA and DNA

Abstract

Thermodynamic parameters for duplex formation were determined from CD melting curves for r(GGACGAGUCC)₂ and d(GGACGAGTCC)₂, both of which form two consecutive ‘sheared’ A:G base pairs at the center [Katahira et al. (1993) Nucleic Acids Res. 21, 5418–5424; Katahira et al., (1994) Nucleic Acids Res. 22, 2752–27591. The parameters were determined also for r(GGACUAGUCC)₂ and d(GGACTAGTCC)₂, where the A:G mismatches are replaced by Watson-Crick A:U(T) base pairs. Thermodynamic properties for duplex formation are compared between the sheared and the Watson-Crick base pairs, and between RNA and DNA. Difference in the thermodynamic stability is analyzed and discussed in terms of enthalpy and entropy changes. The characteristic features in CD spectra of RNA and DNA containing the sheared A:G base pairs are also reported.