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PubMed2003

Monitoring cell cycle distributions in living cells by videomicrofluorometry and discriminant factorial analysis.

BACKGROUND: The study of the cell cycle of living cells is often based on quantification of nuclear DNA. These studies may be improved by multifactorial analysis evaluating several parameters for each cell. METHODS: Single lymphoblastoid living cells were labeled with three fluorescent markers: Hoechst 33342 for nuclear DNA, Rhodamine 123 for mitochondria, and Nile Red for plasma membrane. Numerical image analysis allowed us to obtain, for each cell, morphological parameters (e.g., cell size, nuclear size, and shape) and functional information (e.g., nuclear DNA content, level of mitochondria energetic state, and the amount and properties of the plasma membrane) by fluorescence intensity. These parameters were used in a typological analysis that separated control cells into four groups. RESULTS: A discriminant factorial analysis (DFA) confirmed the four groups: G0-G1, S, G2+M, and polyploid cells called Gn. These groups were significantly different, with a classification probability of 0.9999; these control cells defined a learning population. Different populations of untreated and adriamycin-treated cells were analyzed as additional individuals within a DFA and were classified into the G0-G1, S, G2+M, and Gn groups by their probability of belonging to each of the groups. CONCLUSIONS: This approach is particularly efficient when it is used to determine variations in cellular properties and to objectively study cellular populations.

Cell Cycle↗
PubMed1983

Pulmonary function and symptoms of Nigerian workers exposed to Carbon black in dry cell battery and tire factories.

The pulmonary function and symptoms of 125 workers exposed to carbon black in dry cell battery and tire manufacturing plants were investigated. There was no significant difference in the pulmonary function of the subjects in the two plants. There was good agreement in the symptoms reported in the two different factories: cough with phlegm production, tiredness, chest pain, catarrh, headache, and skin irritation. The symptoms also corroborate those reported in the few studies on the pulmonary effects of carbon black. The suspended particulate levels in the dry cell battery plant ranged from 25 to 34 mg/m3 and the subjects with the highest probable exposure level had the most impaired pulmonary function. The pulmonary function of the exposed subjects was significantly lower than that of a control, nonindustrially exposed population. The drop in the lung function from the expected value per year of age was relatively constant for all the study subgroups but the drop per year of duration of employment was more severe in the earlier years of employment. This study has underscored the need for occupational health regulations in the industries of developing countries.

Air↗
PubMedDate not supplied

A factorial design to optimize cell-based drug discovery analysis.

Drug discovery is dependent on finding a very small number of biologically active or potent compounds among millions of compounds stored in chemical collections. Quantitative structure-activity relationships suggest that potency of a compound is highly related to that compound's chemical makeup or structure. To improve the efficiency of cell-based analysis methods for high throughput screening, where information of a compound's structure is used to predict potency, we consider a number of potentially influential factors in the cell-based approach. A fractional factorial design is implemented to evaluate the effects of these factors, and lift chart results show that the design scheme is able to find conditions that enhance hit rates.

Anti-HIV Agents↗
PubMed1998

DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

Amino Acid Sequence↗
PubMed2006

Mercury levels and liver pathology in feral fish living in the vicinity of a mercury cell chlor-alkali factory.

Twenty-three barbels (Barbus graellsii), 30 bleaks (Alburnus alburnus) and 4 sediment samples were collected from four areas of the Cinca River (NE Spain), upstream and downstream of a mercury cell chlor-alkali plant. Mercury concentration in sediments downstream of the plant was about 25 times higher than the concentration upstream. Mercury levels in the muscle and liver of barbels downstream of the chlor-alkali plant were 10 and 30 times higher than those upstream. The average mercury concentration in the muscle and liver of barbels downstream of the factory was 1.48 and 1.78mg/kgw.w., with maximal concentrations of 2.43 and 3.64mg/kgw.w, respectively. Liver/muscle ratio of mercury was significantly higher in barbels sampled downstream of the factory. Bleak showed a similar pattern, with a significantly higher whole-body mercury concentration downstream of the chlor-alkali plant. Nevertheless, a progressive decrease in mercury concentration downstream of the impact area was found in bleak, but not in barbel. Histopathological analysis showed that fish downstream of the factory had significantly higher prevalence of liver pathologies (p<0.05). Although there are no specific histopathological markers of mercury exposure, barbels with the highest Hg levels in the liver also showed the most severe pathologies, and the processes found in these barbels were consistent with the pro-oxidant effect of heavy metals.

Animals↗
PubMed2025

Hierarchical metabolic engineering for rewiring cellular metabolism.

Metabolic engineering is a key enabling technology for rewiring cellular metabolism to enhance production of chemicals, biofuels, and materials from renewable resources. However, how to make cells into efficient factories is still challenging due to its robust metabolic networks. To open this door, metabolic engineering has realized great breakthroughs through three waves of technological research and innovations, especially the third wave. To understand the third wave of metabolic engineering better, we discuss its mainstream strategies and examples of its application at five hierarchies, including part, pathway, network, genome, and cell level, and provide insights as to how to rewire cellular metabolism in the context of maximizing product titer, yield, and productivity. Finally, we highlight future perspectives on metabolic engineering for the successful development of cell factories.

Metabolic Engineering↗
PubMed2006

Nutrient sensing in plant meristems.

Plants need nutrient to grow and plant cells need nutrient to divide. The meristems are the factories and cells that are left behind will expand and differentiate. However, meristems are not simple homogenous entities; cells in different parts of the meristem do different things. Positional cues operate that can fate cells into different tissue domains. However, founder/stem cells persist in specific locations within the meristem e.g. the quiescent centre of root apical meristem (RAM) and the lower half of the central zone of the shoot apical meristem (SAM). Given the complexity of meristems, do their cells simply respond to a diffusing gradient of photosynthate? This in turn begs the question, why do stem cell populations tend to have longer cell cycles than their immediate descendants given that like all other cells they are directly in the path of diffusing nutrient? In this review, we have examined the extent to which nutrient sensing might be operating in meristems. The scene is set for sugar sensing, the plant cell cycle, SAMs and RAMs. Special emphasis is given to the metabolic regulator, SnRK1 (SNF1-related protein kinase 1), hexokinase and the trehalose pathway in relation to sugar sensing. The unique plant cell cycle gene, cyclin-dependent kinase B1;1 may have evolved to be particularly responsive to sugar signalling pathways. Also, the homeobox gene, STIMPY, emerges strongly as a link between sugar sensing, plant cell proliferation and development. Flowering can be influenced by sucrose and glucose levels and both meristem identity and organ identity genes could well be differentially sensitive to sucrose and glucose signals. We also describe how meristems deal with extra photosynthate as a result of exposure to elevated CO2. What we review are numerous instances of how developmental processes can be affected by sugars/nutrients. However, given the scarcity of knowledge we are unable to provide uncontested links between nutrient sensing and specific activities in meristems.

Carbohydrate Metabolism↗
PubMed2001

Etoposide induces the dispersal of DNA ligase I from replication factories.

In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA--cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.

Antineoplastic Agents, Phytogenic↗
PubMed2004

Localisation of human Y-family DNA polymerase kappa: relationship to PCNA foci.

DNA polymerases of the Y-family are involved in translesion DNA synthesis past different types of DNA damage. Previous work has shown that DNA polymerases eta and iota are localised in replication factories during S phase, where they colocalise one-to-one with PCNA. Cells with factories containing these polymerases accumulate after treatment with DNA damaging agents because replication forks are stalled at sites of damage. We now show that DNA polymerase kappa (pol(kappa)) has a different localisation pattern. Although, like the other Y-family polymerases, it is exclusively localised in the nucleus, pol(kappa) is found in replication foci in only a small proportion of S-phase cells. It does not colocalise in those foci with proliferating cell nuclear antigen (PCNA) in the majority of cells. This reduced number of cells with pol(kappa) foci, when compared with those containing pol(eta) foci, is observed both in untreated cells and in cells treated with hydroxyurea, UV irradiation or benzo[a]pyrene. The C-terminal 97 amino acids of pol(kappa)are sufficient for this limited localisation into nuclear foci, and include a C2HC zinc finger, bipartite nuclear localisation signal and putative PCNA binding site.

Amino Acid Sequence↗
PubMed1993

Relative involvement of protein kinase C and of the estrogen receptor in the cytotoxic action of a population of triphenylethylenes on MCF7 cells as revealed by correspondence factorial (CF) analysis.

A multivariate statistical method, correspondence factorial (CF) analysis, was used to examine the correlations among the protein binding and cell proliferation effects of a series of 36 di- and triphenylethylenes (DPEs and TPEs). The analysis was applied to a study which measured their competition for estradiol binding to cytosol estrogen receptor (ER), their influence on protein kinase C (PKC) activity under different conditions of enzyme activation, their ability to promote the growth of a breast cancer cell line and to inhibit growth at high concentrations (cytotoxicity). The CF analysis revealed several levels of correlation. First, it distinguished those molecules within the population that stimulated rather than inhibited PKC activity. Second, it made apparent a strong correlation between cytotoxicity and inhibition of Ca++ and phosphatidylserine-dependent PKC activity, which was most marked when the enzyme had been activated by diacylglycerol indicating that PKC inhibition under physiological conditions might contribute to the overall cytotoxicity of these compounds. Third, a lower level of correlation was established between competition for ER binding and cytotoxicity. Taken together, the results suggest that MCF7 cells might be most sensitive to a cytotoxic effect of TPEs (via PKC and other targets) when they at the same time decrease estrogen-stimulated proliferation via an ER-mediated antiestrogenic effect.

Animals↗
PubMedDate not supplied

Guidelines for the design and statistical analysis of experiments in papers submitted to ATLA.

In vitro experiments need to be well designed and correctly analysed if they are to achieve their full potential to replace the use of animals in research. An "experiment" is a procedure for collecting scientific data in order to answer a hypothesis, or to provide material for generating new hypotheses, and differs from a survey because the scientist has control over the treatments that can be applied. Most experiments can be classified into one of a few formal designs, the most common being completely randomised, and randomised block designs. These are quite common with in vitro experiments, which are often replicated in time. Some experiments involve a single independent (treatment) variable, while other "factorial" designs simultaneously vary two or more independent variables, such as drug treatment and cell line. Factorial designs often provide additional information at little extra cost. Experiments need to be carefully planned to avoid bias, be powerful yet simple, provide for a valid statistical analysis and, in some cases, have a wide range of applicability. Virtually all experiments need some sort of statistical analysis in order to take account of biological variation among the experimental subjects. Parametric methods using the t test or analysis of variance are usually more powerful than non-parametric methods, provided the underlying assumptions of normality of the residuals and equal variances are approximately valid. The statistical analyses of data from a completely randomised design, and from a randomised-block design are demonstrated in Appendices 1 and 2, and methods of determining sample size are discussed in Appendix 3. Appendix 4 gives a checklist for authors submitting papers to ATLA.

Animal Testing Alternatives↗
PubMedDate not supplied

Modifications of p53 protein and accumulation of p21 and gadd45 mRNA in TGF-beta 1 growth inhibited cells.

Transforming growth factory beta (TGF-beta) is a potent growth inhibitor of epithelial cells. One of the strategies used to elucidate the anti-proliferative mode of action of TGF-beta is to find out whether the receptor-generated signals interact with components of the basic machinery of the cell cycle. In this study we examined whether p53 and two other cycle inhibitory genes that can be transactivated by p53 are affected by TGF-beta 1 in epithelial cells. We show that TGF-beta 1 signalling controls the intracellular localization as well as the phosphorylation pattern and the stability of p53 protein. TGF-beta signalling also elevates the expression of p21/waf-1 and gadd45. The observed modifications in the protein suggest that p53 is involved in mediation of TGF-beta 1 growth inhibition. However, in TGF-beta 1 growth inhibited cells, wild type p53 is not required for the accumulation of the two p53 downstream targets p21/waf-1 and gadd45.

Animals↗
PubMed1997

Identification of transcription factories in nuclei of HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1.

Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated. Silver grains were also scattered over the remaining nucleoplasm but not in the clusters of interchromatin granules, and over the dense fibrillar component of the nucleolus as in control, nontransfected HeLa cells. Pulse-chase experiments revealed the transient presence of migrating RNA in the clusters of interchromatin granules. RNA polymerase II was revealed by immunogold labeling following the use of two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells.

Cell Nucleus↗
PubMed1995

Effect of pregnancy-specific protein B on luteal cell progesterone, prostaglandin, and oxytocin production during two stages of the bovine estrous cycle.

This experiment was designed to evaluate the effects of pregnancy-specific protein B (PSPB) on luteal cell progesterone (P4), PGF2 alpha, PGE2, and oxytocin secretion. Corpora lutea were collected during the mid (d 10 to 12; n = 5) or late luteal (d 17 to 18; n = 5) stage of the estrous cycle. Large and small cells (1.5 x 10(5)/well) were treated with PSPB (0, 2.5, or 5.0 micrograms) and LH (0, 50, or 100 ng) in a 3 x 3 factorial arrangement. Cells were incubated for 18 h before adding treatments; after treatments, medium was collected at 6 and 12 h. During the 18-h pretreatment period, P4, PGF2 alpha, PGE2, and oxytocin production was similar between the prospective treatment groups. The PSPB did not affect P4 production. Stage of the cycle (stage) x time interaction (P < .001) indicated that mid-stage luteal cells produced more P4 than late-stage cells; regardless of stage, P4 decreased with time. The time x LH interaction (P < .001) revealed that at 6 and 12 h the 50- and 100-ng doses of LH increased P4 to greater than the 0-ng dose. Production of PGF2 alpha by mid-stage cells was similar among the three PSPB treatments; however, PGF2 alpha production by late-stage cells increased (P < .01) in response to the 5.0-micrograms dose of PSPB. The LH did not affect PGF2 alpha production. Late-stage luteal cells produced more (P < .001) PGF2 alpha than mid-stage cells during the 18-h pretreatment period and at 6, but not 12, h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Rearrangement of intermediate filament network of BHK-21 cells infected with vaccinia virus.

Association between vaccinia virus (VV) structures and intermediate filaments in specific areas of the cytoplasm of infected cells (virus "factories") suggests that VV infection interferes with the cellular architecture by modifying the intermediate filament network. To analyse this question, we examined the array of intermediate filaments of BHK-21 cells infected with VV by laser scanning confocal microscopy using an anti-vimentin mouse monoclonal antibody. We observed a marked reorganization of intermediate filaments around the nucleus of infected cells. Bidimensional analysis of 32PO4-labeled intermediate filament proteins revealed that the acidic isoform of vimentin and two isoforms of desmin have increased phosphorylation levels in infected cells. Our results suggest that the reorganization of intermediate filaments observed during VV infection could be promoted by an increase in the phosphorylation level of the intermediate filament proteins, vimentin and desmin.

Animals↗
PubMed2005

[Coordination inhibition of the proliferation of MCF-7 by enhancing expression of p53,p21 and decreasing expression of c-myc].

To study the coordination inhibition of the proliferation of breast cancer cell line MCF-7 by enhancing expression of p53,p21 and decreasing expression of c-myc, as well as the relationship among these genes when they express in the cells, eukaryotic expression plasmids of sense p53,p21 and antisense c-myc were constructed first, different concentrations of three plasmids were determined and the combinations were designed according to factorial design. Cells were transfected by the combination plasimids, then the inhibition rate of proliferation of the transfected cells were tested. The results were analyzed using the methods of statistics including Jin's Q-test, Least significant difference (LSD), cluster analysis. It was shown that all of them (Sense p21,p53, antisense c-myc) could inhibit the proliferation of MCF-7 in different levels of concentration, while the degrees of inhibition were different among them. In the aspect of coordination inhibition, the proliferation of MCF-7 was inhibited much strongly by p21 coordination with antisense c-myc and p53 coordination with antisense c-myc, but p53 coordination with p21 did not show inhibition effect. Rusults of cluster analysis showed that the first combination of three genes showed the best coordination, the ninth combination had the highest rate of inhibition. In conclusion, we suppose that when p53 as a cancer suppressor gene and p21 as a CDK suppressor gene express highly, and the expression of c-myc as a cancer gene is inhibited meantime could obviously enhance the inhibition on the proliferation of MCF-7 cells by coordination effect.

Blotting, Western↗
PubMed2003

Progress and challenges for cell encapsulation in brain tumour therapy.

Cell encapsulation provides a method to circumvent the host immune system by encapsulating cells or tissues in immunoisolating, semipermeable membranes before implantation. The technology has been widely studied with an aim of developing bio-organs transplantable into patients without the need of immunosuppression, and in cancer therapy, the principle of cell encapsulation may be further exploited. Encapsulated recombinant cells represent factories or bioreactors for the production of therapeutic proteins. By implanting the bioreactors in the vicinity of the tumour, long-term local de novo delivery of the therapeutic proteins may be achieved. Malignant brain tumours such as glioblastoma multiforme (GBM) remain highly lethal neoplasms, refractory to current therapies. Researchers and medical professionals are working on developing translational therapies to combat these aggressive tumours. Numerous clinical trials on gene therapy for glioma patients have been conducted over the last decade, but the results have largely been disappointing. Cell encapsulation represents an alternative method for local delivery of therapeutic proteins with antineoplastic properties to glioma patients. The concept has not yet reached clinical trials, but encouraging results have been achieved in rats bearing gliomas when implanting endostatin-secreting encapsulated cells into the rat brain. This review primarily focuses on the recent progress that has been made with cell encapsulation technology. In addition, the challenges this field faces before clinical application in brain tumour patients is discussed.

Alginates↗