P. Van Oostveldt

Telomere length versus hormonal and bone mineral status in healthy elderly men.

Telomeres, the termini of linear chromosomes, exert a key role in the process of cellular ageing. Progressive telomere shortening is implicated in senescence in vitro and ample evidence exists to support the hypothesis that telomere length is correlated with chronological age and ageing phenotypes in vivo. In this study, we assessed whether mean telomere length of peripheral blood leukocytes predicts age-associated bone loss and/or is related to sex steroid status in an elderly healthy male population (71-86 years). Out of this population, we selected 110 samples for telomere restriction fragment (TRF) length analysis. Fasting blood was analysed for testosterone, estradiol, sex hormone binding globulin and biochemical markers of bone turnover. Also, the bioavailable fractions of sex steroids were calculated. Bone mineral density was measured at baseline and longitudinal follow-up was available for 84 men. We found that mean TRF length was inversely correlated with age (r=-0.19; P=0.049). Although no correlations were found with sex steroids or BMD at baseline, age corrected mean TRF length was associated with longitudinal bone loss for different distal forearm sites (P<0.05). Further studies are required to confirm our results, yet in this study, the predictive value of telomere length for bone loss appears to be substantial, hence underscoring the role of telomere length as a biomarker of ageing phenotypes.

Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle.

Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV‐304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially positioned at the interface of chromatin domains. TRF1 and TRF2 are abundantly present in these territories but not firmly bound. At the onset of mitosis, the bulk of TRF protein dissociates from telomere regions, territories disintegrate and individual telomeres become faintly visible. The combination of stable cell lines, CLEM and cytometry proved essential in providing novel insights in compartment‐based nuclear organization and may serve as a model approach for investigating telomere‐driven genome‐instability and studying long‐term nuclear dynamics.

Effect of Light on Nucleic-acid Synthesis and Polyploidy Level in Elongating Epicotyl Cells of Pisum sativum

The synthesis of DNA and RNA and the increase in dry matter were followed in the elongating cells of the epicotyl of peas (Pisum sativum L.) germinating in total darkness or in continuous light. The amounts of DNA and RNA per epicotyl were estimated by chemical methods, the amount of DNA per cell was measured by histophotometric techniques. The increase in DNA, RNA and dry matter in the epicotyl cells is much higher during germination in darkness than in light. During elongation in the dark most cortical epicotyl cells reach the 8C polyploidy level, in the light only the 4C polyploidy level is reached. A decrease in RNA synthesis is in agreement with a reduction in nuclear volume.SummaryThe synthesis of DNA and RNA and the increase in dry matter were followed in the elongating cells of the epicotyl of peas (Pisum sativum L.) germinating in total darkness or in continuous light. The amounts of DNA and RNA per epicotyl were estimated by chemical methods, the amount of DNA per cell was measured by histophotometric techniques. The increase in DNA, RNA and dry matter in the epicotyl cells is much higher during germination in darkness than in light. During elongation in the dark most cortical epicotyl cells reach the 8C polyploidy level, in the light only the 4C polyploidy level is reached. A decrease in RNA synthesis is in agreement with a reduction in nuclear volume.

Cytoplasmic dynein colocalizes with melanosomes in normal human melanocytes

Background Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin‐based and microtubule‐based motor proteins co‐operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast‐growing plus end and a slow‐growing minus end . Microtubule‐associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end‐directed movement of melanosomes.  Objectives We aimed to investigate the in vitro expression of the minus end‐directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts.  Methods Reverse transcription–polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed.  Results The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co‐localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and α‐tubulin with the melanosome surface was detected using immunogold electron microscopy.  Conclusions The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end‐directed melanosome movement along microtubules.

High content image cytometry in the context of subnuclear organization.

The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fast‐growing need for image‐based cytometry. Simultaneously, the massive amount of data that is generated by image‐cytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA damage repair patterns in response to genotoxic stress and radiation, and we show the potential of data mining in pinpointing specific phenotypes after transient transfection. The presented approach allowed for systematic analysis of subnuclear features in large image data sets and accurate classification of phenotypes at the level of the single cell. Consequently, this type of nuclear fingerprinting shows potential for high‐throughput applications, such as functional protein assays or drug compound screening.

Underreplication of repetitive DNA in polyploid cells of Pisum sativum

Abstract Earlier reported results of a difference in the amount of DNA between 2C meristematic plumula cells and 2C cortex cells of the epicotyls are confirmed by fluorometric Feulgen-DNA determinations. Renaturation experiments of isolated DNA suggest that this difference in DNA is probably correlated with an underreplication of some highly repetitive DNA sequences in cells preparing endomitosis.

Quantification of microparticle coating quality by confocal laser scanning microscopy (CLSM).

In this work, a novel protocol was developed for determining film coating thickness and coating quality of microparticles, based on the use of confocal laser scanning microscopy (CLSM). CLSM was found to be an adequate non-destructive technique for the quantification of the coating thickness and coating quality of individual thin-coated small particles. Combined with image analysis, it was possible to derive with high accuracy the coating thickness distribution of a representative number of microparticles. The performance of the novel methodology was assessed by the quantification of the coating thickness and coating quality of protein-coated microparticles produced by fluidized bed coating. It was found that the CLSM data on coating layer thickness were generally in good agreement with the results from chemical analysis, down to a thickness of 1-1.5 microm. Using CLSM the importance of setting up the appropriate distance between the coating nozzle and the powder bed with respect to microparticle coating quality in fluidized bed processing was illustrated. Coating quality was found to decrease with increasing distance the coating droplets have to travel before impinging onto the core particles as a result of spray-drying of the coating droplets. Also, coating quality decreased with increasing viscosity of the coating droplets, resulting in reduced spreading on the cores.

Four-dimensional imaging and computer-assisted track analysis of nuclear migration in root hairs of Arabidopsis thaliana

Nuclear migration is a fundamental mechanism necessary for the proper growth and development of many eukaryotic organisms. In this study root hairs of Arabidopsis thaliana were used as a research model to gain insight into the dynamics of nuclear migration. Root hairs are long tubular outgrowths of epidermal cells and are responsible for the uptake of water and nutrients. During the development of root hairs, the nucleus migrates into the hair after the bulge is formed. The position of the nucleus relative to the tip plays an essential role in the growth process. However, what is happening to the nucleus in full‐grown root hairs is still unclear. To study nuclear dynamics in living root hair cells, stably transformed plants with the fusion proteins Histone2B‐YFP and NLS‐GFP‐GUS were used. Four‐dimensional confocal laser scanning microscopy made it possible to monitor the exact position of the nucleus in different root hairs. To analyse the sequential positions of the nuclei in the root hairs, a new computer‐assisted method was developed. After track analysis a number of parameters could be extracted from the movies, such as the average speed, the amplitude, direction factor and the range of movement in the root hairs. Our results show that nuclei do not reach a final position in full‐grown root hairs and this sustained movement seems to be more similar in root hairs lying close to each other. Moreover, with this methodology it could be quantitatively demonstrated that the integrity of actin is necessary for nuclear movement.

Microtubule turnover in ooplasm biopsy reflects ageing phenomena in the parent oocyte.

Oviductal oocytes retrieved from superovulated B6D2F1 mice at 13.5, 16 and 19 h after human chorionic gonadotrophin (HCG) (groups A, B and C respectively, n = 382) were micromanipulated to obtain 12-20 mum sized ooplasm biopsy fragments. Experiments were divided into three sets. Ooplasmic microtubule dynamics were studied in ooplasm biopsy specimens and parent oocytes (set 1) and ooplasm biopsy specimens (set 2), whilst zona pellucida dissolution time, cortical granule loss and spindle/chromatin morphology using confocal microscopy were also studied in parent oocytes (set 2). Oocytes withstood oocyte biopsy with a high survival rate (98.2%) and the biopsied oocytes underwent successful fertilization and development (set 3). An absolute one-to-one correlation was seen between the oocyte biopsy specimens and the parent oocytes in terms of ooplasmic microtubule dynamics (set 1), and increased ooplasmic microtubule dynamics in oocyte biopsy specimens paralleled ageing phenomena in the parent oocytes (set 2). Zona pellucida dissolution time was significantly lower in parent oocytes from group A versus groups B (P = 0.032), and C (P < 0.001). (Groups A, B, C include minimal, moderate, increased ooplasmic microtubule dynamics in oocyte biopsy specimens respectively.) Oocyte cortical granule loss and spindle/chromatin abnormalities were mainly seen in group C (P < 0.001). Oocyte biopsy can thus be applied to judge age-related changes in the parent oocytes.

The prognostic significance of the DNA content in Ewing's sarcoma: a retrospective cytophotometric and flow cytometric study.

The DNA content of the cell nuclei of Ewings sarcoma was analysed by means of cytophotometry in situ with image analysis in Feulgen‐stained sections in 37 patients, and by retrospective flow cytometry according to the method of Hedley in 26 patients. Different histogram patterns were obtained: normal unimodal or bimodal DNA distributions and abnormal DNA distributions with one or two stem lines, or an abnormal DNA distribution with no stem lines. Both methods enabled us to make a distinction between two groups of Ewings sarcomas with a different prognosis. All patients with aneuploid tumours died within 5 years after the initial diagnosis. Eleven of 19 (58%) patients with a normal DNA distribution in their tumour, as determined by cytophotometry, are still alive and in good health with a mean survival period of 7.5 years, ranging from 2 to 19 years. Of the group of patients in which flow cytometry revealed a normal DNA pattern, eight of 15 (53%) are still alive and in good health, with a mean survival period of 8 years. These results indicate that both techniques are reliable methods for obtaining prognostic information in Ewings sarcomas. However, cytophotometry in situ yielded a better discrimination for the overall survival (P < 0.01) than did flow cytometry (P <0.05). In 19% of the cases there was a discrepancy between the DNA histograms obtained with the two techniques. In five of 26 cases the DNA distributions were classified as normal by one method and aneuploid by the other. Tumour cell representation or selective loss of cells during enzymatic treatment may be responsible for this discrepancy.