The relation between cell size,chromosome length and the orientation of chromosomes in dividing root cortex cells

Cells in the root meristem are organised in longitudinal files. Repeated transverse cell divisions in these files are the prime cause of root growth. Because of the orientation of the cell divisions, we expected to find mitoses with an spindle axis parallel to the file axis. However, we observed in the root cortex ofVicia faba large number of oblique chromosome orientations. From metaphase to telophase there was a dramatic increase of the rotation of the spindle axis. Measurements of both the size of the cortex cells and the chromosome configurations indicated that most cells were too small for an orientation of the spindle parallel to the file axis. Space limitation force the spindle into an oblique position. Despite this spindle axis rotation, most daughter cells remained within the original cell file. Only in extremely flat cells did the position of the daughter nuclei forced the cell to set a plane of division parallel to the file axis, which result in side-by-side orientation of the daughter cells. Telophase spindle axis rotations are also observed inCrepis capillaris andPetunia hybrida.. These species have respectively medium and small sized chromosomes compared toVicia. Since space limitation, which causes the rotation, depends both on cell and chromosome size, the frequency and extent of the phenomenon in former two species is comparatively low.     

Independent ATPase activity of Hsp90 subunits creates a flexible assembly platform

The ATPase activity of the molecular chaperone Hsp90 is essential for its function in the assembly of client proteins. To understand the mechanism of human Hsp90, we have carried out a detailed kinetic analysis of ATP binding, hydrolysis and product release. ATP binds rapidly in a two-step process involving the formation of a diffusion-collision complex followed by a conformational change. The rate-determining step was shown to be ATP hydrolysis and not subsequent ADP dissociation. There was no evidence from any of the biophysical measurements for cooperativity in either nucleotide binding or hydrolysis for the dimeric protein. A monomeric fragment, lacking the C-terminal dimerisation domain, showed no dependence on protein concentration and, therefore, subunit association for activity. The thermodynamic linkage between client protein binding and nucleotide affinity revealed ATP bound Hsp90 has a higher affinity for client proteins than the ADP bound form. The kinetics are consistent with independent Michaelis-Menten catalysis in each subunit of the Hsp90 dimer. We propose that Hsp90 functions in an open-ring configuration for client protein activation.     

BioPEPR: a system for the automatic prescreening of cervical smears.

A feasibility study has indicated that a Prescion Encoding and Pattern Recognition (PEPR) cathode ray tube prescreening system for cervical smears can be both accurate and fast. Smears are prepared using a syringing technique and are stained with a Feulgen-type nuclear stain and a protein counter-stain. The use of film as an intermediate step between the cells and Bio PEPR allows the scanning of fields as large as 8 x 8 mm. The morphological features of the cells are measured as directed by a hierarchical decision strategy. Additional programs detect artifacts, overlaps, and leukocytes. For clean samples, false positive and false negative rates on the cell level have been obtained that will allow acceptable smear level rates (10% false positive, 1% false negative). These rates have been reached without compromising the required speed goals of 120 to 180 smears per hr. The efficiency of the system is dependent on the quality of the smears. Measurements on a set of 192 routinely prepared smears indicate acceptable false negative rates and a false positive rate of about 18%. A reduction of this rate is expected with small improvements in cell preparation and measuring software, leading to the overall system efficiency required for commercial feasibility.     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

DNA Methylation Landscapes of Human Fetal Development

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.     

Influence of culture heterogeneity in cell surface charge on adhesion and biofilm formation by Enterococcus faecalis

Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms. Enterococcus faecalis strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of E. faecalis strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein (esp) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.