Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.     

Free nonimmobilized ligands as a tool for purification of proteins

Purification of proteins on a large scale is a complex multistep process, and alternative economic strategies are required. This study presents a novel approach (Affinity Sinking, AS) for purification of native proteins utilizing nonimmobilized modified ligands. The nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports. Therefore, purification from large volumes can be accomplished without the use of industrial-scale affinity columns. The mechanism of product capture is formation and precipitation of a specific [target-protein/modified-ligand] complex by using a soluble interconnecting entity that generates an insoluble [target-protein/modified-ligand/interconnecting entity] sediment containing the target protein. Rabbit IgG and two glycoproteins were purified accordingly, utilizing free avidin (as the interconnecting entity) and either desthiobiotinylated-protein A (DB-ProA) or desthiobiotinylated-concanavalin A (DB-ConA) as the modified ligand. The recovery yields for the IgG and the two glycoproteins were 80-86% and 70-75%, respectively. Target proteins are eluted from the generated pellet nearly without disrupting the [modified-ligand/interconnecting entity] macro-complex, thus enabling a practical procedure of recovering target proteins. Leaching of the DB-ProA ligand under eluting conditions (pH 3) was found to be lower than 1%. The two modified ligands, DB-ProA and DB-ConA, were regenerated without any chromatographic procedure in 80% and 85%-89% yield, respectively. The advantage of excluding the polymeric component from the purification process and obtaining highly purified proteins has been demonstrated, and it implies that other contaminants (e.g. endotoxins, prions, host DNA) could be excluded as well, thereby reducing the number of purification steps in a typical downstream process.     

On invariants of a 2-D proteome map derived from neighborhood graphs

We consider the problem of the construction of invariants for characterization of 2-D maps, such as 2-D proteome maps, 2-D NMR spectral maps, etc., that in addition to facilitating cataloguing such maps, can be used for comparison of maps and numerical evaluation of their degree of similarity. A novel approach, based on the concept that the nearest neighborhood of points (spots) on a map are sufficiently flexible to allow one not only to vary the number of points used for characterization of the map but also the density of information on their relative positions, is put forward. The method is illustrated with the Coomassie brilliant blue stained 2-D gel electrophoresis patterns of the proteomes from liver cells of healthy male Fisher F344 rats and the rats treated with four peroxisome proliferators.     

Fate of sperm organelles during early embryogenesis in the rat

The report is part of a continuing study in which we employ monoclonal antibodies to membrane domains and internal organelles of rat spermatozoa in order to trace events during maturation, capacitation, fertilization, and early development. In the present study, we have used immunocytochemistry at the light and EM levels to localize one antibody, 5A5, to the fibrous sheath and a second, 3D5, to the outer mitochondrial membrane. Antibody 5A5 does not stain the fibrous sheath of spermatozoa of rodents other than the rat, while 3D5 can be localized to the outer mitochondrial membrane of rat, hamster, and mouse spermatozoa. In order to follow these antibodies during fertilization and early embryogenesis, we developed a method to stain internal components of zygotes and early embryos. Our findings suggests that the fibrous sheath disappears prior to the first cleavage and that mitochondria can be detected up to the 2-cell stage in mouse and the 4-cell stage in rat. © 1994 Wiley-Liss, Inc.     

Intensity dependent estimation of noise in microarrays improves detection of differentially expressed genes

Background  

In many microarray experiments, analysis is severely hindered by a major difficulty: the small number of samples for which expression data has been measured. When one searches for differentially expressed genes, the small number of samples gives rise to an inaccurate estimation of the experimental noise. This, in turn, leads to loss of statistical power.     

Advances in using MRI probes and sensors for in vivo cell tracking as applied to regenerative medicine

The field of molecular and cellular imaging allows molecules and cells to be visualized in vivo non-invasively. It has uses not only as a research tool but in clinical settings as well, for example in monitoring cell-based regenerative therapies, in which cells are transplanted to replace degenerating or damaged tissues, or to restore a physiological function. The success of such cell-based therapies depends on several critical issues, including the route and accuracy of cell transplantation, the fate of cells after transplantation, and the interaction of engrafted cells with the host microenvironment. To assess these issues, it is necessary to monitor transplanted cells non-invasively in real-time. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task, given its ability to image deep inside tissue with high temporal resolution and sensitivity. Extraordinary efforts have recently been made to improve cellular MRI as applied to regenerative medicine, by developing more advanced contrast agents for use as probes and sensors. These advances enable the non-invasive monitoring of cell fate and, more recently, that of the different cellular functions of living cells, such as their enzymatic activity and gene expression, as well as their time point of cell death. We present here a review of recent advancements in the development of these probes and sensors, and of their functioning, applications and limitations.KEY WORDS: Regenerative medicine, Stem cells, Magnetic resonance imaging, Paramagnetic contrast agents, CEST, Perfluorocarbon particles, Biosensor, Cell labeling, Cellular function     

Programmed cell death occurs asymmetrically during abscission in tomato

Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.