Chronophin is a glial tumor modifier involved in the regulation of glioblastoma growth and invasiveness.

Glioblastoma is the most aggressive primary brain tumor in adults. Although the rapid recurrence of glioblastomas after treatment is a major clinical challenge, the relationships between tumor growth and intracerebral spread remain poorly understood. We have identified the cofilin phosphatase chronophin (gene name: pyridoxal phosphatase, PDXP) as a glial tumor modifier. Monoallelic PDXP loss was frequent in four independent human astrocytic tumor cohorts and increased with tumor grade. We found that aberrant PDXP promoter methylation can be a mechanism leading to further chronophin downregulation in glioblastomas, which correlated with shorter glioblastoma patient survival. Moreover, we observed an inverse association between chronophin protein expression and cofilin phosphorylation levels in glioma tissue samples. Chronophin-deficient glioblastoma cells showed elevated cofilin phosphorylation, an increase in polymerized actin, a higher directionality of cell migration, and elevated in vitro invasiveness. Tumor growth of chronophin-depleted glioblastoma cells xenografted into the immunodeficient mouse brain was strongly impaired. Our study suggests a mechanism whereby the genetic and epigenetic alterations of PDXP resulting in altered chronophin expression may regulate the interplay between glioma cell proliferation and invasion.

Structural organization of the hepatitis B virus minichromosome.

The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA, is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins. In this study we investigated the architecture of the HBV minichromosome in more detail. In contrast to cellular chromatin the nucleosomal spacing of the HBV minichromosome has been shown to be unusually reduced by approximately 10 %. A potential candidate responsible for an alteration in the chromatin structure of the HBV minichromosome is the HBV core protein. The HBV core protein has been implicated in the nuclear targeting process of the viral genome. The association of the HBV core protein with nuclear HBV replicative intermediates could strengthen this role. Our findings, confirmed by in vivo and in vitro experiments indicate that HBV core protein is a component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes by 10 %. From this model of the HBV minichromosome we propose that the HBV core protein may have an impact on the nuclear targeting of the HBV genome and be involved in viral transcription by regulating the nucleosomal arrangement of the HBV regulatory elements, probably in a positive manner.

TAT results in a longitudinal study of bereaved college students.

The authors analyzed projective data obtained from 141 college students who wrote stories on three separate occasions to selected cards from the Thematic Apperception Test (TAT). The students included 46 members of support groups for bereaved college students, 34 members of a bereavement control group, and 61 nonbereaved students. The study used a repeated-measures pretest-posttest control group design to gather longitudinal data about the trajectory of bereavement with and without support group intervention. Coders, who reached consistently high interrater reliability, looked for themes of death, grief, coping, and affiliation in the stories. Multivariate analysis of variance (MANOVA) and repeated-measures MANOVA tests were applied to analyze coding results. Overall MANOVA results indicated significant group differences in the responses to the TAT cards. Repeated-measures MANOVA found group differences in use of themes of death and grief and found Group x Time differences in maintaining a sense of self-efficacy while in a crisis. A majority of the stories contained affiliation imagery but without any group differences in the use of such imagery.

Localization of hepatitis B virus core protein and viral DNA at the nuclear membrane.

An early step in the replication of the hepatitis B virus (HBV) genome is the transport of the viral DNA into the nucleus of the infected cell. So far only little is known about the events and mechanisms at the nuclear membrane required for entry of the viral genome into the nucleus. Using a hepatoblastoma cell line that constitutively produces hepatitis B virions and in so doing displays intracellular viral amplification, we showed that nonparticulated HBV core protein is associated with nuclear membrane pore complexes. Additionally, viral DNA has been detected firmly attached to the nuclear membrane. Small amounts of viral core protein, as well as viral DNA, were detectable within the cell nucleus. However, core particles could not be shown at the nuclear membrane or within the nuclei of these cells. Our observations on localization of HBV DNA and core protein at the nuclear membrane thus provide a suggestion for further examinations of the transfer of the viral genome from the cytoplasm into the nucleus of the infected cell.

Diheteroduplex formation using gold labeled single-stranded PCR fragments and its application in electron microscopy.

Heteroduplex analysis is commonly used to map homologous sequences in DNA:DNA or DNA:RNA hybrids in spread preparations by electron microscopy. However, the standard procedures are not suitable to detect the orientation of a fragment with a defined sequence in a hybrid molecule. Here, we describe an alternative protocol for the visualization of DNA:DNA "diheteroduplex" structures based on digoxigenin/anti-digoxigenin gold labeling that allows determination of the position and orientation of a fragment. Single-stranded polymerase chain reaction (PCR) generated fragments labeled at their 3' ends are hybridized to double-stranded plasmid DNA. Electron microscopy of spread preparations visualizes the gold label and, in combination with morphometric measurements, it is possible to determine the position and orientation of the fragment with the diheteroduplex molecule.