Pore membrane and/or filament interacting like protein 1 (POMFIL1) is predominantly expressed in the nervous system and encodes different protein isoforms

We have isolated and characterized a novel differentially spliced gene predominantly expressed in the nervous system, which encodes protein isoforms with significant homology to the alpha-actinin protein superfamily, the Caenorhabditis elegans UNC-53 protein and weak homology to the nuclear membrane protein POM121. Similar to POM121 the primary structures show a hydrophobic region that is likely to form one or more adjacent transmembrane segment(s). Indirect immunofluorescence with antibodies against a synthetic peptide gave staining of the nucleus. Target experiments with EGFP (enhanced green fluorescent protein)-fusion proteins confirmed the nuclear localization. Two further members of this gene family could be isolated. All three pore membrane and/or filament interacting like (POMFIL) genes are differentially expressed in neuronal tumor cell lines. In 40% of tested primary neuroblastomas expression of POMFIL1 is strongly reduced and after brain injury POMFIL1 protein expression is upregulated, indicating that POMFIL1 is involved in the process of neuron growth and regeneration, as well as in neural tumorigenesis.     

Genomewide comparison of DNA sequences between humans and chimpanzees

A total of 8,859 DNA sequences encompassing ~1.9 million base pairs of the chimpanzee genome were sequenced and compared to corresponding human DNA sequences. Although the average sequence difference is low (1.24%), the extent of changes is markedly different among sites and types of substitutions. Whereas ~15% of all CpG sites have experienced changes between humans and chimpanzees, owing to a 23-fold excess of transitions and a 7-fold excess of transversions, substitutions at other sites vary in frequency, between 0.1% and 0.5%. If the nucleotide diversity in the common ancestral species of humans and chimpanzees is assumed to have been about fourfold higher than in contemporary humans, all possible comparisons between autosomes and X and Y chromosomes result in estimates of the ratio between male and female mutation rates of ~3. Thus, the relative time spent in the male and female germlines may be a major determinant of the overall accumulation of nucleotide substitutions. However, since the extent of divergence differs significantly among autosomes, additional unknown factors must also influence the accumulation of substitutions in the human genome.     

A comparison of N-methyl-N-nitrosourea-induced chromatid aberrations and micronuclei in barley meristems using FISH techniques

Individuals can be exposed to high doses (more than 5Gy) during radiation accidents. It is, of course, helpful to the physician to have biological indicators also for such high doses. The problem with most cytogenetic indicators is, that the response levels off at doses starting around 5-7Gy of low LET radiation and that the dose-response curve even declines after doses exceeding about 10Gy. Thus, it may be difficult to decide, whether the dose was, for example, 8 or 14Gy. We studied how the micronucleus assay can be used to give information also in the high dose range. It turned out that micronucleus frequency itself cannot be used for the estimation of doses exceeding about 5-7Gy. There are, however, at least three other endpoints that can be determined in the cytochalasin B assay that can assist the decision in the high dose range: (1) the number of mononucleated cells; (2) the ratio of tri- to tetranucleated cells; (3) the average micronucleus frequency in micronucleus positive binucleated cells.     

Myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class I and Fas-CD95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins

Down-modulation of major histocompatibility class I (MHC-I) molecules is a viral strategy for survival in the host. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, can down-modulate the expression of MHC-I molecules, but the viral factor(s) has not been described. We cloned and characterized a gene coding for an endoplasmic reticulum (ER)-resident protein containing an atypical zinc finger and two transmembrane domains, which we called myxoma virus leukemia-associated protein (MV-LAP). MV-LAP down-regulated surface MHC-I and Fas-CD95 molecules upon transfection; the mechanism probably involves an exacerbation of endocytosis and was lost when the ER retention signal was removed. In addition, the lytic activity of MHC-I-restricted antigen-specific cytolytic T lymphocytes (CTL) against myxoma virus-infected antigen-presenting target cells was significantly reduced, revealing a strong correlation between MHC-I down-regulation by MV-LAP and CTL killing in vitro. In vivo experiments with a knockout virus showed that MV-LAP is a virulence factor, potentially involved in the immunosuppression characteristic of myxomatosis. Data bank analysis revealed that MV-LAP has homologs in herpesviruses and other poxviruses. We propose the name "scrapins" to define a new group of ER-resident surface cellular receptor abductor proteins. The down-regulation of cell surface molecules by scrapins probably helps protect infected cells during viral infections.     

Modeling of mixing in 96-well microplates observed with fluorescence indicators

Mixing in 96-well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double-disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 microL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition.     

Phosphorylated inositol compounds in beta -cell stimulus-response coupling

Pancreatic beta-cell function is essential for the regulation of glucose homeostasis in humans, and its impairment leads to the development of type 2 diabetes. Inputs from glucose and cell surface receptors act together to initiate the beta-cell stimulus-response coupling that ultimately leads to the release of insulin. Phosphorylated inositol compounds have recently emerged as key players at all levels of the stimulus-secretion coupling process. In this current review, we seek to highlight recent advances in beta-cell phosphoinositide research by dividing our examination into two sections. The first involves the events that lead to insulin secretion. This includes both new roles for inositol polyphosphates, particularly inositol hexakisphosphate, and both conventional and 3-phosphorylated inositol lipids. In the second section, we deal with the more novel concept of the autocrine role of insulin. Here, released insulin initiates signal transduction cascades, principally through the activity of phosphatidylinositol 3-kinase. This new round of signal transduction has been established to activate key beta-cell genes, particularly the insulin gene itself. More controversially, this insulin feedback has also been suggested to either terminate or enhance insulin secretion events.     

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.