The abundance of alpha-tubulin mRNA increases during ciliary regeneration in Tetrahymena, and this occurs independently of the soluble tubulin content

Control mechanisms of tubulin synthesis are analyzed during ciliary regeneration of the ciliate Tetrahymena. Titration of the alpha-tubulin mRNA concentrations during the regeneration period reveal that enhancement of tubulin synthesis is preceded and accompanied by increased concentrations of tubulin mRNA molecules. Stimulation of tubulin synthesis is independent of the pool size of soluble tubulin molecules, as suggested by at least two independent lines of evidence: First, like cells of normal phenotype a temperature sensitive size mutant enhances tubulin synthesis as well as tubulin mRNA concentration during ciliary regeneration, although these large mutant cells have a much higher concentration and amount of soluble tubulin molecules in the cytoplasm. Second, slowly regenerating cells of normal phenotype shift-up their concentration of tubulin mRNA molecules already before a time, when ciliary outgrowth might cause a significant depletion of the pool of soluble tubulin molecules. Thus, neither an induction of tubulin synthesis nor an increase in tubulin mRNA molecules is mediated via changes in the pool size of soluble tubulin molecules.     

Intraoperative isolation and processing of BM-derived stem cells

To improve tissue regeneration of ischemic myocardium, autologous bone marrow-derived stem cells have been injected intramyocardially in five patients undergoing coronary artery bypass grafting and transmyocardial laser revascularization. An innovative method for the intraoperative isolation of CD133(+)-stem cells in less than 3 hours has been established. After induction of general anesthesia, approx. 60-240 ml of bone marrow were harvested from the posterior iliac crest and processed in the operating room under GMP conditions using the automated cell selection device Clini-MACS. Following standard CABG surgery, LASER channels were shot in predefined areas within the hibernating myocardium. Subsequently, autologous CD133(+)-stem cells (1.9-9.7 x 10(6) cells; purity up to 97%) were injected in a predefined pattern around the laser channels. Through the intraoperative isolation of CD133(+)-cells, this effective treatment of ischemic myocardium can be applied to patients scheduled both for elective and for emergency revascularisation procedures.     

Vector space classification of DNA sequences

Revisiting the problem of intron-exon identification, we use a principal component analysis (PCA) to classify DNA sequences and present first results that validate our approach. Sequences are translated into document vectors that represent their word content; a principal component analysis then defines Gaussian-distributed sequence classes. The classification uses word content and variation of word usage to distinguish sequences. We test our approach with several data sets of genomic DNA and are able to classify introns and exons with an accuracy of up to 96%. We compare the method with the best traditional coding measure, the non-overlapping hexamer frequency count, and find that the PCA method produces better results. We also investigate the degree of cross-validation between different data sets of introns and exons and find evidence that the quality of a data set can be detected.     

Use of CpG island microarrays to identify colorectal tumors with a high degree of concurrent methylation

Oligonucleotide-targeted RNase H protection assays are powerful means to analyze protein binding domains in ribonucleoprotein particles (RNPs). In such an assay, the RNA component of a RNP and, in an essential control reaction, the corresponding deproteinized RNA are targeted with an antisense DNA oligonucleotide and RNase H. If the oligonucleotide is able to anneal to the complementary sequence of the RNA, RNase H will cleave the RNA within the double-stranded DNA/RNA region. However, protein binding to a specific RNA sequence may prevent hybridization of the DNA oligonucleotide, thereby protecting the RNA molecule from endonucleolytic cleavage. An RNase H protection analysis can usually be carried out with crude cell extract and does not require further RNP purification. On the other hand, purified RNP fractions are preferable when a crude extract contains RNase activity or a heterogenous RNP population of a specific RNA. The cleavage pattern of RNase H digestion can be analyzed by Northern blotting or primer-extension assays. In addition, the investigation of RNP fragments, for example, by native gel electrophoresis, may reveal important structural information about a RNP. In this article, we describe procedures for RNP and RNA preparation, the oligonucleotide-targeted RNase H protection assay, and methods for the analysis of RNA and RNP cleavage products. As an example, we show oligonucleotide-targeted RNase H protection of the Trypanosoma brucei U1 small nuclear RNP.     

Notch1 enhances B-cell receptor-induced apoptosis in mature activated B cells without affecting cell cycle progression and surface IgM expression

The transmembrane receptor Notch1 plays a crucial role in differentiation and apoptosis of hematopoietic cells. To investigate the influence of Notch1 on apoptosis and cell growth of mature murine B cells, we transduced the murine B-lymphoma line NYC 31.1 with a constitutively active, intracellular form of human Notch1 (Notch1-ICT). NYC cells represent mature activated B cells that can be induced to undergo apoptosis by crosslinking of the B-cell receptor (BCR). In contrast to investigations in immature chicken B-cell lines, transduced Notch1-ICT did not affect cell cycle progression, cell growth or surface IgM levels in NYC cells and resulted only in a slight induction of apoptosis. However, BCR-crosslinking enhanced apoptosis, but did not influence cell cycle progression in Notch1-ICT-positive NYC cells. These data imply a distinct function of Notch1 in mature murine B-cells as compared to immature chicken B cells and provide further evidence for Notch1's involvement in B-cell differentiation and development.     

The Ca2+-binding protein calretinin is selectively enriched in a subpopulation of the epithelial rests of Malassez

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca²⁺-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca²⁺-binding proteins in the ERM has not so far been characterized. Among the three Ca²⁺-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca²⁺ is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca²⁺ is regulated only by calretinin. The expression of Ca²⁺-binding proteins is restricted in a developmental manner in the ERM.     

After shrinkage apoptotic cells expose internal membrane-derived epitopes on their plasma membranes

Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.