Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230.

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids. The C-terminal part of ca. 260 amino acids represents the catalytic domain which is highly conserved in all recently cloned PtdIns 4-kinases. N-terminal motifs indicate multiple heterologous protein interactions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a specific activity of 58 micromol mg-1min-1. The enzyme expressed in Sf9 cells is essentially not inhibited by adenosine, it shows a high Km for ATP of about 300 microM and it is half-maximally inactivated by approximately 200 nM wortmannin. These data classify this enzyme as type 3 PtdIns 4-kinase. Antibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activity. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI4K230, in several human tissues, including brain, suggests that these enzymes are involved in distinct basic cellular functions.

The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine.

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parallel to their inhibitory potency in activity measurements. The specific binding of FSBA to PI4K230 was demonstrated also by Western-blot experiments. These results suggest that FSBA-reactive group(s) involved in the enzyme activity are located near to the ATP-binding site in a hydrophobic region of native PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role in the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-directed and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA suggest that these sites are located spatially close to each other.

[Telomerase activity and telomere length of human mesenchymal stem cells. Changes during osteogenic differentiation]. / Telomeraseaktivität und Telomerlänge humaner mesenchymaler Stammzellen. Veränderungen während der osteogenen Differenzierung.

Human mesenchymal stem cells (hMSC) exhibit properties of self-renewal and differentiation. Assuming that telomerase activity is associated with self-renewal, it might be useful to identify and define hMSC on the basis of their telomerase status. However, telomerase activity in hMSC remains a controversial issue. Therefore, the aim of our study was to investigate telomerase activity in proliferating and highly proliferating hMSC and to measure telomerase activity and changes in telomere restriction fragment (TRF) length of confluent hMSC and of osteogenically differentiated hMSC. For tissue engineering applications scaffolds should be seeded with cells that have not lost their ability to self-replicate and differentiate during in vitro cell culture. Telomerase activity could be used to characterise and isolate these cells.

[Tissue engineering of bone. Integration and migration of human mesenchymal stem cells in colonized contructs in a murine model]. / Tissue Engineering von Knochen. Integration und Migration von humanen mesenchymalen Stammzellen in besiedelten Konstrukten im Mausmodell.

Tissue engineering opens up new ways for therapy of bone defects. Therefore, the aim of this study was to establish a mouse model to investigate local cell growth of human mesenchymal stem cells (hMSC) on the scaffold in vivo. Moreover, migration of cells to other organs should be excluded.hMSC (Cambrex, USA) were cultivated according to supplier's recommendations. After inoculation on cylindric scaffolds, one matrix cell construct and one scaffold without hMSC were implanted subcutaneously left and right paravertebrally in athymic nude mice. After 2, 4, 8, and 12 weeks constructs and organs were harvested for immunohistological evaluation and PCR. In conclusion, we found integration of scaffolds loaded with hMSC implanted ectopically. HMSC seeded on 3D scaffolds survived for a period of up to 12 weeks. In addition, we could not detect hMSC in any other organ of the host.

Immunohistochemical localisation of two phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, in the central nervous system of rats.

The distribution and cellular localisation of the phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, that are believed to play important roles in the intracellular signalling mechanisms were studied in the rat brain (cortex, cerebellum, hippocampus and spinal cord) using immunocytochemistry with light and electron microscopy. PI4K230 was detected with a specific antibody purified by affinity chromatography from the egg yolk of chicken immunised with a 33-kDa fragment of bovine PI4K230, comprising amino acids 873-1175 of the native protein. PI4K92 was immunostained with a commercially available antibody raised in rabbit against amino acid residues 410-537 of human PI4K92. At the light microscopic level, the immunostaining of PI4K230 and PI4K92 showed a very similar distribution throughout the neurons and appeared as dense punctate labelling in the cytoplasm of perikarya and stem dendrites of various neurons. In addition to neurons, a strongly stained cell population was observed in the molecular layer of the cerebellar cortex that resembled Bergmann glia cells. Electron microscopy of neurons in the ventral horn of the spinal cord showed dense granular immunoprecipitates for both PI4K230 and PI4K92, mostly associated with the outer membrane of mitochondria and membranes of the rough endoplasmic reticulum. In addition, immunostaining of PI4K92 was also frequently found on the outer surface of cisterns and vesicles of Golgi complexes, whereas PI4K230 immunoreactivity was colocalised with some multivesicular bodies. Neither nuclear localisation nor a regular attachment to the cell membrane of these enzymes were observed. Our findings indicate that PI4K230 and PI4K92 are not involved directly in the ligand-stimulated turnover of phosphoinositides at the plasma membrane of neurons. However, they may provide regulatory phosphoinositides for intracellular vesicular traffic being associated with various organelles.