Reinhold Hofbauer

Selective downregulation of VEGF-A(165), VEGF-R(1), and decreased capillary density in patients with dilative but not ischemic cardiomyopathy.

Cardiomyopathy (CM) comprises a heterogeneous group of diseases, including ischemic (ICM) and dilative (DCM) forms. The pathogenesis of primary DCM is not clearly understood. Recent studies in mice show that vascular endothelial growth factor (VEGF) is involved in ICM. Whether VEGF plays a role in human CM is unknown. We examined the mRNA and protein expression of VEGF and its receptors in hearts of patients with end-stage DCM and ICM and in healthy individuals using real-time polymerase chain reaction and Western blotting. Number of capillaries, area of myocytes, and collagen were calculated in cardiac biopsies using transmission electron microscopy. In DCM, except for VEGF-C, mRNA transcript levels of VEGF-A165, VEGF-A189, and VEGF-B and the protein level of VEGF-A and VEGF-R1 were downregulated compared with controls (P <0.05). However, in ICM, mRNA transcript levels of VEGF isoforms and protein levels of VEGF-C were upregulated. The vascular density was decreased in DCM but increased in ICM compared with controls (P <0.05). Muscular hypertrophy was not different for ICM and DCM, although DCM had more collagen (P <0.05). Blunted VEGF-A and VEGF-R1 protein expression and downregulated mRNA of the predominant isoform of VEGF-A, VEGF-A165, to our knowledge shown here for the first time, provide evidence that the VEGF-A defect in DCM is located upstream. Whether downregulation of certain VEGF isoforms in DCM is a cause or consequence of this disorder remains unclear, although upregulated VEGF levels in ICM are most likely the result of ischemia.

Selective upregulation of vascular endothelial growth factor receptors neuropilin-1 and -2 in human neuroblastoma

Recent studies show that vascular endothelial growth factor (VEGF) and its receptors Flt‐1 and KDR, and a series of other angiogenic molecules, are upregulated in advanced but not low stage human neuroblastoma. Neuropilin‐1 and 2 (NRP) are novel specific receptors of VEGF165, whose role is unknown in human neuroblastoma.

Altered Substrate and Inhibitor Specificity of Purified Human Adult Thymidine Kinases (TK2) from Leukemic Cells

The two thymidine kinases in mammalian cells, TK1 and TK2, are important for the balanced supply of deoxypyrimidine nucleotides for DNA replication and repair1, 2, 3. TK1 is cell cycle regulated and has the most restricted substrate specificity4, 5. TK2 is constitutively expressed and differs substantially from the cell cycle regulated TK1 regarding kinetic reaction mechanisms and substrate specificity6, 7. The inhibition with TTP is cooperative with TK1, but non-cooperative and competitive with TK2. The substrate kinetics with ATP are positive cooperative with TK1, but non-cooperative with TK2. Finally, thymidine is phosphorylated by TK2 with a characteristic, negative cooperative mechanism.

Gene technology-based antimetabolite design: The use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with its biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to its structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzymes biological properties.

Increased levels of dihydrofolate reductase mRNA can be measured in normal, growth-stimulated mouse fibroblasts

Levels of mRNA for the enzyme dihydrofolate reductase (EC 1.5.1.3) were determined in growth-stimulated 3T6 cells which contained wild-type dosage of the gene coding for this enzyme. As in the case of methotrexate-resistant cells having highly amplified levels of genes for dihydrofolate reductase, an increase in dihydrofolate reductase mRNA by a factor of 2-4 can be determined when cells enter the S phase. This increase is inhibited by sodium butyrate (which inhibits growth-stimulated 3T6 cells in mid G1 phase) but not by hydroxyurea (which inhibits in early S phase). We conclude that with the available methods it is possible to study the regulation of S phase-specific enzymes after growth stimulation at the level of the mRNA, even if gene amplification is not possible or desirable.