A new method for radiochemical separation of arsenic from irradiated germanium oxide.

Radioarsenic labelled radiopharmaceuticals could be a valuable asset to Positron Emission Tomography (PET). In particular, the long half-lives of (72)As (T(1/2)=26 h) and (74)As (T(1/2)=17.8 d) allow to investigate slow physiological or metabolical processes, like the enrichment and distribution of antibodies in tumor tissue. This work describes the direct production of no-carrier-added (nca) arsenic isotopes *As, with *=71, 72, 73, 74 or 77, the reaction to [*As]AsI(3) and its radiochemical separation from the irradiated solid germanium oxide via polystyrene-based solid-phase extraction. The germanium oxide target, irradiated at a cyclotron or a nuclear reactor, is dissolved in concentrated HF and Ge is separated almost quantitatively (99.97%) as [GeF(6)](2-). [*As]AsI(3) is formed by addition of potassium iodide. The radiochemical separation yield for arsenic is >90%. [*As]AsI(3) is a versatile radioarsenic labelling synthon.

Polyploidization and centrosome hyperamplification in inflammatory bronchi.

OBJECTIVE AND DESIGN: Inflammatory and tumorous bronchi were screened in order to obtain new tumor relevant cytogenetic parameters. MATERIAL OR SUBJECTS: Bronchial cells of 32 patients were cultivated by standard cell culture procedures. METHODS: Tetraploidy and aneuploidy was determined by enumeration of chromosome 7 and 8 versus the number of centrosomes. The resulting data were correlated with histopathological data. RESULTS: Tetra- and aneuploidy of epithelial cells were detectable in 76% of tumor cell cultures, 75% of high grade inflammatory tissues and 40% of non- and low grade-inflammatory tissues. Additionally, we observed centrosome hyper-amplification and multipolar mitoses not only in the tumor but also in the early stages of inflammation. CONCLUSION: Inflammatory bronchi already show tumor-specific features and may consequently represent the preliminary genetic stage of cancer development in bronchi.

Centrosome multiplication accompanies a transient clustering of polyploid cells during tissue repair.

Cells from different human wounds were analyzed concerning their degree of ploidy. The experiments showed an increased tetraploidization rate in well-healing wounds especially during inflammation and proliferation. Recent data described a polyploidization in different tissues, which is accompanied and maybe caused by the multiplication of the centrosome. We show here for the first time that cells from nonmalignant tissue, namely human wound cells, are characterized by an extensive centrosome multiplication. In an effort to identify a certain mechanism, by which the centrosome may act as a modulator of the cells' ploidy, we focused our interest on p53, whose interaction with the centrosome was recently described. Applying a wound model onto p53-wildtype (wt) and p53-knockout (ko) mice, we could show that polyploidization was not reversible in p53-ko mice during wound healing. The lack of p53, the centrosome multiplication, and the polyploidization therefore may contribute to the physiological process of tissue repair in physiologically "normal" tissue.

Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic effects of proton and phenylalanine on the regulation of muscle pyruvate kinase.

Steady-state kinetic studies of muscle pyruvate kinase were conducted as a function of pH and phenylalanine concentrations. Results show that at a pH below 7.0, there is no observable effect of phenylalanine on the kinetic properties of muscle pyruvate kinase. When the results at a pH below 6.5 are used as the state for comparison, the kinetic results show that phenylalanine and proton exert a synergistic effect on the allosteric properties of the enzyme. A significantly greater change in Hill coefficients at high pH can be detected in the presence of phenylalanine than in its absence. To pinpoint the specific mechanism that leads to the synergistic effect, the kinetic data were resolved into the five equilibrium and two rate constants that characterize the basic two-state model. It can be shown that KTI, the binding constant of phenylalanine to the inactive T state, is strongly proton-linked. The affinity of phenylalanine for the T state increases with increasing pH. When the pH dependence of KTI was analyzed by the linked-function theory [Wyman, J. (1964) Adv. Protein Chem. 19, 224-285], it was shown that deprotonation favors phenylalanine binding to the T state. KRI (the binding constant of phenylalanine to the active R state), KTS (the binding constant of substrate to the T state), and L (the isomerization constant of the two states) not only are all weakly proton-linked but also it was shown that protonation favors the ligand-pyruvate kinase complex. KRS, the binding constant of substrate for the R state, shows no observable linkage to proton concentration.(ABSTRACT TRUNCATED AT 250 WORDS)