Cell Type Specific Signalling by Hematopoietic Growth Factors in Neural Cells

Correct timing and spatial location of growth factor expression is critical for undisturbed brain development and functioning. In terminally differentiated cells distinct biological responses to growth factors may depend on cell type specific activation of signalling cascades. We show that the hematopoietic growth factors thrombopoietin (TPO) and granulocyte colony-stimulating factor (GCSF) exert cell type specific effects on survival, proliferation and the degree of phosphorylation of Akt1, ERK1/2 and STAT3 in rat hippocampal neurons and cortical astrocytes. In neurons, TPO induced cell death and selectively activated ERK1/2. GCSF protected neurons from TPO- and hypoxia-induced cell death via selective activation of Akt1. In astrocytes, neither TPO nor GCSF had any effect on cell viability but inhibited proliferation. This effect was accompanied by activation of ERK1/2 and inhibition of STAT3 activity. A balance between growth factors, their receptors and signalling proteins may play an important role in regulation of neural cell survival.     

Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies

pH is one of the key parameters that affect the stability and function of proteins. We have studied the effect of pH on the pyridoxal-5'-phosphate-dependent enzyme phosphoserine aminotransferase produced by the facultative alkaliphile Bacillus circulans ssp. alkalophilus using thermodynamic and crystallographic analysis. Enzymatic activity assay showed that the enzyme has maximum activity at pH 9.0 and relative activity less than 10% at pH 7.0. Differential scanning calorimetry and circular dichroism experiments revealed variations in the stability and denaturation profiles of the enzyme at different pHs. Most importantly, release of pyridoxal-5'-phosphate and protein thermal denaturation were found to occur simultaneously at pH 6.0 in contrast to pH 8.5 where denaturation preceded cofactor's release by approximately 3 degrees C. To correlate the observed differences in thermal denaturation with structural features, the crystal structure of phosphoserine aminotransferase was determined at 1.2 and 1.5 A resolution at two different pHs (8.5 and 4.6, respectively). Analysis of the two structures revealed changes in the vicinity of the active site and in surface residues. A conformational change in a loop involved in substrate binding at the entrance of the active site has been identified upon pH change. Moreover, the number of intramolecular ion pairs was found reduced in the pH 4.6 structure. Taken together, the presented kinetics, thermal denaturation, and crystallographic data demonstrate a potential role of the active site in unfolding and suggest that subtle but structurally significant conformational rearrangements are involved in the stability and integrity of phosphoserine aminotransferase in response to pH changes.     

Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr-DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH-chromate reactions were unable to cause SSB unless activated by H2O2. SSB repair-deficient XRCC1(-/-) and proficient XRCC1+ EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1(-/-) cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1(-/-) and XRCC1+ cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H2O2, and is suppressed by high GSH concentrations.     

A GCUA tetranucleotide loop found in the poliovirus oriL by in vivo SELEX (un)expectedly forms a YNMG-like structure: Extending the YNMG family with GYYA

The cloverleaf structure in the 5'-untranslated region of enterovirus RNA that regulates viral RNA replication contains an evolutionarily conserved YNMG tetraloop closed by a Y-G base pair. This loop is believed to interact specifically with the viral protease 3C. To further characterize the specificity of this interaction, the tetraloop and two flanking base pairs of the poliovirus RNA were randomized, and viable viral clones were obtained using in vivo SELEX. Among many different mutants with the canonical YNMG sequences to be described elsewhere, a large-plaque-forming clone contained a deviating uGCUAg sequence. The NMR structure of a small hairpin capped with uGCUAg that we present here shows that the GCUA tetraloop adopts a novel fold, which is highly similar to that of the YNMG tetraloop with common stacking properties and hydrogen-bond interactions including an unusual syn conformation of the adenosine. Thermodynamic studies show moderate stabilities of hairpins with canonical YNMG and the novel GCUA loops, which, together with the similarity of spatial structures, illustrates that the tetraloop structure itself is crucial for the RNA-protein interaction required for the viral replication. A re-evaluation of the ribosomal secondary structure database reveals a hairpin containing a GCUA loop, which covaries with YNMG and is involved in a tertiary interaction, and in the 50S ribosomal subunit from Haloarcula marismortui the structurally comparable apex of stem-loop 35a is a recognition site for protein L2. These observations show a more general occurrence and importance of the so-far unrecognized GYYA hairpin loops.     

Childhood thyroid cancer, radiation dose from Chernobyl, and dose uncertainties in Bryansk Oblast, Russia: a population-based case-control study

A population-based case-control study was conducted to estimate the radiation-related risk of thyroid cancer in persons who were exposed in childhood to (131)I from the Chernobyl accident of April 26, 1986 and to investigate the impact of uncertainties in individual dose estimates. Included were all 66 confirmed cases of primary thyroid cancer diagnosed from April 26, 1986 through September 1998 in residents of Bryansk Oblast, Russia, who were 0-19 years old at the time of the accident, along with two individually matched controls for each case. Thyroid radiation doses, estimated using a semi-empirical model based on environmental contamination data and individual characteristics, ranged from 0.00014 Gy to 2.73 Gy and had large uncertainties (median geometric standard deviation 2.2). The estimated excess relative risk (ERR) associated with radiation exposure, 48.7/Gy, was significantly greater than 0 (P = 0.00013) but had an extremely wide 95% confidence interval (4.8 to 1151/Gy). Adjusting for dose uncertainty nearly tripled the ERR to 138/Gy, although this was likely an overestimate due to limitations in the modeling of dose uncertainties. The radiation-related excess risk observed in this study is quite large, especially if the uncertainty of dose estimation is taken into account, but is not inconsistent with estimates previously reported for risk after (131)I exposure or acute irradiation from external sources.     

Synthesis and luminescent properties of lanthanide homoleptic mercaptothi(ox)azolate complexes: Molecular structure of Ln(mbt)3 (Ln = Eu, Er)

The thiolate complexes of rare earth metals Ln(SR)₃ (La, HSR = 2-mercaptothiazoline (1); La, HSR = 2-mercaptobenzoxazole (2); Y, La, Sm, Eu, Tb, Gd, Er, Tm, HSR = 2-mercaptobenzothiazole (3)) were synthesized in 84-97% yield by the reactions of silylamides Ln[N(SiMe₃)₂]₃ with respective thiols. The products were characterized by elemental analysis, IR and UV/Vis spectroscopy. The structures of 3(Eu) and 3(Er) were determined by single-crystal X-ray diffraction. All obtained compounds revealed efficient luminescence in the region 400-550 nm at 293 K assigned to the ligands emission. Besides, the luminescent spectra of thiolates 3 at 77 K displayed the phosphorescent band of the ligand at 550 nm and in the cases of 3(Eu) and 3(Tb) the sets of emissions bands characteristic for Eu³⁺ and Tb³⁺ ions.     

Electrostatic properties of promoter recognized by E. coli RNA polymerase Esigma70

A comparative analysis of electrostatic patterns for 359 sigma70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase alpha-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.