Volume 385, Issue 1

This review addresses many of the chemical aspects of nitrosative stress mediated by N(2)O(3). From a cellular perspective, N(2)O(3) and the resulting reactive nitrogen oxide species target specific motifs such as thiols, lysine active sites, and zinc fingers and is dependant upon both the rates of production as well as consumption of NO and must be taken into account in order to access the nitrosative environment. Since production and consumption are integral parts of N(2)O(3) generation, we predict that nitrosative stress occurs under specific conditions, such as chronic inflammation. In contrast to conditions of stress, nitrosative chemistry may also provide cellular protection through the regulation of critical signaling pathways. Therefore, a careful evaluation of the chemistry of nitrosation based upon specific experimental conditions may provide a better understanding of how the subtle balance between oxidative and nitrosative stress may be involved in the etiology and control of various disease processes.

Like many juggernauts in biology, the elusive nature of nitric oxide (NO) sprints through the fields, sometimes the savior, at other times the scimitar. In the liver, which is the metabolic center of the organism, hepatocytes and immune cells trade blows using the reactive diatomic molecule NO to induce cellular damage under toxic conditions. In response, hepatocytes can utilize several mechanisms of NO to their protective advantage by prohibiting the activation of programmed cell death, a.k.a. apoptosis. The balance of these effects in this reactive milieu set the stage for the homeostatic response to cellular injury that determines whether hepatocytes will live, die, or regenerate. Insights that we and others have gained from the liver under pathologic conditions of stress can be applied to the understanding of cellular death mechanisms in other organs and tissues.

The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a watersoluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkalilabile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acidbinding residue within the ligand-binding pocket, I323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.

Thermus thermophilus L11 protein has previously been reported to be resistant against tryptic and chymotryptic proteolysis under native conditions. With a single amino acid substitution, namely Trp101Arg, conformational changes were induced that resulted in the exhibition of specific amino acids that served as targets for tryptic and chymotryptic action and rendered the protein highly unstable even during purification. This unexpected process was evidenced by the isolation with size exclusion gel chromatography of the wellstructured chymotryptic N-terminal domain in a high amount and its characterization both by Edman degradation and QTOF-EMS spectroscopy. On the other hand, the substitution of Val38Cys, which did not contribute to structural changes, indicates a very possible implication of this amino acid in the protein methylation process. The data reported in this work illustrate the distinctive amino acid dynamics in a thermophilic protein, which, while serving the function common to its counterparts from mesophilic organisms, has had to adapt to the extreme environmental conditions typical of thermophilic organisms.

In higher organisms, the replacement of GDP bound to Ras proteins with GTP, under the participation of an exchange factor, is an important step in the initiation of cell division. Ras-GTP activates kinases and other effectors, which pass signals to the cell nucleus and to the cytoskeleton. The active state of Ras is terminated by hydrolysis of the bound GTP with the assistance of an activating protein (GAP). Knowledge of these regulatory events is based on extensive experimental data, but many aspects of their interpretation are still controversial. It is assumed here that a significant part of the free energy released when two partners associate is stored in a kinetic equilibrium of forces (KEF), and used to facilitate the separation from a third partner. The activation of the Raf kinase is explained primarily in terms of an allosteric effect of Ras-GTP on the phosphate transfer in the catalytic region of the kinase. A mechanism is proposed for the modification of GAP by Ras-GTP, which is believed to be a prerequisite for the wellknown crosstalk between the Ras- and Rho-dependent signalling pathways. The cell, by meeting the requirements for KEF, manages to reduce activation barriers, thus significantly accelerating the regulatory events and other complex biological reaction sequences.

To investigate the glycosylation of the human bradykinin B2 receptor and the functional significance of this modification, we studied receptors mutated at single or multiple combinations of the three potential N-linked glycosylation sites, asparagines N3, N12 and N180, in COS-7, HEK 293 and CHOK1 cells. Western blot experiments demonstrated that all three extracellular asparagines are glycosylated. The kinetics of bradykinin binding and receptor sequestration remained unchanged after glycosylation had been suppressed. However, the glycosylated receptors were expressed at the cellsurface to a much greater extent than the nonglycosylated receptor and coupling to phospholipase C was less efficient for receptor lacking N-terminal glycosylation. These results indicate that, for the human bradykinin B2 receptor, glycosylation is not required for optimal ligand binding, but plays an important role in cell-surface addressing and receptor function.

The presence of Neu5Ac on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, has been reported recently. Here we report the occurrence of Neu5Ac as a major component on amastigotes, as well as Neu5Gc, Neu5,9Ac2 and Neu9Ac5Gc as indicated by fluorimetric high performance liquid chromatography and gas liquid chromatography/electron impact mass spectrometry. Furthermore, binding studies with Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and various Siglecs, showed the presence of both (alpha2-6) and (alpha2-3)linked sialic acids; their binding was reduced after sialidase pretreatment. Western blotting of amastigote membrane glycoproteins with SNA demonstrated the presence of two sialoglycoconjugates of Mr values of 164 000 and 150 000. Similarly, binding of MAA demonstrated the presence of five distinct sialoglycans corresponding to molecular masses of 188, 162, 136, 137 and 124 kDa. Achatinin-H, a lectin that preferentially identifies 9-Oacetylated sialic acid (alpha2-6)linked to GalNAc, demonstrated the occurrence of two 9-Oacetylated sialoglycans with Mr 158 000 and 150 000, and was corroborated by flow cytometry; this binding was abolished by recombinant 9-Oacetylesterase pretreatment. Our results indicate that Neu5Ac w(alpha2-6) and (alpha2-3)linked, as well as Neu5Gc and their 9-Oacetyl derivatives, constitute components of the amastigote cell surface of L. donovani.

Unregulated uptake of low density lipoprotein (LDL) in macrophages is the hallmark of early atherogenic lesions, and Chlamydia pneumoniae infection of macrophages induces this process by an unknown mechanism. It was therefore aimed in this study to investigate (i) the role of C. pneumoniae in macrophage expression of the lipoprotein lipase (LpL) gene, (ii) the probable role of Ca[2+] influx signals and (iii) the effect of the process on LDL uptake. Lipoprotein lipase mRNA expression and LpL activity in infected RAW-264.7 cells were significantly upregulated. A biphasic Ca[2+] influx signal was observed in infected cells with a moderate influx (303 nM Ca[2+]) favoring optimal LpL gene expression. Also, the antagonists of Ltype Ca[2+] channel in macrophages significantly downregulated LpL gene expression and the biomolecular content of C. pneumoniae responsible for the observed events was in part found to be Chlamydia lipopolysaccharide (cLPS). Investigations aimed at determining the specific relevance of Ca[2+]dependent lipoprotein lipase gene expression in C. pneumoniae -infected macrophages showed that the condition caused enhanced uptake of LDL which was abrogated by Calphostin-C-mediated downregulation of LpL. This discovery of a specialized Ca[2+] influx signal-mediated LpL upregulation in C. pneumoniae -infected macrophages provides a mechanistic insight into early events involving C. pneumoniae in macrophage foam cell formation resulting from LDL uptake.

Ultraviolet radiation may cause non-melanoma skin cancer by genetic and epigenetic events. In this study, we investigated in a squamous cell carcinoma cell line, SCL-1, whether UV irradiation modulates the expression of matrix metalloproteinases, known to be involved in tumor progression and metastasis by degradation of extracellular matrix components. UVA or UVB irradiation of SCL-1 resulted in a rapid transcriptional upregulation and increased secretion of two members of the matrix metalloproteinase family, MMP-10 (stromelysin-2) and MMP-1 (interstitial collagenase). The increase in MMP-10 steadystate mRNA levels was detected 1 hour after UVA and 4 h after UVB irradiation, whereas MMP-1 was upregulated 4 h after UVA and 16 h after UVB irradiation of tumor cells. UVinduced phosphorylation of extracellular regulated kinases (ERK-1/2) and p38 stress kinase and increased binding of AP-1 transcription factor preceded the rapid stimulation of MMPs in SCL-1 cells. Incubation of cells with the MEK1/2 inhibitor U0126 or the p38 inhibitor SB202190 abolished the UVA and UVB mediated induction of MMP-1 and MMP-10. In conclusion, this study shows that UV irradiation of squamous cell carcinoma results in a rapid upregulation of MMPs. Our results suggest that the time course of induction of target genes, like MMPs, differs between cell types depending on the stimulus.

Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and pastimmunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7x10exp-7 cm(2)s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5x10exp-8 cm(2)s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a pastimmunity serum sample gave an average diffusion coefficient of 3.5x10exp-8 cm(2)s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7x10exp-7 to 1.6x10exp-7 cm(2)s(-1), which is much smaller than that observed with acute phase or pastimmunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and pastimmunity sera. FCS measurement of VLPimmune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.

The transcription factor p53 acts as major tumor suppressor and is inactivated by mutation in more than 50% of all human tumors. We have established an efficient procedure for the in vitro folding and purification of the p53 DNA binding domain (p53DBD) using a modified factorial matrix approach that supplies large amounts of homogeneous (isotope-labeled) p53DBD for application in biochemical, crystallographic and NMR spectroscopic studies. We further show with biophysical methods that in vitro folded p53DBD is fully functional and that its conformation is identical to that obtained from the soluble fraction.