Cytokine polymorphisms in silicosis and other pneumoconioses

Silicosis and coal workers' pneumoconiosis are complex multifactorial lung diseases whose etiopathogenesis are not well defined. It is generally accepted that fibrotic lung disorders are mediated by macrophage-derived cytokines and growth factors. There is evidence showing a crucial role for tumor necrosis factor-a (TNF-alpha) and interleukin-1 (IL-1) in inflammation caused by silica dust and in the transition from simple to progressive massive fibrosis. In this review we discuss genetic polymorphisms responsible for regulating the production of these proinflammatory cytokines and their role in modifying silicosis severity.     

Oxidative stress in the chronic phase after stroke

The spontaneous and the stimulated extracellular generation of reactive oxygen species (ROS) by peripheral phagocytes, the blood antioxidant capacity and the degree of oxidative damage were evaluated in patients with severe ischemic and hemorrhagic stroke in the chronic phase of disease. It was found in patients compared to the control group that: (i) the spontaneous phagocyte oxidative activity was enhanced independently of the type of stroke and the time elapsed after stroke onset; (ii) there was no difference in the extracellular ROS generation stimulated by opsonin-dependent and independent receptor mechanisms; (iii) there was no change in the indices of blood antioxidant capacity; (iv) the concentration of plasma lipid peroxides was enhanced regardless of the type of stroke, but it significantly increased over time; and (v) the concentration of blood thiobarbituric acid-reactive material was also enhanced. It was independent of the type of stroke and remained elevated during the whole period studied. We have demonstrated an enhanced spontaneous phagocyte oxidative activity and oxidative damage to lipids in patients in the chronic phase after stroke. The elimination of generated ROS and products of lipid peroxidation from the circulation could prevent the aggravation of chronic vascular injury in patients and could reduce the possibility of a subsequent stroke. This suggests the need for complex therapy, including antioxidant treatment directed to exclude the effects of free radicals, after the oxidative stress of stroke.     

Chemical fingerprinting of plants

The chemical fingerprinting of a plant demands the determination of a large number of elements at background level but also in polluted regions and in relation to other species. The most important stages of the development of plant fingerprints are mentioned in this paper and a short survey of the state of the art is presented. Examples on fingerprinting of lichens, mosses, Taraxacum officinale and Populus nigraItalica are presented.     

Subcellular heterogeneity of mitochondrial membrane potential in anther tapetum

Studies on animal material have revealed that changes in the mitochondrial permeability transition pore (PTP), which cause a reduction in the mitochondrial transmembrane potential (_m) followed by release of cytochrome c, belong to the earliest manifestations of some types of apoptosis. We have attempted to monitor the _m of mitochondria during programmed cell death (PCD) of the secretory tapetum using JC-1, a fluorochrome dye that detects mitochondrial membrane potential and to relate changes in this potential to mitochondrial ultrastructure. Analysis of tapetal cells isolated from Ornithogalum virens anthers revealed that the _m of mitochondria in the tapetal cells alters during development; the change, however, is not uniform in the mitochondrial population within a single tapetal cell. In young tapetal cells, at the tetrad stage, we detected only the red fluorescence of JC-1 aggregates in all tapetal mitochondria, which indicates highly negative _m. In an advanced stage of PCD at the late microspore stage, in each tapetal cell we detected both mitochondria with red (as formerly) and mitochondria with green fluorescence. The green fluorescence of JC-1 monomers indicates mitochondria with depolarised membranes. These changes in _m are related to observed changes in mitochondria ultrastructure. This is the first documentation of intracellular heterogeneity of _m during anther tapetum development. Alteration in _m suggests a relationship between mitochondrial function and PCD processes in tapetal cells.     

In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.The Arabidopsis (Arabidopsis thaliana) cell division cycle protein CDC48A was previously shown to interact with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) (Rienties et al., 2005) and to coimmunoprecipitate with SERK1 in Arabidopsis cultured cells and seedlings (Karlova et al., 2006). In living cells the CDC48A protein colocalizes with SERK1 at peripheral endoplasmic reticulum (ER)-based membranes and the plasma membrane (PM). Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) showed that CDC48A interacts with SERK1 at the PM (Aker et al., 2006).CDC48A is a member of the family of AAA ATPases (ATPases associated with various cellular activities), shown to have various functions in cell division, membrane fusions, and in proteasome- and ER-associated degradation (ERAD) of proteins (Woodman, 2003). The role of AAA ATPases is to generate mechanical force to disrupt or fuse molecular structures by means of ATP binding and hydrolysis. The AtCDC48A protein was shown to play a role in ERAD and in membrane fusions (Rancour et al., 2002; Müller et al., 2005). AAA proteins are present as stacked hexameric rings that are stabilized by the binding of ATP. They are only reported to be active in hexameric form. This study aims to determine the predominant form of the fluorescently tagged CDC48A protein in Arabidopsis protoplasts at the peripheral ER and the PM domains where interactions between CDC48A and the SERK1 receptor take place, and, therefore, to determine if SERK1 is associated with the active form of CDC48A.CDC48A is 77% identical to the mammalian homolog vasolin-containing protein (VCP) or p97. Recently, conformational and dynamic changes during the ATP hydrolysis cycle of p97 have been studied by cryo-electron microscopy, crystallography, and small-angle x-ray scattering (for review, see Pye et al., 2006). The monomeric protein contains an N-terminal domain, two AAA domains, and a C-terminal domain (Figs. 1 and ​and3).3). The N domain is important for binding of adaptor proteins and substrates. The two AAA domains D1 and D2 are responsible for binding and hydrolysis of ATP. The D1 and D2 domains form stacked hexameric rings above each other that are connected by a linker. The ATP-binding site in the D1 domain and the D1-D2 linker are responsible for hexamerization, while the ATP-binding site in the D2 domain possesses the major ATPase activity (Wang et al., 2003a). The N domain is flexibly attached to the D1 domain, and projects out of the ring, facilitating binding to other proteins. During ATP hydrolysis, large conformational changes are transmitted from the D2 domain via the D1 domain onto the N domains, due to binding, hydrolysis, and release of the nucleotide (DeLaBarre and Brunger, 2003, 2005). In addition, the pores within the D1 and D2 rings narrow and widen in different nucleotide states. This causes the rings to rotate relative to each other (Davies et al., 2005). The C-terminal tail was not ordered within the crystal structure, and it is therefore unknown whether it stays close to the D2 ring or projects from the core structure toward the N domain.Open in a separate windowFigure 1.Expression in Arabidopsis protoplasts of CDC48A, CDC48A^A1A2, CDC48A^Ndel, and CDC48A^N-D1 mutants all tagged to YFP. YFP is replaced by CrFP. In A the various mutants are shown schematically. Lysates of transfected cells were submitted to SDS-PAGE in B, to native PAGE in C, and probed with anti-YFP anti-serum. The expected sizes of the denatured proteins are 125 kD for full-length (FL) CDC48A- and CDC48A^A1A2-YFP, 96 kD for CDC48A^Ndel-YFP, and 77 kD for CDC48A^N-D1-YFP. Nondenatured hexameric CDC48A-YFP is expected to be between 650 and 750 kD depending on the number of YFP tags.Open in a separate windowFigure 3.Models of the expected hexamers of CDC48A after coexpression of two different protomers. A, Monomeric CDC48A. B, CDC48A fused with CrFP and with YFP (both at the C terminus) are combined and depicted as a hexamer. For simplicity only four fluorophores are drawn at adjacent positions at the C terminus, but in fact these can be randomly positioned at each C domain. C, YFP fused to the N terminus and CrFP to the C terminus. D, YFP and CrFP fused to the N terminus. E and F, CDC48A^N-D1 and CDC48A^Ndel, each depicted as a monomer.For Arabidopsis CDC48A the crystal structure is not known, but in vitro-produced proteins form a hexamer while PUX1 (plant UBX domain-containing protein) facilitates CDC48A oligomer disassembly (Rancour et al., 2004). Up to now hexamerization of AAA ATPases was only shown in vitro or in total cell lysates, studies that do not reveal any spatial information on the oligomerization status of proteins in a living cell. Therefore, the protein was fused to the GFP variant monomeric cerulean fluorescent protein (CrFP) or to yellow fluorescent protein (YFP), and the hexamerization of CDC48A proteins in living cells was monitored by FRET-FLIM. Employing this technique, oligomerization of the CDC48A proteins in living cells was shown to be nonuniform. In addition, intersubunit distances in the oligomeric CDC48A protein could be calculated, revealing that the C-terminal domains stayed in close proximity rather than protruding out of the molecule.It was also shown that SERK1 interacts with the N terminus as well as the C terminus of CDC48A (Aker et al., 2006). After performing a trans-phosphorylation reaction with the SERK1 kinase domain, only Ser-41, a Ser residue found to be in the N domain of CDC48A, was phosphorylated.To investigate the diffusion of the CDC48A protein in cells, fluorescence correlation spectroscopy (FCS), in which fluorescence intensity fluctuations caused by diffusion of fluorescent molecules in and out of a femtoliter volume are monitored in time, was employed. These fluctuations give information about diffusion times of proteins through the volume and, hence, about the size of protein complexes.Our results show that the oligomeric form of CDC48A in living cells is primarily hexameric and that the fluorescently tagged CDC48A is still able to form hexamers. The SERK1 receptor interacts with CDC48A at the same locations where oligomerization of the CDC48A protein is shown. We conclude therefore that SERK1 interacts with the hexameric form of CDC48A. Using FCS the presence of CDC48A in larger protein complexes in vivo was predicted.     

First solid state alkaline-earth complexes of monensic acid A (MonH): crystal structure of [M(Mon)<Subscript>2</Subscript>(H<Subscript>2</Subscript>O)<Subscript>2</Subscript>] (M = Mg,Ca), spectral properties and cytotoxicity against aerobic Gram-positive bacteria

Alkaline-earth metal complexes of the monoanionic form of the polyether ionophore monensin A were isolated for the first time in solid state and were structurally characterized using various spectroscopic methods (IR, NMR, FAB-MS). The stoichiometric reaction of monensic acid (MonH) with M²⁺ (M = Mg, Ca) in the presence of an organic base leads to the formation of mononuclear complexes of composition [M(Mon)₂(H₂O)₂]. The structures of magnesium (1) and calcium (2) monensin complexes in the solid state were established by single crystal X-ray crystallography. The complexes crystallize as [Mg(Mon)₂(H₂O)₂]·5MeCN (1) and [Ca(Mon)₂(H₂O)₂]·H₂O·5MeCN (2) in the monoclinic P21 space group. The alkaline-earth metal ion is placed in a distorted octahedral environment, defined by two monensin anions acting as bidentate ligands in the equatorial plane of the complex as well as by two water molecules occupying the axial positions of the inner coordination sphere. The bactericidal activity of 1 and 2 was evaluated against aerobic Gram-positive microorganisms applying the double layer agar hole diffusion method.     

Arsenic carcinogenicity: Relevance of c-Src activation

Environmental and occupational exposure to arsenic is associated with increased risk of skin, urinary bladder and respiratory tract cancers. Increasing evidence indicates that arsenic acts at the level of tumor promotion by modulating the signaling pathways responsible for cell growth. One of this pathways might include c-Src dependent EGFR and MAPK activation.     

Hepatoprotective effects of saponarin, isolated from Gypsophila trichotoma Wend. on cocaine-induced oxidative stress in rats

The antioxidant effect of saponarin, which is the main flavone isolated from Gypsophila trichotoma Wend., and its protection against cocaine hepatotoxicity were investigated in male Wistar rats. The animals were treated with cocaine (40 mg/kg i.p.) alone and also after 3 consecutive days of pretreatment with saponarin (80 mg/kg p.o.). After 18 hours the rats were sacrificed by decapitation. The production of thiobarbituric acid reactive substances, reduced glutathione (GSH) and the activity of the following antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were assessed in liver homogenate. Administered alone, cocaine induced significant hepatotoxicity manifested with GSH depletion and reduced antioxidant defences. Saponarin pretreatment, however, decreased cocaine toxicity both by increasing GSH levels and antioxidant enzyme activities. The results of this study proved the antioxidant activity of saponarin and its protective effect against cocaine-induced oxidative stress and hepatotoxicity.     

Diminished genotoxicity of mitomycin C and farmorubicin included in polybutylcyanoacrylate nanoparticles.

The genotoxic effects of mitomycin C (MMC) and farmorubicin (FR) in a free form and included in polybutylcyanoacrylate nanoparticles (PBCN) were studied employing the Salmonella/microsome mutagenicity assay and the micronucleus test in mouse bone marrow as well as in mouse fetal liver. The data obtained clearly indicated that MMC (0.25-2.00 micrograms/plate) was a strong mutagen in S. typhimurium TA102, while the same concentrations of this compound in PBCN were ineffective in inducing his+ revertant mutations in bacterial cells. A similar total suppression of mutagenic activity of FR (1.0-20.0 micrograms/plate) was registered in S. typhimurium TA98 when the drug was included in PBCN. Furthermore, the incorporation of MMC (2.0 or 4.0 mg/kg, i.p.) into PBCN strongly diminished or even abolished its clastogenic activity in the bone marrow of virgin and pregnant mice as well as in mouse fetal liver, respectively. In addition, a lack of genotoxic effect of PBCN only was also established. The toxic activity of MMC in mouse bone marrow was significantly reduced or completely abolished after its inclusion in PBCN. A conclusion might be drawn that the genotoxic activity of some antitumor drugs might be markedly diminished or even abolished after their incorporation in PBCN.