Magnetic phagosome motion in J774A.1 macrophages: influence of cytoskeletal drugs

The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome.     

Immunohistochemical distribution of connexin 43 in the cartilage of rats and mice

Using fluorescence immunohistochemistry, the distribution of connexin 43 was examined in hyaline cartilage and in the perichondrium of mouse and rat knee joints. In addition, rat chondrocytes were shown to be coupled in dye transfer studies with Lucifer Yellow. Connexin 43 was detected between chondrocytes in the outer layer of knee joint cartilage, between chondrocytes of the growth plate and between fibrocartilage-like cells at tendon and ligament insertions and in the tendons and ligaments proper. However, in the hyaline cartilage of the hind limbs of mature rats, the degree of connexin 43 immunoreactivity was diminished. These data suggest a possible involvement of connexins in cartilage development. © 1998 Chapman & Hall     

Crystal structure of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from a PCB degrader at 2.0 A resolution.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.     

Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).In eukaryotes, the reversible phosphorylation of serine, threonine, and tyrosine residues within proteins is a wide-spread post-translational modification, essential for controlling a multitude of cellular processes. During the last decade, sequencing projects unexpectedly unraveled that plant genomes encode for a considerable larger number of protein kinases than the other kingdoms of life. Arabidopsis thaliana contains 1112 PKs (4% of all genes), twice the number encoded by the human genome (518 or 2% of all genes) and other plants have an even higher number of kinases (1).Phosphatidyl inositol 3′ kinase related kinases are important players in DNA damage response (DDR)¹ and crucial for genome integrity (2). Key to DNA double strand break (DSB) repair is a chain of events starting with detection of the lesion, activation of a signaling cascade, cell cycle arrest, and recruitment of the repair machinery. The cascade is triggered by the Phosphatidyl inositol 3′ kinase related kinases family kinases ataxia telangiectasia-mutated (ATM) (3) and Ataxia telangiectasia-mutated and Rad3-related (ATR) (4). Both kinases are conserved across eukaryotes. Their downstream targets have been systematically identified in yeast (5) and human cells (6, 7). Their essential role in mediating DNA repair in higher plants has been established (8–10). In Arabidopsis, loss of function mutants are viable (11); however, atm mutants are highly sensitive to genotoxic stress and have a reduced fertility. atr mutant plants have a cell-cycle checkpoint defect upon exposure to genotoxic chemicals (12). Somatic growth under nonchallenging conditions is not affected in the double mutant but plants are sterile, highlighting the role of both kinases coordinating meiotic DNA repair. In plants, systematic phosphoproteomic studies of the involved pathways have not been reported but would contribute to further elucidating the molecular mechanism of the observed phenotypes. Interestingly, plants lack clear homologs for many downstream regulatory components in the signaling cascade (e.g. CHK1, CHK2, p53, and MDC1) (13). In this context, it should be noted that DNA-PKcs (DNA-dependent protein kinase), another Phosphatidyl inositol 3′ kinase related kinases family member involved in DNA repair, has not been identified in plant genomes (14, 15), underscoring the significance of ATM and ATR as master regulators.ATM is recruited to DSBs via its interaction with NBS1/XRS2, a member of the MRN/X complex (MRE11/RAD50/NBS1-XRS2). In plants, the detailed molecular base for ATM recruitment has remained unknown. The complex acts as damage sensor in yeast, first to be detected at DNA double strand break (DSB) sites and essential for resection of DNA (16). In all organisms analyzed, the MRN/X complex is required for genotoxic stress resistance (17). The Mre11 endonuclease activity is critical for ATM activation, likely triggered by the generation of short oligo-nucleotides (18). In higher eukaryotes, ATM activation relies on MRN binding to DSBs via MRE11, subsequent tethering of DSB ends via RAD50 and recruitment of ATM. This interaction leads to monomerisation of inactive ATM dimers, followed by autophosphorylation. The MRN subcomplex member NBS1 interacts with monomeric ATM leading to its localization in close proximity of the DSB site (19). NBS1, H2AX, the checkpoint kinase CHK2, and the trimeric replication protein A (RPA) are important downstream targets of ATM (6).In yeast, ATR is activated by RPA coated single-stranded DNA (ssDNA) that is generated by 5′ resection mediated by MRX/N, Exo1, Sgs1, and Dna2 during DSB processing (20). Furthermore, ssDNA may become exposed because of replication fork break down during DNA replication or nucleotide excision repair (21). Exposed ssDNA is rapidly bound by RPA, attracting ATRIP, and the Rad17-RFC complex. ATRIP interacts with ATR and is essential for its activation and function (22). The Rad17-RFC complex is functional in loading the 9–1-1 protein complex (Rad9, Rad1, and Hus1) to 5′ dsDNA-ssDNA junctions, in turn stimulating ATR activity at the site of the exposed ssDNA (23). In human cells, TopBP1 is required for activation of ATR and localizing to DNA lesion sites. Rad17, TopBP1, RPA, and the checkpoint kinase CHK1 are known downstream targets of activated ATR.The core effectors of DNA repair (e.g. RAD51), the proteins detecting DNA damage and mediating initiation of repair (e.g. MRX) and the two master regulators ATM and ATR are conserved in plants but many downstream components have diverged considerably. Yet, a comprehensive model for DDR in plants requires identification of all components to delineate the involved signaling pathways and their cross-talk with other regulatory processes.Mass spectrometry-based methods are powerful and hypothesis-free approaches for protein characterization, enabling high-throughput studies of protein complexes (24), protein expression profiling (25), or large-scale identification of protein kinase targets (26). Also, the identification and quantification of thousands of phosphopeptides has become feasible by technological and methodological advances. As a consequence, system-wide analyses of signaling networks has become possible (27). Despite above mentioned advances, comprehensive phosphoproteomic studies remain challenging in regards to sample preparation and phosphopeptide enrichment. An additional complication in large-scale studies is imposed by the requirement for correct automatic localization of phosphorylation sites (28). Abundant metabolites make sample preparation in plants especially difficult; however, a number of large-scale phosphoproteomic studies have been reported (29–34).Phosphorylation sites in proteins are in most cases substoichiometric. As a consequence, the comprehensive analysis requires enrichment of phosphopeptides prior to LC-MS/MS. From the large number of developed methods (35), metal-based affinity chromatography such as immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) have become most widely used. Both materials have different specificities, resulting in a substantial increase of identified phosphopeptides when employed consecutively (36).Here we delineate a methodology to identify and relatively quantify phosphorylation events proteome-wide in higher plants (Fig. 1). We demonstrate its applicability in the context of ATM and ATR dependent DNA damage repair in Arabidopsis thaliana. The approach combines filter assisted sample preparation (FASP) (37), isobaric labeling via iTRAQ, phosphopeptide enrichment using IMAC and TiO₂, strong cation exchange (SCX) chromatography, followed by LC-MS/MS. We compared the relative differences between the phosphoproteomes of wild type plants with the double mutant (atm atr), studying both irradiated and nonirradiated plants. In addition, we performed an independent analysis based on peptide generation after protein precipitation.Open in a separate windowFig. 1.Workflow for identification of ATM/ATR dependent and independent phosphorylations. Wild type and atm atr double mutant plants were either exposed to irradiation or grown under regular conditions. Extracted proteins were purified via FASP and labeled with iTRAQ. Phosphopeptides were enriched by consecutive application of IMAC and TiO₂. Both, the phosphopeptide-enriched fraction and the flow-through of the TiO₂ chromatography, were separated by SCX chromatography and fractions were analyzed by reversed phase LC-MS/MS.All together, we identified 10,831 proteins. Four-hundred and 13 phosphoproteins are phosphorylated upon ionizing radiation, among them 108 in an ATM/ATR dependent manner. The acquired data-set represents a unique resource for plant researchers and extends the current knowledge on ATM/ATR dependent DNA repair pathways.     

Enhanced survival of lethally irradiated adenosine A3 receptor knockout mice. A role for hematopoietic growth factors?

Adenosine A₃ receptor knockout (A₃AR KO) mice and their wild-type (WT) counterparts were compared from the point of view of their abilities to survive exposures to lethal doses of γ-radiation belonging to the range of radiation doses inducing the bone marrow acute radiation syndrome. Parameters of cumulative 30-day survival (experiment using a midlethal radiation dose) or cumulative 11-day survival (experiment using an absolutely lethal radiation dose), and of mean survival time were evaluated. The values of A₃AR KO mice always reflected their higher survival in comparison with WT ones, the P values being above the limit for statistical significance after the midlethal radiation dose and standing for statistical significance after the absolutely lethal radiation dose. This finding was considered surprising, taking into account the previously obtained findings on defects in numbers and functional properties of peripheral blood cells in A₃AR KO mice. Therefore, previous hematological analyses of A₃AR KO mice were supplemented in the present studies with determination of serum levels of the granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Though distinct differences in these parameters were observed between A₃AR KO and WT mice, none of them could explain the relatively high postirradiation survival of A₃AR KO mice. Further studies on these mice comprising also those on other than hemopoietic tissues and organs can help to clarify their relative radioresistance.     

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta)

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an “in-house” epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of ³H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using β-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because β-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used β-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.     

Cytochemical and X-ray microanalytical studies of calcium in the extending zone, particularly in the rhizodermis of Zea mays roots

Calcium was detected in CaCl (10 m M )-pretreated roots of Zea mays L. (cv. LG 11) by means of electron probe X-ray microanalysis with energy-dispersive spectrometry (EDS) using embedded samples, fixed by the antimonate staining procedure. A high level of calcium was found where large amounts of antimonate precipitates were observed by light or transmission electron microscopy. In the elongation zone, after 20 h in humid air following a 2 h CaCl₂ pretreatment, the level of calcium was higher in trichoblasts than in atrichoblasts. In these cells it was detected mainly in the walls and nucleus, and antimonate staining was observed in the walls. Abundant precipitates containing calcium were associated with the nucleus, vacuoles, mitochondria and endoplasmic reticulum of non-differentiated cells, whereas they were confined to the walls of these cells just after the CaCl₂ pretreatment. The involvement of calcium in the formation of root hairs is discussed.