Light-induced Trpin/Metout Switching During BLUF Domain Activation in ATP-bound Photoactivatable Adenylate Cyclase OaPAC.

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

Mix-and-extrude: high-viscosity sample injection towards time-resolved protein crystallography.

Time-resolved crystallography enables the visualization of protein molecular motion during a reaction. Although light is often used to initiate reactions in time-resolved crystallography, only a small number of proteins can be activated by light. However, many biological reactions can be triggered by the interaction between proteins and ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D-printed microchannels in conjunction with a micronozzle. The diffusive mixing enables the study of the dynamics of samples in viscous media. The device design allows mixing of the ligands and protein crystals in 2 to 20 s. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within lipidic cubic phase, can be mixed efficiently. The potential of this approach for time-resolved membrane protein crystallography to support the development of new drugs is discussed.

Vulvovaginal-gingival Lichen Planus: Association with Lichen Planopilaris and Stratified Epithelium-specific Antinuclear Antibodies.

Vulvovaginal-gingival lichen planus (VVG-LP) consists of a triad of symptoms: vulval, vaginal and gingival lichen planus lesions. The aim of this study was to analyse the prevalence of lesions in various anatomical locations in patients with VVG-LP. The study included 126 consecutive patients with lichen planus. Sixteen (12.7%) patients fulfilled the criteria of VVG-LP. In 12/16 (75%) patients with VVG-LP scalp lesions were also observed. Stratified epithelium-specific antinuclear antibodies (SES-ANA) and anti-ΔNp.3α antibodies were detected in 10/16 (75%) patients with VVG-LP and in 15/110 (13.6%) patients with other forms of lichen planus (p < 0.05). In conclusion, VVG-LP is frequently associated with lichen planopilaris. The new entity may be termed "vulvovaginal-gingival-pilar lichen planus" and our study indicates that SES-ANA is a marker of this type of lichen planus with extensive, severe and refractory-to-therapy involvement of the mucous membranes, skin and scalp.

Gold nanoparticle-enhanced target for MS analysis and imaging of harmful compounds in plant, animal tissue and on fingerprint.

Gold nanoparticle-enhanced target (AuNPET) was used for detailed investigation of various materials of biological origin - human fingerprint, onion bulb and chicken liver. Analysis of these objects was focused on toxic and harmful compounds - designer drug containing pentedrone, diphenylamine in onion and potentially cancerogenic metronidazole antibiotic in liver. Detection of large quantity of endogenous compounds from mentioned objects is also shown. Most of analyzed compounds were also localized with MS imaging and relationship between their function and location was discussed. Detected compounds belong to a very wide range of chemical compounds such as saccharides, ionic and non-ionic glycerides, amino acids, fatty acids, sulfides, sulfoxides, phenols etc. Fingerprint experiments demonstrate application of AuNPET for detection, structure confirmation and also co-localization of drug with ridge patterns proving person-drug contact.

High levels of RAD51 perturb DNA replication elongation and cause unscheduled origin firing due to impaired CHK1 activation.

In response to replication stress ATR signaling through CHK1 controls the intra-S checkpoint and is required for the maintenance of genomic integrity. Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double strand breaks and interstrand crosslinks. In addition, HR, with its key player RAD51, provides critical support for the recovery of stalled forks during replication. High levels of RAD51 are regularly found in various cancers, yet little is known about the effect of the increased RAD51 expression on intra-S checkpoint signaling. Here, we describe a role for RAD51 in driving genomic instability caused by impaired replication and intra-S mediated CHK1 signaling by studying an inducible RAD51 overexpression model as well as 10 breast cancer cell lines. We demonstrate that an excess of RAD51 decreases I-Sce-I mediated HR despite formation of more RAD51 foci. Cells with high RAD51 levels display reduced elongation rates and excessive dormant origin firing during undisturbed growth and after damage, likely caused by impaired CHK1 activation. In consequence, the inability of cells with a surplus of RAD51 to properly repair complex DNA damage and to resolve replication stress leads to higher genomic instability and thus drives tumorigenesis.

Impact of homologous recombination on individual cellular radiosensitivity.

PURPOSE: Individual radiosensitivity as measured with in vitro irradiated lymphocytes using metaphase analysis can predict the risk of normal tissue effects after radiotherapy. This parameter is considered to be primarily determined by the cellular repair capacity of DNA double-strand breaks (DSBs). It is now tested to which extent this capacity also depends on homologous recombination (HR), which is a pathway available when cells are in S/G2 phase. METHODS: Experiments were performed with CHO K1 cells, in which HR was suppressed via knock-down of RAD51 using RNA interference (RNAi). RAD51 was measured via western and foci formation, cell survival by colony forming, DSBs by gammaH2AX foci formation, and chromosomal damage using PCC, G0 or G2 assay. RESULTS: In quiescent G1 cells DSB repair is completed 6h after irradiation. But there is still a substantial fraction of non-repaired DSBs. Most of these DSBs are repaired when G1 cells are stimulated into cell cycle. Suppression of HR by down-regulation of RAD51 did not affect this repair. In contrast, repair was inhibited when cells were irradiated in late S/G2. In line with these data down-regulation of HR did affect survival of cells irradiated in late S/G2, but not in G1. CONCLUSIONS: Individual radiosensitivity as measured for G0/1 cells using metaphase analysis does not depend on homologous recombination.

Lymphoblastoid cell lines differing in p53 status show clear differences in basal gene expression with minor changes after irradiation.

BACKGROUND AND PURPOSE: The genetic profile as determined by microarray is considered to be an ideal marker of the individual radiosensitivity. However, it is still an open question, whether this profile has to be determined prior to or only after irradiation, since the expression of some genes is affected by irradiation. These changes are induced mainly due to a p53-dependent transactivation. MATERIALS AND METHODS: In this study gene expression profiles were measured for 3 lymphoblastoid cell lines differing in p53 status (p53 wt: TK6; p53null: TK6E6, p53mut: WTK1) measured either prior to or 3h after exposure to 2Gy. The gene expression profile was determined using the Affymetrix Human HG U133A GeneChip and for selective genes, variation in gene expression was validated by qRT-PCR. In addition, different assays were used to characterize the radioresponse of these three strains. RESULTS: The three strains were found to be different in all aspects of radiosensitivity studied. Cells with p53wt showed more apoptosis, slightly stronger arrest in G1, but less lethal aberrations and a lower viability when compared to cells with mutated p53, whereas cells absent in p53 are characterized by an intermediate response. The gene expression profile measured prior to irradiation already revealed huge differences. Significance analysis of microarrays (SAM) identified 141 genes that changed expression twofold or more with a false discovery rate (FDR) of 5.4%. When compared to p53null cell line with p53wt showed a twofold difference in up- or down-regulation in 28 genes. A much higher variation was even found when p53mut cells were compared with p53null cells with a twofold difference in even 123 genes. The respective genes were found to be involved mainly in apoptosis, cell cycle regulation, metabolisms and signalling but with only one gene relevant for DNA repair. Radiation was found to affect this profile solely for cells with p53wt with a twofold significant up-regulation in only five genes. For selective genes (BCL2, CASP1, CCND2, DDB2, XPC, RAD51C, SESN1, FUCA1, CDKN1A, MDM2, XPC) array data were confirmed by qRT-PCR. CONCLUSION: The result, that the gene expression profile of lymphoblastoid cells differing in p53 status already displayed clear differences when measured prior to irradiation with only few changes after irradiation, which are solely seen for p53wt cells, suggests, that the differences in radiosensitivity observed for these cells are primarily determined by the variation in expression profile present already prior to irradiation.

[Promoter variants of aldosterone synthase gene (CYP11B2) and salt-sensitivity of blood pressure]. / Warianty promotora genu syntazy aldosteronu (CYP11B2) i sodowrazliwosc cisnienia tetniczego.

The T(-344)C polymorphism in promoter of CYP11B2 gene encoding aldosterone synthase has been associated with differences in plasma aldosterone (ALDO) concentrations. In addition, the results of recent study carried out in Japan suggest that C(-344) allele of CYP11B2 may be a genetic marker of salt-sensitive hypertension characterized by low plasma renin activity (PRA) and high ALDO/PRA ratio. Therefore, it raises the question of whether the T(-344)C polymorphism of CYP11B2 gene may be associated with salt-sensitive hypertension in Caucasians. The DNA samples were obtained from 68 Polish hypertensives. During 3 subsequent 1-week periods each subject received diets of normal, low and high sodium content (120-140, 20-40 and 240-260 mmol Na+/day, respectively). Salt sensitivity was expressed as the difference between mean arterial pressure (MAP) on high salt diet and MAP on low salt one (delta MAPH-L). Genomic DNA isolated from peripheral blood nuclear cells was amplified by PCR method with primers flanking the polymorphic region and C(-344) allele was identified by gain of Hae III restriction site. There were 14 TT homozygotes (20.6%), 35 TC heterozygotes (51.5%) and 19 CC homozygotes (27.9%) in the studied group. No significant differences in delta MAPH-L, glomerular filtration rate, natriuresis, excreted fraction of filtered sodium, PRA, ALDO and ALDO/PRA ratio determined on each diet have been found in subjects according to CYP11B2 genotype. Our preliminary results suggest the lack of association of the T(-344)C CYP11B2 polymorphism with salt-sensitive hypertension as well as with activity of plasma renin-angiotensin-aldosterone system in Caucasian patients.

For X-irradiated normal human fibroblasts, only half of cell inactivation results from chromosomal damage.

PURPOSE: To study the relationship between residual double-strand breaks (dsbs), chromosomal damage, and cell inactivation for X-irradiated normal human fibroblasts. METHODS AND MATERIALS: The experiments were performed with 12 normal human fibroblast strains and, for comparison, a fibroblast line from a LiFraumeni patient (LFS2800), a squamous cell carcinoma line (FaDu), and CHO cells. Cells were irradiated in plateau phase, which was followed by immediate or delayed (14 h) plating. Chromosomal damage was measured by metaphase technique and loss of proliferative capacity by colony-forming assay. The data obtained were compared with residual double-strand breaks measured previously (Dikomey et al. IJROBP 2000;46:481-490). RESULTS: For each fibroblast strain, the number of lethal chromosome aberrations (CAs) increased with dose, but with a substantial variation among the strains (coefficient of variation = 20%-26%). The number of lethal aberrations was significantly correlated with the number of residual dsbs measured for the same strain (r(2) = 0.71, p = 0.0006). The residual dsbs were assumed to represent both non- and also mis-rejoined dsbs. There was a significant correlation between lethal aberrations and cell survival, but only for delayed and not immediate plating (r(2) = 0.69, p < 0.0008 vs. r(2) = 0.19, p = 0.16). For delayed plating, the ratio between lethal events (LEs) and CAs amounted to LE:CA = 2.0 +/- 0.05:1, indicating that on average, only half of cell inactivation resulted from chromosomal damage. The other 50% was attributed to the p53-dependent permanent G1 arrest, because cells lacking in functional p53 (LFS2800, FaDu, CHO) showed a ratio of LE:CA = 1.01 +/- 0.02:1. CONCLUSION: On average, up to 50% of the inactivation of X-irradiated normal human fibroblasts is a result of lethal chromosome aberrations, whereas the rest is due to a p53-dependent process, probably permanent G1 arrest.