Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase

Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein-chromophore interactions we developed a new procedure to prepare apo-photolyase. MTHF-free photolyase was obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. Under these conditions the flavin remains bound and the defolated enzyme can be released from the column with 0.5 M imidazole pH 7.2. The MTHF-free protein was still capable of DNA repair, showing 70% activity of native enzyme. Fluorescence polarization experiments confirmed that MTHF binding is weakened at low ionic strength. Apo-photolyase was obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0. The apo-photolyase thus obtained was highly reconstitutable and bound nearly stoichiometric amounts of FAD(ox). Photolyase reconstituted with FAD(ox) had about 34% activity of native enzyme, which increased to 83% when FAD(ox) was reduced to FADH(-). Reconstitution kinetics performed at 20 degrees C showed that apo-photolyase associates with FADH(-) much faster (k(obs) approximately 3,000 M(-1) s(-1)) than with FAD(ox) (k(obs)=16 [corrected] M(-1) s(-1)). The dissociation constant of the photolyase-FAD(ox) complex is about 2.3 microM and that of E-FADH(-) is not higher than 20 nM (pH 7.2).     

Characterization of germination receptors of Bacillus cereus ATCC 14579

Specific amino acids, purine ribonucleosides, or a combination of the two is required for efficient germination of endospores of Bacillus cereus ATCC 14579. A survey including 20 different amino acids showed that l-alanine, l-cysteine, l-threonine, and l-glutamine are capable of initiating the germination of endospores of B. cereus ATCC 14579. In addition, the purine ribonucleosides inosine and adenosine can trigger germination of the spores. Advanced annotation of the B. cereus ATCC 14579 genome revealed the presence of seven putative germination (ger) operons, termed gerG, gerI, gerK, gerL, gerQ, gerR, and gerS. To determine the role of the encoded putative receptors in nutrient-induced germination, disruption mutants were constructed by the insertion of pMUTIN4 into each of the seven operons. Four of the seven mutants were affected in the germination response to amino acids or purine ribonucleosides, whereas no phenotype could be attributed to the mutants with disrupted gerK, gerL, and gerS loci. The strain with a disrupted gerR operon was severely hampered in the ability to germinate: germination occurred in response to l-glutamine but not in the presence of any of the other amino acids tested. The gerG mutant showed significantly reduced l-glutamine-induced germination, which points to a role of this receptor in the l-glutamine germination signaling pathway. gerR, gerI, and gerQ mutants showed reduced germination rates in the presence of inosine, suggesting a role for these operons in ribonucleoside signaling. Efficient germination by the combination of l-glutamine and inosine was shown to involve the gerG and gerI operons, since the germination of mutants lacking either one of these receptors was significantly reduced. Germination triggered by the combination of l-phenylalanine and inosine was lost in the gerI mutant, indicating that both molecules are effective at the GerI receptor.     

Direct detection of human Staphylococcus aureus carriage in the nose using the Lightcycler Staphylococcus kit

The Lightcycler Staphylococcus kit is a diagnostic tool for direct real-time detection of Staphylococcus aureus in clinical materials. We show here that detection of S. aureus nasal carriage using this test is hampered by competition of DNA from coagulase-negative staphylococci. However the test is well suited for species identification after culture and the identification of high-load S. aureus carriers.     

Identification and characterization of human cardiolipin synthase

The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Delta yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Delta expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate-diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed.     

Determination of linear viscoelastic behavior of abdominal aortic aneurysm thrombus

The objective of this study is to determine whether the linear viscoelastic properties of an abdominal aortic aneurysm thrombus can be determined by rheometry. Although large strains occur in the in vivo situation, in this work only linear behavior is studied to show the applicability of the described methods. A thrombus exists of several layers that vary in composition, structure and mechanical properties. Two types of thrombus are described. In discrete transition thrombi the layers are not or at most weakly attached to each other and the structure of each layer is different. Continuous transition thrombi consist of strongly attached layers whose structure changes gradually throughout the thickness of the thrombus. Shear experiments are performed on samples from both types of thrombus on a rotational rheometer using a parallel plate geometry. In the discrete type the storage modulus G' cannot be assumed equal for the different layers. In the continuous thrombus, G', changes gradually throughout the layered structure. In both types the loss modulus, G', does not vary throughout the thrombus. Furthermore, it was found that Time-Temperature Superposition is applicable to thrombus tissue. Since results were reproducible it can be concluded that the method we used to determine the viscoelastic properties is applicable to thrombus tissue.     

Structure-function analysis of the coxsackievirus protein 3A: identification of residues important for dimerization, viral rna replication, and transport inhibition

The coxsackievirus B3 3A protein forms homodimers and plays important roles in both viral RNA (vRNA) replication and the viral inhibition of intracellular protein transport. The molecular determinants that are required for each of these functions are yet poorly understood. Based on the NMR structure of the closely related poliovirus 3A protein, a molecular model of the coxsackievirus B3 3A protein was constructed. Using this structural model, specific mutants were designed to study the structure-function relationship of 3A. The mutants were tested for their capacity to dimerize, support vRNA replication, and block protein transport. A hydrophobic interaction between the monomers and an intermolecular salt bridge were identified as major determinants required for dimerization. We show that dimerization is important for both efficient vRNA replication and inhibition of protein transport. In addition, determinants were identified that were not required for dimerization but that were essential for either one of the biological functions of 3A. The combination of the in silico and in vivo results obtained in this study provides important insights in both the structural and functional aspects of 3A.     

Fluorescent T7 display phages obtained by translational frameshift

Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein. By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3′ end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a −1 shift in reading frame during translation. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. The perspectives of fluorescent display phages in the fast emerging field of single molecule detection and sorting technologies are discussed.     

Annexin A5-conjugated quantum dots with a paramagnetic lipidic coating for the multimodal detection of apoptotic cells

Apoptosis, or programmed cell death, plays an important role in the etiology of a variety of diseases, including cancer. Visualization of apoptosis would allow both early detection of therapy efficiency and evaluation of disease progression. To that aim we developed a novel annexin A5-conjugated bimodal nanoparticle. The nanoparticle is composed of a quantum dot that is encapsulated in a paramagnetic micelle to enable its use both for optical imaging and MRI. Multiple recombinant human annexin A5 protein molecules were covalently coupled to the nanoparticle for targeting. In this study the specificity of the annexin A5-conjugated nanoparticles for apoptotic cells was demonstrated both with fluorescence microscopy and MRI, which confirms its potential for the detection of apoptosis with both imaging modalities in vivo.     

Altered chemokine receptor profile on circulating leukocytes in human heart failure

Chemokines and their receptors have been implicated in the pathogenesis of different forms of heart failure (HF). We examined CC-and CXC-chemokine receptor expression in fresh peripheral blood leukocyte populations from 24 end-stage HF patients consisting of coronary artery disease (CAD; n=6) and hypertrophic cardiomyopathy (HCM; n=7) or idiopathic dilated cardiomyopathy (IDCM; n=8) or valvular disease (VD; n=3) and compared the data with 18 healthy controls. Levels of CCR1, 2, 3, 4, 5, and 7, and CXCR1, 2, 3, and 4 were measured by flow cytometry, and the expression profile was assessed as molecules of equivalent soluble fluorochrome units as well as frequency (percentage) of CD3⁺, CD4⁺, and CD8⁺ T cells and monocytes or granulocytes. Frequency of CD3⁺ CXCR4⁺, CD3⁺ CXCR1⁺, and CD3⁺ CXCR3⁺ cells was significantly increased in HF patients, whereas only CCR7 and CXCR4 expression levels were elevated on CD3⁺ cells. Both CD4⁺ CXCR4⁺ and CD8⁺ CXCR4⁺ cell frequencies were significantly increased irrespective of cardiac disease etiology. Elevated CCR7 expression was less pronounced on CD4⁺ than CD8⁺ cells in patients with CAD and IDCM. Expression of CXCR4 on CD8⁺ cells was upregulated substantially, regardless of the cause of disease. CD8⁺ CXCR1⁺ and CD8⁺ CXCR3⁺ but not CD4⁺ CXCR1⁺ or CD4⁺ CXCR3⁺ cells were increased in the HF patients with IDCM and CAD, respectively. Expression of CXCR1 or CXCR3 on both CD4⁺ and CD8⁺ cells did not differ in all the groups. For monocytes, frequency of CD14⁺ CCR1⁺ and CD14⁺ CCR2⁺ cells was significantly decreased in CAD patients, whereas, increase in CD14⁺ CXCR4⁺ cell frequency was accompanied with elevated CXCR4 expression. On granulocytes, CXCR1 and CXCR2 receptors were downregulated in all patients, compared with controls. Our results suggest that the altered expression profile of CC- and CXC-chemokine receptors on circulating leukocyte populations involves enhanced activation of the immune system, perhaps as part of the pathogenic mechanisms in HF. Modulation of the chemokine network could offer interesting novel therapeutic modalities for end-stage HF.     

Stratospheric Ozone Depletion: High Arctic Tundra Plant Growth on Svalbard is not Affected by Enhanced UV-B after 7 years of UV-B Supplementation in the Field

The response of tundra plants to enhanced UV-B radiation simulating 15 and 30% ozone depletion was studied at two high arctic sites (Isdammen and Adventdalen, 78° N, Svalbard).The set-up of the UV-B supplementation systems is described, consisting of large and small UV lamp arrays, installed in 1996 and 2002. After 7 years of exposure to enhanced UV-B radiation, plant cover, density, morphological (leaf fresh and dry weight, leaf thickness, leaf area, reproductive and ecophysiological parameters leaf UV-B absorbance, leaf phenolic content, leaf water content) were not affected by enhanced UV-B radiation. DNA damage in the leaves was not increased with enhanced UV-B in Salix polaris and Cassiope tetragona. DNA damage in Salix polaris leaves was higher than in leaves of C. tetragona. The length of male gametophyte moss plants of Polytrichum hyperboreum was reduced with elevated UV-B as well as the number of Pedicularis hirsuta plants per plot, but the inflorescence length of Bistorta vivipara was not significantly affected. We discuss the possible causes of tolerance of tundra plants to UV-B (absence of response to enhanced UV-B) in terms of methodology (supplementation versus exclusion), ecophysiological adaptations to UV-B and the biogeographical history of polar plants