The Rad5 Helicase and RING Domains Contribute to Genome Stability through their Independent Catalytic Activities.

Genomic stability is compromised by DNA damage that obstructs replication. Rad5 plays a prominent role in DNA damage bypass processes that evolved to ensure the continuation of stalled replication. Like its human orthologs, the HLTF and SHPRH tumor suppressors, yeast Rad5 has a RING domain that supports ubiquitin ligase activity promoting PCNA polyubiquitylation and a helicase domain that in the case of HLTF and Rad5 was shown to exhibit an ATPase-linked replication fork reversal activity. The RING domain is embedded in the helicase domain, confusing their separate investigation and the understanding of the exact role of Rad5 in DNA damage bypass. Particularly, it is still debated whether the helicase domain plays a catalytic or a non-enzymatic role during error-free damage bypass and whether it facilitates a function separately from the RING domain. In this study, through in vivo and in vitro characterization of domain-specific mutants, we delineate the contributions of the two domains to Rad5 function. Yeast genetic experiments and whole-genome sequencing complemented with biochemical assays demonstrate that the ubiquitin ligase and the ATPase-linked activities of Rad5 exhibit independent catalytic activities in facilitating separate pathways during error-free lesion bypass. Our results also provide important insights into the mutagenic role of Rad5 and indicate its tripartite contribution to DNA damage tolerance.

DNA-dependent protease activity of human Spartan facilitates replication of DNA-protein crosslink-containing DNA.

Mutations in SPARTAN are associated with early onset hepatocellular carcinoma and progeroid features. A regulatory function of Spartan has been implicated in DNA damage tolerance pathways such as translesion synthesis, but the exact function of the protein remained unclear. Here, we reveal the role of human Spartan in facilitating replication of DNA-protein crosslink-containing DNA. We found that purified Spartan has a DNA-dependent protease activity degrading certain proteins bound to DNA. In concert, Spartan is required for direct DPC removal in vivo; we also show that the protease Spartan facilitates repair of formaldehyde-induced DNA-protein crosslinks in later phases of replication using the bromodeoxyuridin (BrdU) comet assay. Moreover, DNA fibre assay indicates that formaldehyde-induced replication stress dramatically decreases the speed of replication fork movement in Spartan-deficient cells, which accumulate in the G2/M cell cycle phase. Finally, epistasis analysis mapped these Spartan functions to the RAD6-RAD18 DNA damage tolerance pathway. Our results reveal that Spartan facilitates replication of DNA-protein crosslink-containing DNA enzymatically, as a protease, which may explain its role in preventing carcinogenesis and aging.

Circularly polarized carrier-envelope-phase stable attosecond pulse generation based on coherent undulator radiation.

In this Letter, we present a new method for generation of circularly polarized attosecond pulses. According to our calculations, shape-controlled, carrier-envelope-phase stable pulses of several hundred nanojoule energy could be produced by exploitation of the coherent undulator radiation of an electron bunch. Our calculations are based on an existing particle accelerator system (FLASH II in DESY, Germany). We investigated the energy dependence of the attosecond pulses on the energy of electrons and the parameters of the radiator undulator, which generate the electromagnetic radiation.

The role of hemorheological factors in cardiovascular medicine.

Cardiovascular diseases (CVD) are the most frequent cause of death throughout the world. The coronary vessel system is a special part of the circulation since there is a continuous change in blood flow, perfusion pressure and shear rate during each cardiac cycle. It is also the place of the narrowest capillaries in the human body, therefore the role of rheological alterations may be of greater importance than in the other parts of the circulatory system. During the past decades, our group has investigated hemorheological parameters (HP) in over 1,000 patients diagnosed with various forms of ischemic heart disease (IHD). In one prospective study, we measured the HP of patients with acute coronary syndrome (ACS). On admission, all examined variables were significantly worse than those of control subjects. During the hospital phase, some of the HP showed further deterioration, and HP remained in the pathologic range during the follow-up period. In another study, we showed that HP are in close correlation with the severity of coronary artery disease. In patients treated with percutaneous coronary intervention, changes in HP were very similar to those observed in subjects with ACS. In a recent study, we analyzed HP in patients undergoing CABG surgery. Our data suggest a hemorheological advantage of off-pump surgery. In another study low Hct/WBV ratio can be regarded as a risk factor of cardiac death in IHD. Our data indicate that rheological parameters are significantly altered in patients with IHD: the extent of the alterations is in excellent correlation with the clinical severity of the disease. Our findings prove that HP play a critical role in the pathogenesis of myocardial ischemia. In recent in vitro and in vivo studies we have investigated the effects of red wine on hemorheological parameters. Our results show that moderate red wine consumption has beneficial effects on hemorheological parameters which may contribute to the French paradox.

Reviewing data reduction methods for ektacytometry.

Ektacytometry quantifies erythrocyte deformability by measuring the elongation of suspended red blood cells subjected to a range of shear stresses. Raw shear stress-elongation index plots are difficult to interpret and thus data reduction methods characterizing the relationship using few parameters without loss of information and good reproducibility are essential, especially for the clinician. Two such curve fitting formulas, used widely in the literature for this purpose, are reviewed herein. The Lineweaver-Burke method overestimates maximal deformability if shear stresses below 1 Pa are applied. A modified version of the formula estimates maximal deformation more accurately but gives little weight to data at low shear stresses. Neither method is accurate if negative elongation indices are present (artifact phenomenon when measurement is performed from high to low shear stresses). The Streekstra-Bronkhorst method provides efficient data reduction though the theoretical background of the formula is incorrect. The parameters have expressive meaning; however, both maximal and minimal deformations are slightly underestimated. Moreover, parameters are biased according to the range of measured shear stresses.

Pituitary adenylate cyclase activating polypeptide in the retina: focus on the retinoprotective effects.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects against different neuronal injuries, such as traumatic brain and spinal cord injury, models of neurodegenerative diseases, and cerebral ischemia. PACAP and its receptors are present in the retina. In this study, we summarize the current knowledge on retinal PACAP with focus on the retinoprotective effects. Results of histological, immunohistochemical, and molecular biological analysis are reviewed. In vitro, PACAP shows protection against glutamate, thapsigargin, anisomycin, and anoxia. In vivo, the protective effects of intravitreal PACAP treatment have been shown in the following models of retinal degeneration in rats: excitotoxic injury induced by glutamate and kainate, ischemic injury, degeneration caused by UV-A light, optic nerve transection, and streptozotocin-induced diabetic retinopathy. Studying the molecular mechanism has revealed that PACAP acts by activating antiapoptotic and inhibiting proapoptotic signaling pathways in the retina in vivo. These studies strongly suggest that PACAP is an excellent candidate retinoprotective agent that could be a potential therapeutic substance in various retinal diseases.

In vitro hemorheological effects of red wine and alcohol-free red wine extract.

The French paradox is based on epidemiological evidence which supports that moderate red wine consumption reduces the risk of cardiovascular diseases. A number of experimental animal studies reported favourable cardiovascular effects of alcohol-free red wine extract (AFRW). Our study was designed to determine red wine and AFRW induced changes in various hemorheological parameters. These effects may play a role in the pathophysiology of the French paradox regarding the cardiovascular protective impacts of red wine. Blood samples of healthy volunteers were mixed with red wine to achieve alcohol concentrations of 1 per thousand, 3 per thousand and 10 per thousand, respectively, with equivalent amount of AFRW or physiological saline. Blood samples were pretreated with red wine or AFRW in order to prove the protective effects on erythrocytes from impairment of deformability caused by the free radical generator phenazine methosulfate (PMS). Erythrocyte aggregation (Myrenne and LORCA), deformability (LORCA) and platelet aggregation (Carat TX4) were measured. Erythrocyte aggregation using Myrenne aggregometer was inhibited by red wine and AFRW compared to the saline treated samples. The difference reached already significance at 1 per thousand concentration at the AFRW samples (p < 0.05). Furthermore, red wine caused stronger inhibition than AFRW. The difference between the two agents became significant at 10 per thousand concentration (p < 0.05). LORCA aggregation index and threshold shear rate supported these results at the highest concentration. Erythrocyte deformability of healthy volunteers did not change significantly for any concentrations of red wine and AFRW. On the other hand AFRW at 3 per thousand concentration significantly prevented erythrocytes from impairment of deformability caused by PMS (p < 0.05). Platelet aggregation was significantly inhibited by the highest concentration of AFRW (p < 0.05). Our results show that red wine and AFRW have some beneficial effects on hemorheological parameters that may contribute to the French paradox.

Hemorheological disturbances in patients with chronic cerebrovascular diseases.

Hemorheological disturbances may occur in more than 40% of patients with ischemic cerebrovascular diseases. In this study the changes of rheological factors--hematocrit, plasma fibrinogen concentration, whole blood and plasma viscosity, red blood cell aggregation and deformability were investigated in 297 patients (173 males, 124 females, mean age 60 +/- 11 years) with transient ischemic attack or chronic phase (> 3 months after onset) ischemic stroke, and in 73 healthy volunteers (35 males, 38 females, mean age 38 +/- 7 years). Hematocrit, plasma and whole blood viscosity were significantly (p < 0.0001) elevated in cerebrovascular patients compared to controls. Plasma fibrinogen concentration (p < 0.001), red blood cell aggregation (p < 0.05) and deformability (p < 0.01) were also impaired in stroke patients. Hemorheological disturbances were dominant in stroke patients with diabetes, hyperlipidemia and smoking habits. Hematocrit, plasma viscosity and red blood cell aggregation showed a significant (p < 0.025-0.001) correlation with the severity of carotid artery stenosis. We could not find any characteristic distribution of rheological parameters among the three subtypes of brain ischemia. Our results show that all of the measured rheological parameters are significantly impaired in chronic ischemic cerebrovascular disorders, especially in diabetic, smoking and alcoholic patients. They correlate with the severity of the carotid artery stenosis, but there is no association with the type of ischemic stroke.

Follow-up of hemorheological parameters and platelet aggregation in patients with acute coronary syndromes.

Pathologic hemorheological parameters and increased platelet aggregation in association with other risk factors significantly increase the possibility of the development of myocardial ischemia. Hemorheological parameters and platelet aggregation were investigated in 157 patients (mean age: 65+/-12 years) with acute coronary syndromes and in 68 healthy subjects (mean age: 36+/-6 years). Plasma fibrinogen, plasma and whole blood viscosity, red blood cell aggregation and filterability and platelet aggregation were measured in the hospital phase (after admission, on 2nd and 6th days) and monitored after discharge (at 1, 6 and 12 months). After admission all these parameters were significantly higher in patients than in control subjects (p<0.01) and almost all of them remained in the pathologic range at discharge. Some of the rheologic parameters showed a slight improvement after 1 month, but hematocrit and whole blood viscosity were higher than those after admission and of control subjects (p<0.05). After 6 and 12 months these parameters showed a small, but significant increase. Pathologically altered hemorheological parameters could be observed in patients with classical cardiovascular risk factors and significant improvement was found after elimination of them. Antiplatelet therapy was efficient in about half of the treated patients after admission; and despite a significant improvement, the proportion of ineffectively treated patients was still considerable during the follow-up. Our results support the role of abnormal hemorheological parameters in the development of myocardial ischemia and draw attention to the rheologic risk of these patients. The results of platelet aggregation measurements show the insufficiency of antiplatelet therapy at some cases and confirm the importance of guided secondary prevention.