Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]–[4]. Using a purified DDB2 DRC, we created a probe (“DDB2 proteo-probe”) that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.     

CDC42 and Rac1 are implicated in the activation of the Nef-associated kinase and replication of HIV-1

Background : The negative factor (Nef) of human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV) is required for high levels of viremia and progression to AIDS. Additionally, Nef leads to cellular activation, increased viral infectivity and decreased expression of CD4 on the cell surface. Previously, we and others demonstrated that Nef associates with a cellular serine kinase (NAK) activity. Recently, it was demonstrated that NAK bears structural and functional similarity to p21-activated kinases (PAKs).Results : In this study, we demonstrate that Nef not only binds to but also activates NAK via the small GTPases CDC42 and Rac1. First, the dominant-negative PAK (PAKR), via its GTPase-binding domain, and dominant-negative GTPases (CDC42Hs-N17 and Rac1-N17) block the ability of Nef to associate with and activate NAK. Second, constitutively active small GTPases (CDC42Hs-V12 and Rac1-V12) potentiate the effects of Nef. Third, interactions between Nef and NAK result in several cellular effector functions, such as activation of the serum-response pathway. And finally, PAKR, CDC42Hs-N17 and Rac1-N17 decrease levels of HIV-1 production to those of virus from which the nef gene is deleted.Conclusions : By activating NAK via small GTPases and their downstream effectors, Nef interacts with regulatory pathways required for cell growth, cytoskeletal rearrangement and endocytosis. Thus, NAK could participate in the budding of new virions, the modification of viral proteins and the increased endocytosis of surface molecules such as CD4. Moreover, blocking the activity of these GTPases could lead to new therapeutic interventions against AIDS.     

Beschleunigung der „Dunkeladaptation“ eines UV-Rezeptors durch sichtbare Strahlung

Zusammenfassung Elektrophysiologische Untersuchungen über das adaptive Verhalten der UV-Rezeptoren von Ascalaphus im Frontalauge zeigten, daß die Empfindlichkeit nach UV-Helladaptation durch Strahlung des sichtbaren Bereichs zwischen 420 und 550 nm erheblich beschleunigt wird. Die Wellenlänge 589 erwies sich als nahezu unwirksam, Strahlung im Bereich zwischen 460 und 480 nm war am wirksamsten. Die spektrale Empfindlichkeit für die regenerative Wirkung der sichtbaren Strahlung ist identisch mit der spektralen Absorptionswahrscheinlichkeit des thermostabilen Folgefarbstoffes B. Aus der spektralen Verteilung des Himmelslichtes und der spektralen Absorptionswahrscheinlichkeit für den UV-Sehfarbstoff und seinem Folgefarbstoff wird abgeleitet, daß der UV-Rezeptor ohne jede chemische Resynthese des Sehpigments, allein durch Quantenabsorption, seine Empfindlichkeit zu steuern vermag. Wie beim Tintenfischrezeptor (Eledone) wird das Rezeptorpotential nur durch Treffer am Ausgangsfarbstoff ausgelöst, nicht durch Treffer am Folgefarbstoff B.

---

Acceleration of dark-adaptation by visible light in UV-receptors

Summary The adaptation of the ultra-violet (UV) receptors in the frontaleye of the insect Ascalaphus (Neuroptera) was studied by electrophysiological techniques. The measurements showed that the sensitivity increase after illumination by an UV adapting light is considerably accelerated by exposure to visible light with wavelengths between 420 and 550 nm (Figs. 1, 2). Most effective are the wavelengths between 460 and 480 nm (Table 1). The wavelength 589 nm has almost no effect. The spectral efficiency of the regenerative effect is identical to the probability that light quanta will be absorbed by the thermostable secondary pigment. The results suggest that the sensitivity of the UV receptor is determined solely by the absorption of light quanta, without any chemical resynthesis of visual pigment. As in cuttle-fish, a receptor potential is elicited only when light is absorbed by the primary pigment. Light absorption by the secondary pigment is ineffective.

---

---

Mit Unterstützung durch die DFG, SFB Bionach.

---

Mit Unterstützung durch die jugoslawischen SBK- und SFNR-Fonds.     

An efficient and convenient microwave-assisted chemical synthesis of (thio)xanthones with additional in vitro and in silico characterization

Xanthones and their thio-derivatives are a class of pleiotropic compounds with various reported pharmacological and biological activities. Although these activities are mainly determined in laboratory conditions, the class itself has a great potential to be utilized as promising chemical scaffold for the synthesis of new drug candidates. One of the main obstacles in utilization of these compounds was related to the difficulties in their chemical synthesis. Most of the known methods require two steps, and are limited to specific reagents not applicable to a large number of starting materials. In this paper a new and improved method for chemical synthesis of xanthones is presented. By applying a new procedure, we have successfully obtained these compounds with the desired regioselectivity in a shorter reaction time (50s) and with better yield (>80%). Finally, the preliminary in vitro screenings on different bacterial species and cytotoxicity assessment, as well as in silico activity evaluation were performed. The obtained results confirm potential pharmacological use of this class of molecules.     

Quality of life changes after inguinal hernia repair using anterior rectus sheath--a preliminary study

Chronic pain is the most serious long-term complication after groin hernia repair. The aim of this preliminary research was to assess the quality of life before and after standard tension-free mesh repair and new method of tension-free inguinal hernia repair using anterior rectus sheath. Total of 62 patients were evaluated. Anterior rectus sheath method was performed in 29 patients and in 33 patients standard mesh repair was used (Lichtenstein repair). Quality of life was assessed before and after the surgery using short-form SF-36 questionnaire (QualityMetric Inc.), adjusted for Croatian language. There were statistically significant improvements in bodily pain and general health scores in both groups. Patients operated using mesh technique also demonstrated statistically significant improvements in social functioning and emotional role. Similarly, patients in whom inguinal hernia was repaired using anterior rectus sheath had significantly better postoperative scores for physical functioning and role physical scores. Quality of life assessment demonstrated good ability to differentiate between several independent aspects of quality of life. Anterior rectus sheath repair significantly improved quality of life and was shown to be similar to mesh repair in the aspect of physical functioning.     

Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region

Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purification, and characterization of the intracellular region of a Notch ligand. Starting from a set of synthetic oligonucleotides, we assembled a synthetic gene optimized for Escherichia coli codon usage and encoding the cytoplasmic region of human Jagged-1 (residues 1094-1218). The protein containing a N-terminal His(6)-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His(6)-tag by a dipeptidyl aminopeptidase, the protein was purified to homogeneity and characterized by spectroscopic techniques. Far-UV circular dichroism, fluorescence emission spectra, fluorescence anisotropy measurements, and (1)H nuclear magnetic resonance spectra, taken together, suggest that the cytoplasmic tail of human Jagged-1 behaves as an intrinsically unstructured domain in solution. This result was confirmed by the high susceptibility of the recombinant protein to proteolytic cleavage. The significance of this finding is discussed in relation to the recently proposed role of the intracellular region of Notch ligands in bi-directional signaling.