Identification of a peptide binding motif for secreted frizzled-related protein-1

Secreted Frizzled-related proteins (sFRPs) bind Wnts and modulate their activity. To identify putative sFRP-1 binding motifs, we screened an M13 phage displayed combinatorial peptide library. A predominant motif, L/V-VDGRW-L/V, was present in approximately 70% of the phage that bound sFRP-1. Use of peptide/alkaline phosphatase chimeras and alanine scanning confirmed that the conserved motif was important for sFRP-1 recognition. The dissociation constant for a peptide/sFRP-1 complex was 3.9 microM. Additional analysis revealed that DGR was the core of the binding motif. Although Wnt proteins lack this sequence, other proteins possessing the DGR motif may function as novel binding partners for sFRP-1.     

Prostaglandin E2 and leukotriene C4 levels following different reperfusion periods in rat brain correlated with morphological changes

Prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) are the metabolites of arachidonic acid (AA) that increase in forebrain following global ischemia and reperfusion. These mediators are highly potent vasoconstrictors of cerebral arteries leading to enhanced vascular permeability that induces the formation of vasogenic edema. In this study, after developing and experimental animal model simulating the concept of ischemic penumbra in the rat, the levels of PGE₂ and LTC₄ produced in the forebrain were measured and the effects of these mediators in short duration and prolonged reperfusion were investigated and then correlated with nueropathological findings. We found statistically significant reduction both in PGE₂ and LTC₄-like activities after just 10 min ischemia (p<0.05, p<0.05). PGE₂-like activity significantly increased in the 4th and 60th min of reperfusion (p<0.05, p<0.05). In the 15th min of reperfusion, PGE₂ was found to be significantly reduced (p<0.005) that may be due to the formation of free oxygen radicals by activation of PG hydroperoxidase reaction that inhibits PGE₂ production in the cylooxygenase pathway. LTs were not significantly increased in any reperfused group. Inhibition of the lipoxygenase pathway of AA metabolism may occur as a result of 15-HPETE (15-hydroperoxyeicosatetraenoic acid) production. Pathologically, edema and degeneration of brain tissue were seen beginning from the 4th min of reperfusion that reached a peak in the 60th min of reperfusion which is in accordance with biochemical changes in the damaged tissue. It is concluded that by preventing the formation of AA metabolites in the early hours of ischemia and reperfusion, it could be possible to increase blood flow in the ischemic penumbra that should limit the infarct area.     

Confinement-Dependent Friction in Peptide Bundles

Friction within globular proteins or between adhering macromolecules crucially determines the kinetics of protein folding, the formation, and the relaxation of self-assembled molecular systems. One fundamental question is how these friction effects depend on the local environment and in particular on the presence of water. In this model study, we use fully atomistic MD simulations with explicit water to obtain friction forces as a single polyglycine peptide chain is pulled out of a bundle of k adhering parallel polyglycine peptide chains. The whole system is periodically replicated along the peptide axes, so a stationary state at prescribed mean sliding velocity V is achieved. The aggregation number is varied between k = 2 (two peptide chains adhering to each other with plenty of water present at the adhesion sites) and k = 7 (one peptide chain pulled out from a close-packed cylindrical array of six neighboring peptide chains with no water inside the bundle). The friction coefficient per hydrogen bond, extrapolated to the viscous limit of vanishing pulling velocity V → 0, exhibits an increase by five orders of magnitude when going from k = 2 to k = 7. This dramatic confinement-induced friction enhancement we argue to be due to a combination of water depletion and increased hydrogen-bond cooperativity.     

Systemic lupus-erythematosus: deoxyribonuclease 1 in necrotic chromatin disposal

Systemic lupus-erythematosus is an auto-immune-disease characterized by pathogenic anti-nuclear auto-antibodies. These form immune-complexes that after deposition at basal membranes at various locations initiate inflammatory reactions. There is a clear genetic and gender predisposition (females are affected 10 times more frequently), but also infectious agents and further environmental factors have been shown to be causative for the initiation of the disease. It has been suggested that the auto-antibodies arise after release and/or inefficient removal of nuclear components during cell death (defective cellular "waste disposal" theory). So far, increased apoptotic cell death has been made responsible, but recent data suggest that defective cellular waste disposal during/after necrosis may also lead to the release and prolonged exposure of nuclear components. Here, we concentrate on chromatin disposal during necrosis and the involvement of Deoxyribonuclease 1 in this process with respect to its possible role in the prevention of anti-nuclear auto-immunity.     

Species distribution and detection of Crimean Congo Hemorrhagic Fever Virus (CCHFV) in field-collected ticks in Ankara Province, Central Anatolia, Turkey

Ticks may act as vectors for a number of infectious diseases including Crimean Congo Hemorrhagic Fever (CCHF). The causative agent is Crimean Congo Hemorrhagic Fever Virus (CCHFV), a member of Bunyaviridae, causing extensive ecchymosis, visceral bleeding and hepatic dysfunction with a high fatality rate in the affected individuals. CCHF was initially recognized in Turkey in 2002 and the current number of reported cases exceeds 4,400. This study was conducted to confirm the presence of tick species established as potential CCHFV vectors and investigate CCHFV activity in ticks at Ankara province, Turkey’s second most-densely populated province, where CCHF cases were demonstrated. A total of 1,196 adult ticks, collected from various animals and vegetation in 12 sites located in 5 counties of Ankara during April–July 2010 were identified to species level. Twenty-two tick pools from county K2 were also evaluated for the presence of CCHFV RNA via a one-step real-time RT-PCR assay and reactive results were further confirmed by an in house nested RT-PCR assay. Nine tick species were identified: Rhipicephalus bursa (44.9%), R. sanguineus (18.9%), R. turanicus (18.1%), Haemaphysalis parva (8.3%), Hyalomma marginatum marginatum (5.4%), H. aegyptium (1.4%), H. anatolicum excavatum (1.3%), Hae. punctata (0.3%) and Dermacentor marginatus (0.2%). A total of five tick pools (22.7%) were reactive in real-time and nested RT-PCR assays. The pools included R. bursa, H. m. marginatum and Hae. parva ticks, collected from mammal hosts from two villages in one county. This is the first documentation of CCHFV activity in ticks from Ankara province, which indicates requirement for detailed surveillance to predict high risk zones in the region.     

Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, deaminated and lipid peroxidation-induced purine adducts. MPG from human and mouse has previously been cloned and expressed. However, due to the poor expression level in Escherichia coli (E. coli) and multi-step purification process of full-length MPG, most successful attempts have been limited by extremely poor yield and stability. Here, we have optimized the codons within the first five residues of human MPG (hMPG) to the best used codons for E. coli and expressed full-length hMPG in large amounts. This high expression level in conjunction with a strikingly high isoelectric point (9.65) of hMPG, in fact, helped purify the enzyme in a single step. A previously well-characterized monoclonal antibody having an epitope in the N-terminal tail could detect this codon-optimized hMPG protein. Surface plasmon resonance studies showed an equilibrium binding constant (K_D) of 0.25 nM. Steady-state enzyme kinetics showed an apparent K_m of 5.3 nM and k_cat of 0.2 min⁻¹ of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100 mM KCl. Unlike the previous reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50 °C. Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure–function analysis.     

Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism

Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated beta-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the beta-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.     

An alternative method to minimize pain in the split-thickness skin graft donor site

In the art of plastic surgery, the reconstruction of tissue defects to obtain cosmetic and functional recovery is the major concern. Skin grafting is the most frequently used procedure for reconstructing defects of various size and anatomical localizations. On the other hand, donor-site problems associated with this invaluable procedure are inevitable. Various methods are used in the postoperative management of the partial-thickness donor site created during the harvest of a split-thickness skin graft. Each technique has the potential for complications of fluid loss, excessive pain, prolonged period of healing and immobilization, hypertrophic scarring, and undesirable pigmentation. Donor-site pain is probably the most disturbing complication in the early postoperative period. The aim of this article is to point out the significance of donor-site pain, which has not been emphasized thoroughly in the literature, and to introduce flap skin as a potential graft donor site for patients in whom reduction of donor-site morbidity is of primary concern. The principal goal of the technique described in this article is to eliminate donor-site pain by harvesting the graft from the flap that is insensate after the elevation. In 15 patients, the overlying skin of the flap that had been used for reconstructive purposes was used as the donor site (group I). In the remaining 23 patients, the posterolateral thigh was used as the donor site (group II). Donor-site discomfort was recorded during the first 8 days postoperatively using a visual analogue scale. To analyze the data, we used the Friedman test, Dunn's multiple comparison test, and Mann-Whitney U test. It was observed that the visual analogue scale of both of the groups showed a significant decrease within days (group I, p < 0.0001; group II, p < 0.0001). The mean pain scores were significantly lower in group I than in group II (p < 0.0001). When donor-site pain is of primary concern, this procedure provides uneventful and comfortable healing while avoiding postoperative pain in the donor site. For that reason, this technique might be used in appropriate cases to minimize donor-site pain.