Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula: ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL DIPHOSPHATE SYNTHASE GENE,LAVANDULYL DIPHOSPHATE SYNTHASE*

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent K_m and k_cat values of 208 ± 12 μm and 0.1 s⁻¹, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.     

Evaluation of the effects of weak and moderate static magnetic fields on the characteristics of human low density lipoprotein in vitro

It has been suggested that exposure to electromagnetic fields may be a risk factor for cardiovascular disease in humans. Low density lipoprotein (LDL) modifications such as peroxidation and aggregation have been implicated in the pathogenesis of atherosclerosis. The present study investigated the effects of weak (0.125–0.5 mT) and moderate (1–4 mT) static magnetic fields (SMFs) on LDL oxidation, aggregation and zeta potential in vitro. Our results demonstrated that magnetic flux densities of 0.25 and 0.5 mT decreased, and magnetic flux densities of 3 and 4 mT increased the zeta potential and LDL oxidation in comparison with the control samples. All doses of SMFs increased the LDL aggregation in a time‐ and dose‐dependent manner. It is concluded that SMFs can alter the susceptibility of LDL to oxidation and this alteration is dependent on the applied magnetic flux density. The SMF, in addition to its role in the production and stabilization of free radicals and promotion of lipid peroxidation, may influence the metabolism of lipoproteins and their interaction with other molecules such as apolipoproteins, enzymes and receptors through the alteration of the LDL zeta potential and its particles tendency to aggregation. Bioelectromagnetics 34:397–404, 2013. © 2012 Wiley Periodicals, Inc.     

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

Characterizing a Histidine Switch Controlling pH-Dependent Conformational Changes of the Influenza Virus Hemagglutinin

During the fusion of the influenza virus to the host cell, bending of the HA2 chain of hemagglutinin into a hairpin-shaped structure in a pH-dependent manner facilitates the fusion of the viral envelope and the endosomal membrane. To characterize the structural and dynamical responses of the hinge region of HA2 to pH changes and examine the role of a conserved histidine in this region (the hinge histidine), we have performed an extensive set of molecular dynamics (MD) simulations of 26-residue peptides encompassing the hinge regions of several hemagglutinin subtypes under both neutral and low pH conditions, modeled by the change of the protonation state of the hinge histidine. More than 70 sets of MD simulations (collectively amounting to 25.1 μs) were performed in both implicit and explicit solvents to study the effect of histidine protonation on structural dynamics of the hinge region. In both explicit and implicit solvent simulations, hinge bending was consistently observed upon the protonation of the histidine in all the simulations starting with an initial straight helical conformation, whereas the systems with a neutral histidine retained their primarily straight conformation throughout the simulations. Conversely, the MD simulations starting from an initially bent conformation resulted in the formation of a straight helical structure upon the neutralization of the hinge histidine, whereas the bent structure was maintained when the hinge histidine remained protonated. Finally, mutation of the hinge histidine to alanine abolishes the bending response of the peptide altogether. A molecular mechanism based on the interaction of the hinge histidine with neighboring acidic residues is proposed to be responsible for its role in controlling the conformation of the hinge. We propose that this might present a common mechanism for pH-controlled structural changes in helical structures when histidines act as the pH sensor.     

Epigallocatechin-3-gallate up-regulates tumor suppressor gene expression via a reactive oxygen species-dependent down-regulation of UHRF1

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16^INK4A genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16^INK4A is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16^INK4A and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16^INK4A and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.     

Enhancement of nitrogen release properties of urea–kaolinite fertilizer with chitosan binder

The use of controlled release fertilizer (CRF) has become a new trend to minimize environmental pollution. In this study, urea–kaolinite containing 20 wt% urea after one hour dry grinding was mixed with different concentrations of chitosan as a binder to prepare nitrogen-based CRF. Fourier transform infrared spectroscopy confirmed the hydrogen bonding between urea and kaolinite. Covalent interaction between urea–kaolinite and chitosan make the granules stronger. The nitrogen release was measured in 5 days interval using a diacetylmonoxime calorimetric method at a wavelength of 527 nm. The results illustrated that by increasing the chitosan concentration from 3 to 7.5%, nitrogen release decreased from 41.23 to 25.25% after one day and from 77.31 to 59.27% after 30 days incubation in water. Compressive stress at break tests confirmed that granules with chitosan 6% had the highest resistance and were chosen for ammonia volatilization tests. Ammonia volatilization was carried out using the forced-draft technique for a period of 10 weeks. The results showed that the total amount of ammonia loss for conventional urea fertilizer and urea–kaolinite–chitosan granules was 68.63 and 56.75%, respectively. This controlled release product could be applied in agricultural crop production purpose due to its controlled solubility in the soil, high nutrient use efficiency and potential economic benefits.     

Detection of Vero Cells Infected with Herpes Simplex Types 1 and 2 and Varicella Zoster Viruses Using Raman Spectroscopy and Advanced Statistical Methods

Of the eight members of the herpes family of viruses, HSV1, HSV2, and varicella zoster are the most common and are mainly involved in cutaneous disorders. These viruses usually are not life-threatening, but in some cases they might cause serious infections to the eyes and the brain that can lead to blindness and possibly death. An effective drug (acyclovir and its derivatives) is available against these viruses. Therefore, early detection and identification of these viral infections is highly important for an effective treatment. Raman spectroscopy, which has been widely used in the past years in medicine and biology, was used as a powerful spectroscopic tool for the detection and identification of these viral infections in cell culture, due to its sensitivity, rapidity and reliability. Our results showed that it was possible to differentiate, with a 97% identification success rate, the uninfected Vero cells that served as a control, from the Vero cells that were infected with HSV-1, HSV-2, and VZV. For that, linear discriminant analysis (LDA) was performed on the Raman spectra after principal component analysis (PCA) with a leave one out (LOO) approach. Raman spectroscopy in tandem with PCA and LDA enable to differentiate among the different herpes viral infections of Vero cells in time span of few minutes with high accuracy rate. Understanding cell molecular changes due to herpes viral infections using Raman spectroscopy may help in early detection and effective treatment.