Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6-7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients' genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.     

Binding of bilirubin with albumin-coupled liposomes: implications in the treatment of jaundice

In the present study, we demonstrated the suitability of liposomes as a method of removing plasma bilirubin in hyperbilirubinemic rats. The liposomes have innate tendency to bind with bilirubin through hydrophobic interaction. Among different types of liposomes, the positively charged liposomes were found to have maximum affinity to free bilirubin. However, the entrapment or coupling of serum albumin on the surface of egg phosphatidylcholine liposomes can render a several-fold increase in their bilirubin binding capacity. The proteoliposomes were able to preferentially bind with bilirubin even in the presence of erythrocytes. Interestingly, these liposomes were found to displace bilirubin bound on the surface of erythrocytes as well. The results of the present study further demonstrate that albumin-bearing liposomes were equally effective in removing plasma bilirubin in experimental jaundiced animals. These observations indicate that liposome-mediated selective homing of excess plasma bilirubin to the liver cells (cf. hepatocytes) may help in the development of safer strategy for the treatment of hyperbilirubinemic conditions in the model animals.     

Camelus dromedarius Putative Cytochrome P450 Enzyme CYP2E1: Complete Coding Sequence and Phylogenetic Tree

This study determined the full-length sequence of CYP2E1, one of six cytochrome P450 genes previously examined in camel tissues by western blotting and semi-quantitative PCR. The Camelus dromedarius CYP2E1 has an open reading frame of 1,473 bp, and the cDNA encodes a protein of 490 amino acid residues with a molecular weight of 54.8 kDa. The deduced amino acid sequence showed the highest identity with Bos taurus (88%), Sus scrofa (87%), and Homo sapiens (83%). In a phylogenetic analysis, the C. dromedarius CYP2E1 isoform was located beside cattle and pigs. The deduced amino acid sequence of camel CYP2E1 showed the conserved proline-rich amino terminus and the heme-binding signature localized near the carboxy terminus of the protein.     

Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.     

Astragalus trifoliastrum (Fabaceae), a revived species for the flora of Turkey

As the result of surveying the relevant type specimens, together with macro‐ and micro‐morphological studies, chromosome counting and ITS sequencing, Astragalus trifoliastrum was found to be a species independent of A. laguriformis (with which it has peviously been synonymized). In contrast, A. wanensis, assumed to be a synonym of A. trifoliastrum, indeed appears to be identical with A. trifoliastrum. The diploid chromosome number of 2n = 16 is reported for the first time for A. trifoliastrum.     

Liquid chromatographic assay of abouthiouzine in plasma and its application to pharmacokinetic studies

Abouthiouzine is a newly synthesized antithyroid agent with a proposed less adverse effects profile than other currently used drugs. A simple and rapid reversed phase high performance liquid chromatography assay was developed to determine the concentration of abouthiouzine in human plasma. The procedure involved extraction of the drug and propranolol (internal standard) from the plasma using ethylacetate. The extract was evaporated under nitrogen and the residue was constituted with the mobile phase and injected onto micro-Bondapack phenyl column (10 microm, 3.9 mm x 150 mm). The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer, acetonitrile, and methanol in the ratio of 60:25:15 (v/v/v, pH=3.0), which was delivered at a rate of 1.5 ml/min. Abouthiouzine and the internal standard were monitored using UV detection at 240 nm; the run time was less than 5 min. The detection limit of abouthiouzine is 0.5 microg/ml. The within- and between-day coefficients of variation were less than 7%. Our method has been successfully used to measure abouthiouzine plasma concentrations in a rabbit model following an intravenous administration of the drug.     

Combined millimeter wave and cyclophosphamide therapy of an experimental murine melanoma

The objective of the present studies was to investigate whether millimeter wave (MMW) therapy can increase the efficacy of cyclophosphamide (CPA), a commonly used anti-cancer drug. The effect of combined MMW-CPA treatment on melanoma growth was compared to CPA treatment alone in a murine model. MMWs were produced with a Russian made YAV-1 generator. The device produced 42.2 +/- 0.2 GHz modulated wave radiation through a 10 x 20 mm rectangular output horn. The animals, SKH-1 hairless female mice, were irradiated on the nasal area. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm2, respectively. The maximum skin surface temperature elevation measured at the end of 30 min irradiation was 1.5 degrees C. B16F10 melanoma cells (0.2 x 10(6)) were implanted subcutaneously into the left flank of mice on day 1 of the experiment. On days 4-8, CPA was administered intraperitoneally (30 mg/kg/day). MMW irradiation was applied concurrently with, prior to or following CPA administration. A significant reduction (P < .05) in tumor growth was observed with CPA treatment, but MMW irradiation did not provide additional therapeutic benefit as compared to CPA alone. Similar results were obtained when MMW irradiation was applied both prior to and following CPA treatment.     

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na⁺/H⁺ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via α_v integrin/CD46 in HeLa cells.Human rhinoviruses (HRVs), members of the family Picornaviridae that represent a major cause of the common cold, essentially utilize two different receptor types for host cell attachment. The 12 minor group HRVs, exemplified by HRV2, bind low-density lipoprotein receptor (LDLR), LDLR-related protein (LRP) (20), and very-LDLR (VLDLR) (29) and are internalized via the well-characterized clathrin-dependent endocytic pathway (44); however, these ligands, like others, can switch to different entry portals when the clathrin-dependent pathway is blocked (4). Once the virus arrives in endosomal carrier vesicles or late endosomes, uncoating (i.e., the release of the viral RNA genome) is triggered by the acidic pH (35, 39).The 87 major group HRVs, exemplified by HRV14, bind intercellular adhesion molecule-1 (ICAM-1). Following entry, uncoating is triggered by ICAM-1 itself (3), but the low endosomal pH facilitates this process (37). Based on inhibition of infection by the dominant negative (DN) dynamin-2 mutant dyn^K44A, it was proposed that HRV14 also follows a clathrin-dependent pathway in HeLa-H1 cells (9). However, ICAM-1 lacks a clathrin localization signal and even functions as a viral receptor when its cytoplasmic tail is replaced with a glycosylphosphatidylinositol (GPI) anchor (45). Furthermore, dynamin has also been shown to be involved in pathways other than clathrin-mediated endocytosis (CME), such as caveolae- and lipid raft-dependent entry, as a function of ligand and cell type (reviewed in references 30 and 34). Additionally, dynamin might play a role in formation and closure of circular pinocytic ruffles (31). More recently, a specific entry pathway for ICAM-1 ligands into human umbilical vein endothelial cells was identified and termed “cam-mediated endocytosis”; uptake was found to be triggered upon binding of multivalent ligands, such as immunoconjugates and immunobeads, and to occur independently from clathrin and caveolin. Inhibition by amiloride, actin depolymerization, and protein kinase C inhibitors pointed to macropinocytosis (33). So far, it is not known whether these findings are relevant to the entry pathway of HRVs via ICAM-1 as the uptake kinetics was significantly dependent on particle size. For all these reasons, involvement of clathrin in HRV14 uptake is questionable. Accordingly, we explored entry of HRV14 via ICAM-1 and compared the results with the well-characterized clathrin-dependent entry pathway of HRV2 (44). Employing pharmacological compounds, specific DN inhibitors, immunofluorescence, and electron microscopy, we demonstrate that HRV14 enters rhabdomyosarcoma ICAM-1-expressing (RD-ICAM) cells via a pathway independent of clathrin, caveolin, and flotillin.